RETINOIC ACID RECEPTOR EXPRESSION AND POLYMORPHISMS …
Transcript of RETINOIC ACID RECEPTOR EXPRESSION AND POLYMORPHISMS …
RETINOIC ACID RECEPTOR
EXPRESSION AND
POLYMORPHISMS IN HUMAN
BREAST CANCER
by
Danny Mok BSc(Hons)
This thesis is presented for the degree of
Doctor of Philosophy
of The University of Western Australia
2002
Department of Clinical Biochemistry, PathCentre
Department of Pathology
The University of Western Australia
Sir Charles Gairdner Hospital
Nedlands, Western Australia
Acknowledgments
ACKNOWLEDGMENTS
I would firstly like to thank Dr. John Beilby for his valuable supervision of my
laboratory work, preparation of m y thesis, as well as his tremendous support in times of
trouble and when things were going great.
I gratefully thank Professor John Papadimitriou for his enormous contributions in
reviewing m y thesis, as well as manuscripts for publication and his support of m y PhD
project.
I would also like to thank Professor Roland Hahnel and his wife, Erika, for without
them, this project would not have been possible.
Thanks also to Drs. Peter Garcia-Webb and Chootoo Bhagat for allowing me to use the
facilities within the Department of Clinical Biochemistry, PathCentre. Many thanks to
Dr. David Ingram for allowing m e access to his breast tissue samples.
I wish to thank Associate Professor Theo Gotjamanos for all the support and advice he
has given m e in helping to complete m y PhD and for his mediation of all
correspondence with the University with regards to m y degree.
I am also grateful of the help and advice Associate Professor Ralph Martins and
Guiseppe Verdile offered in the Western blots.
To all my colleagues in the lab: Christine Chin, Clive Hunt, Jodi Burley, Caroline
Chapman, Janeen Bennett, John Prior and Kim Jury, for their great friendship, gossip
sessions and support over the years.
I would like to thank all the staff in the Departments of Clinical Biochemistry,
PathCentre, and Pathology, U W A , for their friendship throughout m y PhD.
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Acknowledgments
To all m y postgraduate student buddies: Anthea Downs, Linda Cunningham, Michael
Swarbrick, Conchita Kuek, David Dunn, Vincent Williams, Sherine Chan, Tane
McNiven, Carlo Pirn, Amerigo Carrello and Peter Mark, for sharing many stories of
failed experiments, disasters in the lab, as well as understanding how difficult a PhD
student's life can be.
Big thanks to all my friends who kept asking the question all PhD students hate hearing:
"how's the PhD going?" Thanks Richie, Tajinder, Pete, Michelle, Marguerite, Dave,
Tracey, Scott, Suzie, Julian, Rebecca, Geoff, Carrie, Angie, Adam, Hassan, Mark, Cath,
Jo and Jurgen.
Very big thanks to my love, Elysia Hollams, for her unending support and
encouragement during the hard times in the laboratory, and for her invaluable effort in
helping m e organise m y thesis writing. Good luck with your PhD, Smurfette!
Finally, I wish to thank my parents, Caroline and David, who always worked hard to
support an education for m e that unfortunately were not available to them. For that, I
dedicate this thesis to m u m and dad. Thanks also to m y brother and sister, Darren and
Davena, just for being there when I needed them.
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Thesis Abstract
THESIS ABSTRACT
Retinoic acid is a natural compound belonging to a group of molecules called
retinoids, which include vitamin A and its biologically active derivatives. Retinoic acid
has been shown to induce cell differentiation both in vivo and in vitro and to inhibit
growth of a wide range of neoplastic cells, including those derived from mammary
carcinomas. The effects of retinoic acid are mediated by nuclear receptors known as the
retinoic acid receptors (RARs) and three isotypes have been identified: RARcc, P and y.
The RARs dimerize with a related nuclear receptor, RXR, and function as ligand-
activated transcription factors by binding to retinoic acid response elements found in the
promoter region of target genes. Loss of expression of RARs has been documented in
various diseases, including malignant cancers, although little information is available
about the status of RARs in breast biopsies. This thesis hypothesised that decreased
expression of RARs and the presence of polymorphisms in their mRNA are involved in
the development and/or progression of human mammary carcinoma. Using PCR, all
three RAR isotypes were detected in normal breast and mammary carcinoma biopsies,
although there was a small loss of RARP expression and a significant loss of RARy
expression observed in mammary carcinoma than in normal breast tissue. This may
suggest that the absence of RARs in breast tissue could lead to malignancy. Further
analysis of RAR mRNA levels in breast tissue using competitive RT-PCR showed
significant decreases in RARP and y expression in mammary carcinoma compared to
normal breast. Semi-quantitative analysis of protein levels also revealed a significant
decrease in the expression of RARP and y in mammary carcinoma compared with
patient-matched normal breast tissue. Statistically, there was no relationship between
Thesis Abstract
RARcc and oestrogen receptor status, in contrast to previous reports in the literature.
However, a positive correlation was observed between ER status and RARy expression,
which has not been previously documented and may have important ramifications on
the use of RARy-selective retinoids in the treatment of breast cancer. No relationship
was observed between RAR protein levels and tumour grade, suggesting that decrease
in RAR expression may be an early event in mammary carcinogenesis. Finally, SSCP
studies to detect polymorphisms in RARs identified a serine to leucine base change at
amino acid 427 in RARy. This polymorphism was observed in 3/72 normal specimens
compared to 7/72 found in mammary carcinoma. Although this was not a significant
difference, analysis of a three-generation family with the S427L polymorphism revealed
it to be heritable. Follow-up studies of the normal specimens could not be performed to
determine if they had developed breast cancer, therefore it is unknown if this
polymorphism predisposes to disease. Whether the S427L polymorphism of the RARy
gene has any role in the development of breast cancer remains to be elucidated. The
results of this thesis show that reduced levels of RARs, as well as genetic
polymorphisms in their exons, may be associated with the initiation or progression of
mammary carcinoma due to insufficient signalling of the normal retinoid response
through its receptors. Clinical trials are currently under way in which breast cancer
patients are given retinoids as treatment of this disease. It is therefore of great
importance that reliable methods are developed for the detection and quantitation of
RARs in breast cancer patients, to determine whether there are sufficient receptors
present in the breast tumour tissue to transduce an effective retinoid response. This
thesis describes such a method, using competitive RT-PCR.
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Statement
STATEMENT
This statement declares that all the work presented in this thesis was performed solely
by the candidate, unless otherwise stated within the thesis.
Danny M o k (Candidate)
Table of Contents
TABLE OF CONTENTS
ACKNOWLEDGMENTS ii
THESIS ABSTRACT iv
STATEMENT vi
TABLE OF CONTENTS vii
LIST OF FIGURES xiii
LIST OF TABLES xviii
ABBREVIATIONS xix
CHAPTER ONE Literature Review 1
1.1 Historical Overview 2
1.2 Metabolism of Retinoic Acid 4
1.2.1 Uptake of Dietary Retinoids 4
1.2.2 Release of Retinol into Cells 6
1.3 Molecular Biology of Retinoid Receptors 7
1.3.1. Types of Retinoid Receptors 10
1.3.2. Structure of RARs 11
1.3.3 Mechanism of RAR-gene targeting 14
1.3.3.1 Up-Regulation of Target Genes 14
1.3.3.2 Down-Regulation of Target Genes 17
1.4 Regulation of Retinoic Acid Receptor Signalling 20
1.5 Retinoids, RARs and Disease 20
1.6 Retinoids, RARs and Mammary Carcinoma 26
1.7 Research Proposal, Aims and Hypothesis of this Study 32
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Table of Contents
CHAPTER TWO Materials and Methods 37
2.1 Specimens 38
2.1.1 Specimens used for PCR Analysis 3 8
2.1.2 Specimens used for Protein Analysis 38
2.1.3 Specimens used for SSCP Analysis 38
2.2 Extraction of Total RNA from Human Breast Tissue 39
2.2.1 Reagents 39
2.2.2 Procedure 40
2.3 Estimation and Integrity Check of Total RNA 41
2.3.1 Reagents 41
2.3.2 Procedure 41
2.4 Synthesis ofcDNA from RNA Samples 42
2.4.1 Reagents 42
2.4.2 Procedure 42
2.5 Extraction of DNA from Whole Blood 43
2.6 Extraction of DNA from Human Serum 43
2.7 Extraction of Protein from Human Breast Tissues 43
2.7.1 Reagents 43
2.7.2 Procedure 44
2.8 Estimation of Total Cell Protein from Breast Tissues 44
2.8.1 Reagents 44
2.8.2 Procedure 44
2.9 Preparation of RARa DNA Competitor for Semi-Quantitative
Competitive Polymerase Chain Reaction (PCR) 45
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2.10 Preparation of Competitor R N A for Competitive RT-PCR 47
2.10.1 Generation of Deletion RAR DNA Competitor Fragments 47
2.10.2 Cloning of Deletion RAR DNA Competitor Fragments 49
2.10.3 Construction of RAR RNA Competitors 49
2.11 PCR Detection of RARs in Human Breast Tissue cDNA 50
2.11.1 Reagents and Equipment 50
2.11.2 PCR Detection of RARs in Human Breast Tissues 51
2.11.3 Semi-Quantitative Competitive PCR Detection of RARa
in Human Breast Cancer Tissue 51
2.11.4 Competitive PCR Detection of RARs in Human Breast
Tissue 53
2.11.5 PCR Detection of RAR Polymorphisms in Human Breast
Tissue 55
2.11.6 PCR Detection of RARy Polymorphism in Serum and
Buffy Coats 59
2.12 Agarose Gel Electrophoresis of PCR Products 59
2.12.1 Reagents 59
2.12.2 Procedure 59
2.13 Preparation of Polyacrylamide Gels for SSCP Analysis 60
2.13.1 Reagents 60
2.13.2 Procedure 60
2.14 SSCP Screening and Sequence Analysis of Polymorphic RARs 61
2.15 Protein Separation on SDS-Polyacrylamide Gels (SDS-PAGE) 62
2.15.1 Reagents 62
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2.15.2 Procedure 63
2.16 Electroblot Transfer of Proteins onto Membranes 63
2.16.1 Reagents 63
2.16.2 Procedure 64
2.17 Immunoblotting (Western Blot) of Breast Tissue Protein for
RARs 64
2.17.1 Reagents 64
2.17.2 Procedure 65
2.18 Quantitation of RAR Competitive PCR Fragments and Protein
Bands 65
CHAPTER THREE PCR Detection of RARs in Breast Tissue 67
3.0 Introduction 68
3.1 PCR Detection of RARs in Breast Tissue 68
3.1.1 Detection of RARs in Normal and Malignant Mammary
Tissue
3.1.2 RAR Status and Tumour Grading in Mammary Carcinoma 69
3.2 Quantitation of RARs using RT-PCR 74
3.2.1 Evaluation of the Competitive RT-PCR Method 74
3.2.1.1 Exponential Amplification of RARa, p and y
mRNA 74
3.2.1.2 Linearity of Competitive RT-PCR 81
3.2.2 Comparison between Semi-Quantitative and Competitive
RT-PCR 81
Table of Contents
3.2.3 Quantitation of RARs in Normal and Malignant Breast
Tissues using Competitive RT-PCR 86
3.2.4 Comparison between RAR mRNA Levels and ER Status
in Mammary Carcinoma 92
3.3 Discussion 96
3.4 Conclusions 104
CHAPTER FOUR Analysis of Retinoic Acid Receptor Protein Levels in
Breast Tissues 106
4.0 Introduction 107
4.1 Distribution of RAR Protein Levels in Breast Tissues 107
4.2 Comparison of RAR and Oestrogen Receptor Protein Levels 111
4.3 Comparison of RAR Protein Levels and Grading of Mammary
Carcinoma Tissue 116
4.4 Discussion 118
4.5 Conclusions 121
CHAPTER FIVE Identification of Retinoic Acid Receptor Polymorphisms 122
5.0 Introduction 123
5.1 Absence of Polymorphisms in RARa and P mRNA in
Normal and Malignant Breast Tissue 124
5.2 Detection of Polymorphisms in RARy mRNA in Normal and
Malignant Breast Tissue 124
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5.3 Detection of RARy Polymorphism in Serum from Females with
Mammary Carcinoma 141
5.4 Detection of RARy Polymorphism in Serum and Whole Blood
from Normal Females 141
5.5 HeritabilityofRARyS427L Polymorphism 142
5.6 Discussion 144
5.7 Conclusions 151
CHAPTER SIX General Discussion 152
6.0 General Discussion 153
6.1 Future Work 159
REFERENCES 161
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List of Figures
LIST OF FIGURES
Figure 1.1.1 Structures of common natural retinoids 3
Figure 1.2.2.1 Metabolism of dietary retinoids 8
Figure 1.3.2.1 Basic structure of retinoic acid receptors 12
Figure 1.7.1 Schematic representation of competitive RT-PCR 35
Figure 2.9.1 Pvu II restriction map of the RARa cDNA region amplified
as described in Section 2.10.2 46
Figure 2.10.1 Restriction maps of the RAR cDNA regions amplified as
described in Section 2.11.2 48
Figure 3.2.1.1.1 Exponential amplification range for RARa using 1 OOng
total sample RNA and 40 pg competitor RNA 75
Figure 3.2.1.1.2 Exponential amplification range for RARP using 1 OOng
total sample RNA and 2 pg competitor RNA 76
Figure 3.2.1.1.3 Exponential amplification range for RARy using 1 OOng
total sample RNA and 40 pg competitor RNA 77
Figure 3.2.1.1.4 Stability of PCR amplification over a number of cycles for
RARa using 100 ng total sample RNA and 40 pg competitor
RNA 78
Figure 3.2.1.1.5 Stability of PCR amplification over a number of cycles for
RARP using 100 ng total sample RNA and 2 pg competitor
RNA 79
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List of Figures
Figure 3.2.1.1.6 Stability of P C R amplification over a number of cycles for
RARy using 100 ng total sample RNA and 40 pg competitor
RNA 80
Figure 3.2.1.2.1 Linearity assay for RARa competitive RT-PCR 82
Figure 3.2.1.2.2 Linearity assay for RARp competitive RT-PCR 83
Figure 3.2.1.2.3 Linearity assay for RARy competitive RT-PCR 84
Figure 3.2.2.1 Semi-quantitative competitive RT-PCR of RARa using a
DNA competitor 85
Figure 3.2.2.2 Comparison between semi-quantitative and competitive
RT-PCR 87
Figure 3.2.3.1 Detection of RARa mRNA levels by competitive RT-PCR 88
Figure 3.2.3.2 Detection of RARp mRNA levels by competitive RT-PCR 89
Figure 3.2.3.3 Detection of RARy mRNA levels by competitive RT-PCR 90
Figure 3.2.3.4 Quantitation of mRNA levels by competitive RT-PCR in
normal breast and primary mammary carcinoma tissue 91
Figure 3.2.4.1 Relationship between ER status and RARa mRNA levels
in mammary carcinoma tissues 93
Figure 3.2.4.2 Relationship between ER status and RARp mRNA levels
in mammary carcinoma tissues 94
Figure 3.2.4.3 Relationship between ER status and RARy mRNA levels
in mammary carcinoma tissues 95
Figure 4.1.1 Detection of RARa protein in normal breast and mammary
carcinoma tissue 108
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List of Figures
Figure 4.1.2 Detection of R A R p protein in normal breast and mammary
carcinoma tissue 109
Figure 4.1.3 Detection of R A R y protein in normal breast and mammary
carcinoma tissue 110
Figure 4.1.4 Comparison of R A R protein levels in normal breast and
mammary carcinoma tissues matched for the same patient 112
Figure 4.2.1 Relationship between R A R a protein levels and E R protein
levels in mammary carcinoma tissues 113
Figure 4.2.2 Relationship between R A R p protein levels and E R protein
levels in mammary carcinoma tissues 114
Figure 4.2.3 Relationship between R A R y protein levels and E R protein
levels in mammary carcinoma tissues 115
Figure 5.1.1 Representative S SCP band patterns observed for R A R a
breast tumour c D N A samples using Alphal primer set 125
Figure 5.1.2 Representative SSCP band patterns observed for R A R a
breast tumour c D N A samples using Alpha2 primer set 126
Figure 5.1.3 Representative SSCP band patterns observed for R A R a
breast tumour c D N A samples using Alpha3 primer set 127
Figure 5.1.4 Representative SSCP band patterns observed for R A R a
breast tumour c D N A samples using Alpha4 primer set 128
Figure 5.1.5 Representative S SCP band patterns observed for R A R a
breast tumour c D N A samples using Alpha5 primer set 129
Figure 5.1.6 Representative SSCP band patterns observed for R A R p
breast tumour c D N A samples using Betal primer set 130
' ^^ = xv
List of Figures
Figure 5.1.7 Representative SSCP band patterns observed for R A R p
breast tumour cDNA samples using Beta2 primer set 131
Figure 5.1.8 Representative SSCP band patterns observed for RARp
breast tumour cDNA samples using Beta3 primer set 132
Figure 5.1.9 Representative SSCP band patterns observed for RARp
breast tumour cDNA samples using Beta4 primer set 133
Figure 5.1.10 Representative SSCP band patterns observed for RARp
breast tumour cDNA samples using Beta5 primer set 134
Figure 5.2.1 Representative SSCP band patterns observed for RARy
breast tumour cDNA samples using Gammal primer set 135
Figure 5.2.2 Representative SSCP band patterns observed for RARy
breast tumour cDNA samples using Gamma2 primer set 136
Figure 5.2.3 Representative SSCP band patterns observed for RARy
breast tumour cDNA samples using Gamma3 primer set 137
Figure 5.2.4 Representative SSCP band patterns observed for RARy
breast tumour cDNA samples using Gamma4 primer set 138
Figure 5.2.5 Representative SSCP band patterns observed for RARy
breast tumour cDNA samples using Gamma5 primer set 139
Figure 5.2.6 Band shift patterns seen for RARy in mammary carcinoma
tissues 140
Figure 5.5.1 Detection of RARy S427L polymorphism in a three-
generation family 143
Figure 5.6.1 Agarose gel showing PCR products of RARy 146
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List of Figures
Figure 5.6.2 Agarose gel of RARy PCR amplification comparing
extraction methods 147
Figure 6.0.1 Model of mammary carcinogenesis due to disruptions
in RAR signalling 160
xvii
List of Tables
LIST OF TABLES
Table 1.3.3.1.1
Table 2.11.2.1
Table 2.11.4.1
Table 2.11.5.1a
Table 2.11.5.1b
Table 2.11.5.2a
Table 2.11.5.2b
Table 2.11.5.3a
Table 2.11.5.3b
Table 3.1.1.1
Table 3.1.1.2
Table 3.1.2.1
Table 4.3.1
Genes known to contain R A R E s in their promoter
regions 16
Sequences of oligonucleotide primers 52
Sequences of oligonucleotide primers for competitive
RT-PCR 54
Sequences of oligonucleotide primers for RARa 56
PCR conditions for amplification of RARa cDNA 56
Sequences of oligonucleotide primers for RARp 57
PCR conditions for amplification of RARp cDNA 57
Sequences of oligonucleotide primers for RARy 58
PCR conditions for amplification of RARy cDNA 58
Detection of RARs in normal human breast tissues 70
Detection of R A R s in primary mammary carcinomas 71
RAR status and tumour grading of mammary carcinoma
biopsies 73
Analysis of RAR protein levels and tumour grading of
mammary carcinoma biopsies 117
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Abbreviations
ABBREVIATIONS
9cRA
AEV
ANOVA
APL
ARAT
ATF
atRA
CBP
CRABP
CRBP
CREB
DCIS
DEPC
DMBA
dNTP
DR
ECL
ER
FABP
GR
GAPDH
IFN
9-cw-retinoid acid
avian erythroblastosis virus
analysis of variance
acute promyelocytic leukemia
acyl coA:retinol acyltransferase
activating transcription factor
all-fran^-retinoic acid
C R E B binding protein
cellular retinoic acid binding protein
cellular retinol binding protein
cyclic A M P response element binding protein
ductal carcinoma in situ
diethylpyrocarbonate
dimethylbenz (a) anthracene
deoxyribonucleotide triphosphate
direct repeat
enhanced chemiluminescence
oestrogen receptor
fatty acid binding protein
glucocorticoid receptor
glyceraldehyde-3-phosphate dehydrogenase
interferon
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Abbreviations
LDL
LRAT
NADP+
NCoR
NPM
NuMA
PCAF
p/CIP
PCR
PLZF
PML
PPAR
PR
PVDF
RAR
RARE
RBP
RT-PCR
RXR
RXRE
RZR
SDS
S.E.M.
SMRT
TBE
low density lipoprotein
lecithimretinol acytransferase
oxidised nicotinamide adenine dinucleotide
nuclear corepressor protein
nucleophosmin
nuclear mitotic apparatus protein
p300/CBP associated factor
p300/CBP interacting protein
polymerase chain reaction
promyelocytic leukaemia zinc finger
promyelocytic leukaemia gene
peroxisome proliferator-activated receptor
progesterone receptor
polyvinylidene difluoride
retinoic acid receptor
retinoic acid receptor response element
retinol binding protein
reverse transcription-polymerase chain reaction
retinoid-X-receptor
retinoid-X-receptor response element
retinoid-Z-receptor
sodium dodecyl-sulphate
standard error of the mean
silencing mediator for retinoid and thyroid hormone receptors
Tris/Borate/EDTA
Abbreviations
TBST
TGF
TIF
TPA
TR
TRE
Tris
Tth
UV
VDR
Tris-buffered saline plus Tween 20
transforming growth factor
transcription intermediary factor
12-o-tetradecanoyl-phorbol-13 -acetate
thyroid hormone receptor
T P A responsive element
Tris(hydroxymethyl)methylamine
Thermus thermophilus
ultra-violet
vitamin D hormone receptor
CHAPTER ONE
LITERATURE
REVIEW
1
Chapter 1 Literature Review
1.1 HISTORICAL OVERVIEW
Retinoids are a family of biologically active molecules that consists of both
natural and synthetic analogues of vitamin A (retinol, see Figure 1.1.1a). The profound
effects of vitamin A on cellular differentiation and proliferation were discovered more
than 70 years ago. Wolbach and Howe (1925) demonstrated that rats fed a vitamin A-
deficient diet developed squamous cell metaplasia in several tissues of epithelial origin,
including epithelial cells of the respiratory, gastrointestinal and genitourinary tracts,
cornea, skin, salivary glands and prostate gland. Not only was hyperproliferation found
in some epithelia, hyperkeratinization was also observed in many tissues. However,
when these rats were fed a normal diet containing vitamin A, the squamous metaplasia
completely reverted to columnar, ciliated and mucous-secreting cells (Wolbach and
Howe, 1933).
The discovery of naturally occurring, biologically active derivatives of vitamin A added
further complexity to our understanding of the retinoid response in the body. It was
found that 11-cw-retinal (Figure 1.1.1b) was the light absorbing chromophore found in
the visual pigment, rhodopsin (Wald, 1968). Furthermore, all-fraH.s-retinoic acid (atRA)
(Figure 1.1.1c) was shown to support growth in vitamin A-deficient rats (Arens, 1946).
Although this experiment used synthetically prepared atRA, it was later discovered to
form naturally from retinol in the rat intestine and liver (Emerick, et al, 1967). Aside
from its effects on vision and growth and differentiation of a variety of cell types,
vitamin A and its derivatives are associated in a wide spectrum of biological activities,
including reproduction, metabolism, as well as serving as morphogenic agents during
embryonic development and formation of limbs (Eichele, 1989;
Chapter 1 Literature Review
C H 2 O H
a. alWraws-retinol (vitamin A )
CHO b. 11-cw-retinal
COOH
c. all-fraw.s-retinoic acid
COOH
d. 9-cw-retinoic acid
Figure 1.1.1 Structures of common natural retinoids
Chapter 1 Literature Review
Brockes, 1989; D e Luca, 1991; Mangelsdorf, etal, 1994). It appears that 11-ds-retinal
is uniquely involved in vision, whereas all-fraws-retinoic acid is recognised as the key
retinoid in essentially all other biological functions of vitamin A.
Although the anti-proliferative and differentiation effects of retinoids in cells
had long been known, the mechanism of action had eluded scientists until recently.
Following studies of other hydrophobic hormones in the 1970s (Evans, 1988), it was
postulated that retinoids act similarly to steroid and thyroid hormones via interaction of
specific protein receptors and segments of DNA, and regulate gene transcription.
Retinoid receptors were discovered a little over 10 years ago and the discovery of these
receptors have provided researchers a greater insight into the signalling effects of
retinoids in biological systems. Of particular interest are studies involving retinoid
receptors and their involvement in human disease, investigations which may help in
identifying causes of disease and perhaps a method of treatment and cure.
1.2 METABOLISM OF RETINOIC ACID
1.2.1 Uptake of Dietary Retinoids
Retinoids cannot be synthesised de novo in the body and are thus required as a
nutrient. They are present in the diet as either provitamin A carotenoids (e.g., P-
carotene) or preformed retinoids. There are over 600 naturally occurring retinoids, of
which approximately 50 are converted to vitamin A (Blaner and Olson, 1994). Upon
uptake of dietary retinoids in the intestine they are hydrolysed and reduced to all-trans-
retinol (vitamin A, "retinol"), presumably by pancreatic carboxyl (cholesteryl) ester
Chapter 1 Literature Review
hydrolase and/or brush border-associated retinyl ester hydrolase (Rigtrup and Ong,
1992).
Most of the retinol is eventually transported from the small intestine into the
liver for storage, which is done in several ways. The majority of the retinol is esterified
in the gut wall with palmitic or stearic acid, incorporated into chylomicrons and secreted
into the lymphatic system (see Figure 1.2.2.1). Upon catabolism of chylomicrons by
lipoprotein lipase into chylomicron remnants, retinyl esters are first taken up by liver
parenchymal cells where they are further hydrolysed to retinol and re-esterified before
being stored in the stellate cells of the liver (Goodman, et al, 1965; Blaner, et al,
1987). Re-esterification occurs by two pathways. One involves the transesterification
of a fatty acyl group from the Ai-position of lecithin to retinol, which is catalysed by
lecithimretinol acyltransferase (LRAT) (MacDonald and Ong, 1988). The second
pathway involves an acyl-coenzyme A dependent esterification of retinol that is
catalysed by acyl-coA:retinol acyltransferase (ARAT) (Ross, 1982).
Retinol can also be transported in the plasma by transthyretin complexed with
retinol binding protein (RBP), a 21 kDa protein containing a single binding site
specifically for retinol (Blaner, 1989). Alternatively, p-carotene can be transported in
plasma by low density lipoprotein (LDL), and retinoic acid, which is present in very low
concentrations in blood and presumably taken up by diffusion in the intestine, can be
transported to the liver for storage by association with albumin (Ross, 1993a). Other
organs also able to store retinoids include adipose tissue, kidney, testis, lung, bone
marrow and the eye (reviewed in Blaner and Olson, 1994).
5
Chapter 1 Literature Review
1.2.2 Release of Retinol into Cells
Retinol is released from storage organs and transported to target cells by binding
to the RBP-transthyretin complex in the plasma. The molecular mechanisms whereby
target cells take up retinol are unknown but presumably involve membrane receptors
specific for RBP (Bavik, et al, 1991). Retinol is a hydrophobic molecule and is present
in the cell bound to a small binding protein called cellular retinol binding protein
(CRBP). There are two types of CRBPs: CRBP-I and CRBP-II, each with a molecular
weight of approximately 15kDa but different tissue distribution (Bashor, et al, 1973;
Ong, 1984). CRBP-I appears to be widely distributed, with highest expression in liver,
kidney and proximal epididymis, while CRBP-II is found only in the small intestine
(Levin, et al, 1987). The esterification of retinol as described previously also occurs in
the presence of CRBP (MacDonald and Ong, 1988). Retinol bound to CRBP can also
undergo reversible oxidation to retinal in the presence of NADP+-dependent retinol
dehydrogenase (Posch, etal, 1991). Retinal can then be used in the visual pathway, or,
still bound to CRBP, be further oxidised to all-fran.s-retinoic acid in the presence of
retinal dehydrogenase (Posch, et al, 1992). The synthesis of retinoic acid releases
CRBP, upon which another binding protein, termed cellular retinoic acid binding
protein (CRABP), binds to retinoic acid. Retinoic acid can thus enter the nucleus and
interact with receptors to initiate transcription of target genes (discussed in next
section). Alternatively, CRABP bound to retinoic acid can be further catabolised into
more polar metabolites, which may or may not be biologically active, via the
cytochrome P450 system. Such polar metabolites include 4-hydroxy- and 4-oxoretinoic
acid, 13-cw-retinoic acid and 3,4-didehydroretinoic acid, all presumably formed by a
class of mixed-function oxidases that contain cytochrome P450 (Skare, et al, 1982;
Leo, et al, 1984; Thaller and Eichele, 1990; Eckhoff, et al, 1991). Another
6
Chapter 1 Literature Review
biologically important metabolite of all-trans-retinoic acid is 9-cis-retinoic acid, which
has been shown to form by isomerization in bovine liver microsomes (Urbach and
Rando, 1994).
Similar to CRBPs, there are also two types of CRABPs, type I and II, each of
approximately 15kDa, which also differ in their tissue distribution (Astrom, et al,
1991). Both types of CRBPs and CRABPs belong to a larger gene superfamily of fatty
acid binding proteins (FABP) that encode for small proteins which function in binding,
transport and metabolism of hydrophobic ligands (Gordon, et al, 1991). As described
above, the primary function of CRBPs and CRABPs is to direct specific enzymatic
reactions involved in retinoid metabolism. Another role of these binding proteins has
been suggested in that they act as cytoplasmic buffers, by sequestering and limiting the
availability of free retinoids in cells (Ross, 1993b). A summary of the metabolism of
retinoids to the common biologically active derivative, all-^raws-retinoic acid, is
depicted in Figure 1.2.2.1.
1.3 MOLECULAR BIOLOGY OF RETINOID RECEPTORS
With the identification and characterisation of retinoid binding proteins in the 1970's, it
was suggested that CRABP-I was the mediator of the biological effects of atRA (Chytil
and Ong, 1987). It was shown that F9 embryonal carcinoma cells, which had detectable
CRABP-I, differentiated upon addition of atRA to the medium. Furthermore, mutant
embryonal carcinoma cells selected for their failure to differentiate in response to atRA
were shown to be lacking in CRABP-I protein (Jetten and Jetten, 1979; Schindler, et al,
1981). However, this theory was later refuted when it was shown that cells of the
human myelocytic leukaemia cell line, HL-60, differentiated into
7
Chapter 1 Literature Review
SMALL INTESTINE Dietary retinyl esters Dietary carotenoids
PLASMA
RBP-ROH
RBP
LIVER
TARGET CELL
RBP-ROH
PLASMA
Figure 1.2.2.1. Metabolism of dietary retinoids. Abbreviations: R O H , retinol; RE, retinyl ester; C M ,
chylomicron; LPL, lipoprotein lipase; CRBP, cellular retinol binding protein; LRAT, lecithin:retinol
acyltransferase; ARAT, acyl-coA:retinol acyltransferase; RBP, retinol binding protein; ROH DHase,
retinol dehydrogenase; RAL, retinal; RAL DHase, retinal dehydrogenase; atRA, all-jVa/w-retinoic acid;
CRABP, cellular retinoic acid binding protein; RAR, retinoic acid receptor
Chapter 1 Literature Review
mature granulocytes in the presence of atRA, yet did not have any detectable CRABP-I
(Breitman, et al, 1982). Moreover, stably transfected F9 teratocarcinoma stem cells
overexpressing CRABP-I protein showed an 80-90% reduction in differentiation-
specific genes upon atRA treatment (Boylan and Gudas, 1991).
Prior to the discovery of retinoic acid receptors, cloning of cDNA sequences for other
nuclear steroid hormone receptors had been accomplished. These included the
oestrogen (ER) (Green, et al, 1986), progesterone (PR) (Misrahi, et al, 1987) and
glucocorticoid receptors (GR) (Weinberger, et al, 1985a), all of which have structural
and sequence similarity with the avian erythroblastosis virus (AEV) v-erbA oncogene
(Weinberger, et al, 1985b; Krust, et al, 1986). Interestingly, the mammalian
counterpart to this oncogene is the non-steroidal thyroid hormone nuclear receptor
(Weinberger, et al, 1986). All these nuclear proteins belong to the steroid/thyroid
hormone superfamily of receptors that function as ligand-activated transcription factors,
modulating gene expression through DNA-specific binding to regulatory regions of
target genes (Evans, 1988). These receptors have a modular structure and share highly
conserved DNA-binding and ligand-binding domains. It was assumed that the effects of
retinoic acid would be mediated via nuclear receptors similar to these steroid/thyroid
hormone receptors. A screening technique was therefore devised on the basis that the
DNA-binding domain of the putative receptor, or 'orphan receptor', would be
homologous to those of the nuclear receptor superfamily.
9
Chapter 1 Literature Review
1.3.1 Types of Retinoid Receptors
In 1987, two independent research groups discovered an orphan receptor that
bound with high affinity to atRA (Giguere, et al, 1987; Petkovich, et al, 1987). This
receptor, termed retinoic acid receptor alpha, or RARa, shared sequence homology in
its DNA-binding domain and ligand-binding domain with other steroid and thyroid
hormone nuclear receptors. This pioneering work provided a cornerstone for
unravelling the mechanisms of action of retinoic acid and other retinoids in cells.
Subsequently, two more nuclear receptors, RARP and RARy, were identified that bound
to atRA with high affinity. All three types of RARs are found in mammals, amphibians
and birds (de The, et al, 1987; Benbrook, et al, 1988; Brand, et al, 1988; Krust, et al,
1989; Mangelsdorf, et al, 1994). The genes for these three RARs map to different
chromosomes, and in humans localise on 17q21.1, 3p24 and 12pl3 for RARa, RARp
and RARy, respectively (Mattei, et al, 1988a, 1988b; Ishikawa, et al, 1990).
Furthermore, various isoforms exist for each type of RAR, which differ structurally only
in their amino-terminal regions. There are two isoforms for RARa (ai, ai), four for
RARP (Pi, P2, P3, P4) and two for RARy (yi, ^2). These isoforms arise by different
mechanisms: differential usage of two promoters in each RAR gene and/or alternative
splicing of the amino-terminal region of each receptor (reviewed in Leid, et al, 1992a).
The ongoing search for orphan receptors that bound to specific ligands led to the
discovery of another retinoid signalling pathway. This receptor, termed retinoid X
receptor, or RXR, was originally found to be activated by atRA (Mangelsdorf, et al,
1990). However, sequence homology revealed that its ligand-binding domain shared
less than 27% identity with that of RARa, suggesting that another retinoid must activate
this receptor. This was later shown to be the case when a natural derivative of all-trans-
Chapter 1 Literature Review
retinoic acid, 9-cw-retinoic acid (9cRA) (Figure Id), was found to bind with high
affinity to RXR (Heyman, et al, 1992; Levin, et al, 1992). As for RARs, three types of
human RXRs have been identified; RXRa, RXRp and RXRy (Mangelsdorf, et al,
1990; Fleischhauer, et al, 1992; Leid, et al, 1992b) which are also found in mammals,
birds and amphibians (Mangelsdorf, et al, 1994).
Other orphan receptors have recently been discovered that are similar to RARs
and RXRs. A receptor termed LXRa was shown to activate gene expression in the
presence of 9cRA (Willy, et al, 1995). It is specifically found in kidney, intestine, liver
and spleen, which thereby restrict its function to certain tissues. This receptor was also
found to form heterodimers (discussed in Section 1.3.3) with RXRa and peroxisome
proliferator-activated receptor a (Miyata, et al, 1996). Another receptor, called RZR
(retinoid-Z receptor), was shown to be structurally similar to RARs and consist of two
types, a and p (Becker-Andre, et al, 1993; Carlberg, et al, 1994). However, retinoids
are not their cognate ligands, nor are they functionally related to RARs.
1.3.2 Structure of RARs
Similar to steroid and thyroid hormone receptors, RARs are modular in structure
and contain six domains, A-F (Figure 1.3.2.1), based on sequence homology studies
(Green and Chambon, 1988). The amino-terminal A domain is highly variable and
gives rise to the type of RAR as well as the isoform. The A/B region contains a ligand-
independent *ra«.s-activation function, termed AF-1 or x\. Domain C contains the
cysteine-rich DNA binding domain and possesses two zinc finger motifs that allow the
11
Chapter 1 Literature Review
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12
Chapter 1 Literature Review
receptor to recognise specific D N A sequences. This 66 amino acid region is highly
conserved (>95% amino acid identity) among the three types of RARs. Conversely, the
central D domain is a hinge region that bears no homology between the various RARs,
and a function is yet to be determined. However, by analogy with other nuclear steroid
hormone receptors, this region may include a nuclear localization signal (Giguere,
1994). The ligand-binding domain is found in domain E and is also highly conserved,
with at least 75% amino acid homology among the RARs. This region is the largest
domain on the RAR and also contains a ligand-dependent trans-activation function,
termed AF-2 or T4 (Mangelsdorf and Evans, 1995). Furthermore, the E domain contains
a heptad repeat motif consisting of a leucine-rich region that is involved in dimerization
of these receptors (Forman, et al, 1989). The final domain, F, is at the carboxyl-
terminal end of the receptor that has an undefined function and bears little homology
with other nuclear receptors.
It is interesting to note that the amino acid sequence homology of any one type
of RAR in different species is far greater than the similarity found between each RAR
type in a given species. This, coupled with the presence of isoforms, suggests that
RARs were derived from a common ancestral receptor and that each RAR performs
separate functions (Chambon, 1994). This is further supported by observations of
differential expression patterns of RARs in various organs as well as during embryonic
development. The RARa receptor appears to be ubiquitously expressed. In the mouse
it has been found in the brain, skin, muscle, kidney, heart, lung, liver and intestine.
Furthermore, the levels of the two RARa isoforms appear to differ between these
organs (Leroy, et al, 1991a). Similarly, murine RARpi and p3 isoforms appear to be
prevalent in the lung, skin and brain, while p2 is found in kidney, liver, lung, heart,
13
Chapter 1 Literature Review
muscle and brain, but at varying levels (Zelent, et al, 1991). The R A R y receptor
appears to have a restricted pattern of expression, being primarily found in the lung and
skin (Zelent, et al, 1989; Kastner, et al, 1990). However, despite these receptors being
differentially expressed, in single knockout studies of each RAR type, the defects
observed are milder than expected, suggesting that there may be some functional
redundancy of transcriptional control between RAR types and/or isoforms (reviewed in
Kastner, et al, 1995).
1.3.3 Mechanism of RAR-gene targeting
1.3.3.1 Up-regulation of Target Genes
As RARs are ligand-inducible transcription factors, they mediate their effects on gene
activation beginning with binding of atRA to the E domain of the receptor. It was
originally thought that RARs, as well as the thyroid hormone (TR) and vitamin D
(VDR) receptors, functioned as homodimers in a similar fashion to their steroid
hormone receptor counterparts. Although they are able to function as homodimers, it
was apparent that an accessory factor was required for high affinity binding of RARs,
TR and VDR to their target genes (Glass, 1994). This accessory factor was later to be
RXR, which binds heterodimerically with other nuclear receptors (RARs, TR, VDR,
peroxisome proliferator-activated receptors (PPAR)) as well as homodimerically with
itself to activate or repress target genes (Lehmann, et al, 1993), (MacDonald, et al,
1993; Mangelsdorf and Evans, 1995). Hence, RXR is commonly referred to as a master
regulator in nuclear receptor signalling pathways. However, it has been shown that
RARa can form heterodimers with VDR to mediate gene activation in response to atRA
(Schrader, etal, 1993).
14
Chapter 1 Literature Review
Once the R A R / R X R heterodimer forms, it directly binds to specific D N A
sequences usually located in the promoter region of target genes. These sequences are
known as retinoic acid response elements (RAREs) for RAR/RXR complex binding,
and 9cRA response elements (RXREs) for RXR homodimer binding. Analysis of the
response element shows a direct repeat of a consensus hexad sequence of the
configuration PuGGTCA(X)„PuGGTCA, where X is a nucleotide spacer and n refers to
the number of nucleotides. This hexad repeat appears to be a common motif for other
non-steroid nuclear receptors. The number of spacer molecules is usually between 1 to
5 nucleotides and this is known as the "l-to-5" rule for non-steroid receptors of the
superfamily (Giguere, 1994). Different nuclear receptors recognise direct repeats of
specific spacing. For example, RAR/RXR heterodimers usually bind to RAREs with a
spacing of 2 or 5 bases (termed DR-2 and DR-5, respectively), while RXR homodimers
bind to RXREs that have half sites separated by one nucleotide (DR-1). This rule
however is not strict, as RAREs have been encountered with DR-1 as well as DR>5,
although they are less frequently found (Mangelsdorf, et al, 1994; Kato, et al, 1995).
For RAR/RXR heterodimers, it appears that RXR occupies the 5'-up-stream half site,
while RAR binds to the 3'-down-stream half site in DR-2 and DR-5 RAREs (Predki, et
al, 1994).
As RARs act as transcription factors for other genes, it is not surprising that
research has been done in identifying such target genes and is still an ongoing task.
There are many genes that have been discovered in humans and other animals so far that
contain RAREs in their promoter regions, although the pattern of the direct repeat
sequence may not be perfect. However, they are similar to the consensus motif. A list
of genes known to contain RAREs in their promoter region is shown in Table 1.3.3.1.1.
15
Chapter 1 Literature Review
Table 1.3.3.1.1. Genes known to contain RAREs in their promoter regions
Gene
a-fetoprotein Alcohol dehydrogenase (ADH3) Alu elements Apolipoprotein(a) Apolipoprotein A-I Apolipoprotein CIII Cathepsin D
Cholesterol 7-a-hydroxylase CRABP-II CRBP-I Cytochrome P4501A1 Fructose-1,6-6r's-phosphatase H I 0 histone HoxAA Insulin-linked polymorphic region Keratin Lactoferrin LamininBl Mitochondrial uncoupling protein Oxytocin Phosphoeno/pyruvate carboxykinase Placental lactogen
R A R p Thyroid-stimulating hormone Tissue transglutaminase type II Tissue-type plasminogen activator
Reference
(Liu, etal, 199A) (Duester, etal, 1991) (Vansant and Reynolds, 1995) (Ramharack, etal, 1998) (Rottman, etal, 1991) (Lavrentiadou, etal, 1999) (Sheikh, et al, 1996)
(Crestani, etal, 1998) (Durand, etal, 1992) (Smith, etal, 1991) (Vecchini, etal, 1994) (Fujisawa, et al, 2000) (Bouterfa^a/., 1995) (Doerksen, etal, 1996) (Clark, et al, 1995) (Tomie-Canie, etal, 1996) (Lee, etal, 1995) (Vasios, etal, 1989) (Alvarez, etal, 1995) (Richard and Zingg, 1991) (Lucas, etal, 1991) (Stephanou and Handwerger, 1995)
(deThe, etal, 1990b) (Breen, etal, 1995) (Nagy, etal, 1996) (Bulens, etal, 1995)
Chapter 1 Literature Review
1.3.3.2 Down-regulation of Target Genes
In addition to their effects on up-regulating target genes, RARs are also capable
of down-regulating genes through interactions with other factors. One such factor is
AP-1 (activating protein-1), which is a complex consisting of the protein products from
the protooncogenes c-jun and c-fos and activating transcription factor (ATF), in which
the Jun protein can bind as a homodimer or as a Jun/Fos heterodimer (Angel and Karin,
1991). Activation of AP-1 can be mediated with the tumour promoter TPA (12-o-
tetradecanoyl-phorbol-13-acetate) and acts as a transcription factor by binding to
specific response elements called TREs (TPA-responsive elements) in promoter regions
of target genes, in much the same way as RARs (Angel and Karin, 1991). A mutual
relationship exists between RARs and AP-1 in that treatment of cells with atRA can
inhibit the action of AP-1, as demonstrated by studies on the human osteocalcin gene
(Schule, et al, 1990). Likewise, activation of AP-1 synthesis by the phorbol ester TPA
effectively blocks the action of atRA. Closer examination of the mechanism behind this
revealed a "cross-coupling" between the two transcription factors where the promoter of
the osteocalcin gene contained an overlapping TRE/RARE site (Schule, et al, 1990).
Thus, simply occupying the response element with either AP-1 or RAR can regulate
each other's action, which is vital in preventing tumourigenesis and invasion. Indeed,
regulation of collagenase and of the metalloproteinase stromerysin, both of which
degrade many components of the extracellular matrix, occur through AP-1/RAR cross-
coupling and if left unbalanced, this may result in tumour invasion (Nicholson, et al,
1990; Schule, et al, 1991). Recently, an alternative mechanism hass been proposed,
where ligand-bound RARa disrupts Jun homodimerization or Jun/Fos
heterodimerizafion, thereby preventing the formation of AP-1 complexes. However,
17
Chapter 1 Literature Review
whether this disruption occurs through direct protein-protein interaction (ie, AP-1-RAR)
or via recruitment of an accessory factor is still unclear (Zhou, et al, 1999).
Recent findings have broadened and refined our understanding of the molecular
mechanisms of RAR signalling. Currently, it is thought that RAR-RXR heterodimers
act via two opposing pathways: activation of target genes in the presence of ligand and
repression in its absence. These transcriptional responses are mediated through
coregulator proteins, which bind with the RAR-RXR heterodimer complex. In the
absence of atRA, a group of cofactors known as corepressors bind to the silencer core of
RARs, which corresponds to the hinge region (domain D) and part of the ligand binding
region (domain E) (Baniahmad, et al, 1992). Such corepressors include the silencing
mediator for retinoid and thyroid hormone receptors (SMRT), nuclear receptor
corepressor (N-CoR) and transcription intermediary factor-1 a and p (TIFla and P)
proteins (Chen and Evans, 1995; Horlein, et al, 1995; Le Douarin, et al, 1995, 1996).
These corepressors function by stabilising the chromatin structure through recruitment
of histone deacetylase into close proximity to the RAR-RXR heterodimer
(Collingwood, et al, 1999). Moreover, these corepressors form part of a multiprotein
complex which mediates gene promoter silencing by RARs (McKenna, et al, 1999).
Conversely, upon binding of atRA to RAR, other cofactors bind to the nuclear
receptor to augment transcription. Several types of coactivators have been identified
which bind to RARs and include p300/cyclic AMP response element (CREB) binding
protein (CBP), p300/CBP associated factor (PCAF) and steroid receptor-coactivator-3,
otherwise known as p/CIP (p300/CBP interacting protein) (Hanstein, et al, 1996;
Kamei, etal, 1996; Torchia, etal, 1997; Blanco, etal, 1998; McKenna, etal, 1999).
All these coactivators belong to a family of molecules related to pi60 (Collingwood, et
18
Chapter 1 Literature Review
al, 1999). W h e n atRA binds to R A R , coactivators bind to the receptor, which in turn
releases corepressors from the RAR-RXR heterodimeric complex, thereby activating
gene transcription via AF-2 control (Minucci and Pelicci, 1999). Although AF-2 is
required for transcriptional activation, it is not required for dissociation of corepressors
from the silencer core (Baniahmad, et al, 1997). Unlike corepressors, these
coactivators modify the chromatin structure by recruiting histone acetyltransferase, once
they are bound to RAR (Torchia, et al, 1998). Interestingly, the pi60 family of
coactivators share a common structural feature, which is the alpha-helical LXXLL
amino acid motif and its integrity is required to mediate binding of coactivators to
ligand-bound RAR-RXR heterodimers (Heery, et al, 1997).
Although the discovery of corepressors and coactivators has defined a dual role
for RAR-RXR heterodimers, which also serves as a useful model for transcriptional
regulation, there are still questions to be answered to validate this model. For example,
it is unclear as to whether there is constitutive binding of RAR-RXR heterodimers to
their cognate RAREs in target genes, which would appear to be a logical requirement
for a dual regulatory role of these receptors involving an active or repressed state.
19
Chapter 1 Literature Review
1.4 REGULATION OF RETINOIC ACID RECEPTOR SIGNALLING
As shown in Table 1.3.3.1.1, there are a large number of genes that can be
regulated by RAR/RXR heterodimers. Similar to other hormone systems, there appear
to be positive and negative feedback autoregulation mechanisms in the retinoid
signalling pathway. The genes encoding RARa, RARp and RARy are known to have
RAREs in one of their promoters (de The, et al, 1990b; Leroy, et al, 1991b; Lehmann,
et al, 1992). This implies that autoinducing the expression of RARs by retinoic acid
could lead to an amplification of the retinoid signal, which has been shown by de The
and others for the RARp gene (de The, et al, 1990b).
In addition to autoregulation of retinoid signalling at the RAR transcript level,
feedback mechanisms are also present in the atRA synthesis pathway. Most
importantly, virtually every enzyme and retinoid binding protein in retinoic acid
metabolism are potentially regulated by RARs in that they contain RAREs and/or
RXREs upstream in their genes (Mangelsdorf, et al, 1994). Thus, activation of CRBP-I
by atRA (Smith, et al, 1991) could potentiate synthesis of atRA or 9cRA. Conversely,
induction of the CRABP-II gene by RARs a, P, or y could lead to an increase in atRA
breakdown, thereby reducing the signal for atRA responses in the cell (Durand, et al,
1992), and acting as a negative regulatory mechanism in retinoid signalling.
1.5 RETINOIDS, RARs AND DISEASE
Since retinoids exert their effects in cells through processes involved in
differentiation, it is not surprising that many studies have been undertaken to ascertain
their roles in disease states, such as carcinogenesis. In tissue culture studies, retinoids
Chapter 1 Literature Review
have been shown to promote differentiation of a wide variety of cells, including
keratinocytes, chondrocytes, preadipocytes, haemopoietic cells and tumour cells such as
F9 teratocarcinoma cells (reviewed in Amos and Lotan, 1990). Studies in animal
models have shown that retinoids are useful chemopreventitive agents against cancers in
different tissues such as mammary gland, skin, lung, urinary bladder, pancreas,
digestive tract, liver and prostate (reviewed in Moon, et al, 1994). Furthermore,
epidemiological studies have examined dietary retinoid uptake and its involvement in
various human cancers derived from lung, oesophagus, head/neck, stomach, pancreas,
colon/rectum, bladder, urinary tract, prostate, cervix and breast (reviewed in Hong and
Itri, 1994). In general, there appears to be an inverse relationship between intake of
retinoids and development of cancer.
The resulting in vitro studies, experiments in animal models, as well as the
epidemiological data have suggested retinoids to be a prime candidate in anti-cancer
treatment. Indeed, clinical trials have been undertaken which show that atRA induced
complete remission in patients with acute promyelocytic leukaemia, as well as showing
high response rates for 13-ci.y-retinoic acid in the treatment of squamous cell carcinomas
of the skin and repression of premalignant oral leukoplakia (Hong, et al, 1986; Huang,
et al, 1988; Lippman and Meyskens, 1989; Castaigne, et al, 1990). Furthermore,
combinations of atRA or 13-cw-retinoic acid with interferon-a-2a produced striking
response rates in patients with squamous cell carcinoma of the skin and cervix
(Lippman, et al, 1992a; Lippman, et al, 1992b). Recently, clinical trials have also
been conducted against Kaposi's sarcoma, as these cells are sensitive to atRA in vitro
(Miller, 1998).
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Chapter 1 Literature Review
Although retinoids demonstrate anti-carcinogenic effects, exceeding
physiological concentrations of atRA can lead to teratogenic results in the developing
embryo. In the mouse model, exposure to atRA during days 8-10 (post-implantation
period) can interfere with development of the brain and other craniofacial organs
(Webster, et al, 1986). Moreover, treatment with atRA later in gestation at days 11-13
led to limb, palate and genitourinary defects (Kochhar, 1973). Conversely, deficiency
(or excess) of atRA in the 1 day old chick embryo resulted in prevention of proliferation
and differentiation in mesenchymal cells, thereby prohibiting the formation of the early
vascular system (Thompson, et al, 1969).
In relation to their growth-inhibitory effects, retinoids have been shown to
induce apoptosis in various cell lines, including those derived from neuroblastoma,
breast, ovarian, lung, head and neck, cervical and malignant haemopoietic cancers
(Delia, et al, 1993; Mariotti, et al, 199A; Kalernkerian, et al, 1995; Oridate, et al,
1995, 1996; Sheikh, et al, 1995; Supino, et al, 1996). The mechanisms by which
retinoids induce apoptosis are not well understood. However, in contrast to the classical
pathway of nuclear receptor gene activation, the apoptotic effects of retinoids have been
reported to involve both RAR-dependent and RAR-independent mechanisms (Sun, et
al, 1999 and references therein). Such a receptor-independent pathway involves
retinoid activation of reactive oxygen species that has been shown to mediate apoptosis.
Caspase-3, part of the family of cysteinyl aspartate-specific proteinases, is involved in
the early stages of apoptosis and has been shown to be activated by retinoids through
induction of RARP expression, in synergism with a cyclic AMP analogue (Srivastava,
et al, 1999). Thus, further understanding of retinoids and their receptors in their
involvement in apoptosis offers clinical potential in treatment of human malignancies.
22
Chapter 1 Literature Review
As R A R s are the ultimate mediators of the anti-carcinogenic effects of retinoic
acid, it is feasible to postulate that altered expression patterns of these receptors may be
involved in promotion and/or development of neoplasia. Indeed, the finding that the
integration site of the hepatitis B virus in human hepatocellular carcinoma was within
the RARp gene provided the first link between alterations in RARs and disease (Dejean,
et al, 1986; Brand, et al, 1988). Studies of RAR expression patterns in various tissues
have been performed and these patterns appear to be tissue-type specific.
In HT29 human colon carcinoma cells, growth inhibition can occur on treatment
with atRA, although this can be blocked by the RARa-selective receptor antagonist, Ro
40-6055. Growth inhibitory effects were not observed with RARp or RARy-selective
antagonists, suggesting that growth inhibition by retinoids in colon carcinoma is most
likely mediated by RARa (Nicke, et al, 1999). The growth inhibitory effect of atRA
was also found in gastric cancer cell lines, which was due to the up-regulation of RARa
mRNA, but sufficient levels of RARa were required for the observed anti-proliferative
effects (Liu, et al, 2001). Furthermore, in clinical samples, premalignant and malignant
gastric tissues had significantly lower levels of RARa expression as detected by in situ
hybridisation, compared to normal gastric mucosa. This suggests a causal role for
lowered RARa expression in gastric carcinoma (Jiang, et al, 1999). Probably the best
evidence that disruption of RAR signalling can lead to neoplasia involves acute
promyelocytic leukaemia (APL). This disease, characterised by an early block in
myeloid maturation, is associated with a specific t(15;17) chromosomal translocation
with breakpoints within the RARa gene and the PML (promyelocytic leukaemia) gene,
a putative transcription factor (Larson, et al, 1984; de The, et al, 1990a; Kakizuka, et
al, 1991). Recently, several rarer variant chromosomal translocations have been
23
Chapter 1 Literature Review
identified in APLs that also involve R A R a . These include a t(ll;17) translocation
involving a fusion of RARa and a Kruppel-like zinc finger protein (PLZF -
promyelocytic leukaemia zinc finger), a t(5;17) translocation involving an RNA-binding
nucleolar phosphoprotein (NPM - nucleophosmin) and another t(ll;17) translocation
involving the nuclear mitotic apparatus protein (NuMA) (Chen, et al, 1993; Redner, et
al, 1996; Wells, et al, 1997). Molecular analysis revealed that the breakpoint in the
RARa gene resulting in the PML/RARa chimeric product occurs within the second
intron (Diverio, et al, 1992). Thus, the PML/RARa fusion protein still retains its
DNA-binding (domain C) and ligand binding (domain E) activities, which may explain
why APL patients are highly sensitive to atRA treatment. Indeed, it has been reported
that the unliganded PML/RARa fusion protein is directly involved in either inhibiting
differentiation or promoting survival of myeloid precursor cells in vitro, giving rise to
the APL phenotype (Grignani, et al, 1993). Although the t(15;17) translocation is
specific for APL and is not found in other leukaemias (Morosetti, et al, 1996), patients
who have complete remission of APL following atRA treatment can relapse even
though they are continuously treated with atRA (Warrell, 1993). This acquired atRA
resistance could be due to a progressive reduction of the retinoid concentration in the
plasma, or further mutations in the E (ligand-binding) domain of the RARa component
of the PML/RARa chimeric gene (Muindi, et al, 1992; Imaizumi M, et al, 1998).
Aberrations in the patterns of expression of RARp have been observed in
various human cancers. In histological specimens containing dysplastic and malignant
head and neck squamous cell carcinomas, there is a significant loss of expression of
RARP compared to adjacent normal and hyperplastic lesions (Xu, et al, 1994a, 1994b).
Furthermore, in oesophageal tissue, there is a gradual loss of RARp expression from
24
Chapter 1 Literature Review
well-differentiated oesophageal carcinoma to moderately-differentiated and poorly-
differentiated specimens, all of which had significantly lower detection levels of RARp
than normal oesophageal tissues (Qiu, et al, 1999). This suggests that loss of RARp
expression is an early event in squamous cell carcinoma of head and neck tissues and
may be involved in its progression through mechanisms that are currently unknown.
Interestingly, studies of head and neck squamous cell carcinoma cell lines have shown
that while reduced or loss of RARP mRNA is observed, this does not correlate with
atRA-induced growth inhibition (Klaassen, et al, 2001; Zou, et al, 2001).
Furthermore, it has been shown that atRA treatment of oral squamous cell carcinoma
cell lines induced a switch from being angiogenic to anti-angiogenic, thus blocking
tumour cell migration. This did not occur by decreased levels of inducers of
angiogenesis such as interleukin-8, but rather by causing tumour cells to secrete
inhibitor(s) of angiogenesis which are presently unknown (Lingen, et al, 1996). Other
studies have shown selective suppression of RARp in human pancreatic carcinoma
tissues and cells, as well as in human lung cancer cell lines (Rosewicz, et al, 1995; Xu,
et al, 1996; Zhang, et al, 1996). This suppression may be a result of hypermethlyation
in the promoter of the RARp gene, as has been shown in gastric carcinoma tissues and
cells (Hayashi, et al, 2001), thereby rendering this receptor in an inactive state.
Some in vitro evidence has linked RARy with control of cancer. It has been
shown that BE(2)-C neuroblastoma cells overexpressing RARy had a reduced growth
rate compared to control cells (Marshall, et al, 1995). The same study also showed that
RARy mRNA levels were significantly higher in early stage neuroblastoma tissue
samples compared to advanced or disseminated tumours. Similarly, overexpression of
RARy in NT2/D1 embryonal carcinoma cells resulted in terminal differentiation that
Chapter 1 Literature Review
was not observed in cells overexpressing R A R a or R A R p (Moasser, et al, 1995). A
strong correlation between RARy mRNA expression and atRA-induced growth
inhibition also has been reported in head and neck squamous cell carcinoma cell lines
(Klaassen, et al, 2001).
Thus it appears that aberrations in RARs, whether it is a simple loss of
expression or by mutation, are a frequent event in many cancers. However, the
abnormal patterns are not uniform and it seems that each RAR may be involved in
specific diseases.
1.6 RETINOIDS, RARs AND MAMMARY CARCINOMA
Retinoids have been extensively studied in terms of their possible therapeutic
benefits in human disease, and much research has been done in relation to breast cancer.
Based on the assumption that cancer is a multi-step process, beginning with an initiation
step (i.e., mutation of a target cell), followed by promotion (i.e., transformation and
immortalisation) and then progression (i.e., metastasis), administration of exogenous
materials (e.g., from diet) may be able to modify the cancer phenotype. This concept
would therefore be independent of the causation of the initiating event. Indeed,
chemoprevention studies showed a reduction of 52% in mammary carcinoma in rats
treated with the carcinogen 7, 12-dimethylbenz (a) anthracene (DMBA) and retinyl
acetate per day versus rats treated with DMBA and a placebo diet (Moon, et al, 1976).
Studies of dietary intake of preformed vitamin A compounds in 89, 494 women
showed a significantly inverse relationship with the risk of breast cancer (/?=0.03), but
not with carotenoid intake (Hunter, et al, 1993). However, in another study, it was
found that increased P-carotene intake was associated with increasingly well-
26
Chapter 1 Literature Review
differentiated tumours in patients with mammary carcinoma (Ingram, et al, 1992). A
follow-up study revealed that those patients with a high intake level of P-carotene in
their diet had significantly reduced breast cancer mortality (/?=0.0093) (Ingram, 1994).
These diet studies, together with the aforementioned animal studies, have prompted
various clinical trials for the use of retinoids in treatment of human mammary
carcinoma, although not all retinoids have resulted in significant responses (Cassidy, et
al, 1982). The synthetic retinoid iV-(4-hydroxyphenyl)retinamide (fenretinide) has been
used in clinical trials because of its low toxicity compared to atRA and its preferential
accumulation in the breast rather than liver (Sporn and Newton, 1979). A large,
randomised study of 2969 women with stage I breast cancer in Milan, Italy, was
initiated in 1987 to evaluate the efficacy of fenretinide in the prevention of second
primaries in breast cancer patients (Costa, et al, 1994). This consisted of 1474 controls
(no retinoid treatment) and 1494 patients who received 200 mg of fenretinide per day
for 5 years. The analysis at a median of 97 months showed no significant differences in
the incidence of a second primary between the two groups, although a beneficial trend
was observed in premenopausal women (Veronesi, et al, 1999). Within this group of
women who were in phase III of the trial, those who received fenretinide after 180 days
had 1.73 times higher natural killer (NK) cell activity than those given a placebo (Villa,
et al, 1993). This indicates that retinoids can modulate cellular immunity and
cytotoxicity by NK cells and offers a mechanism of lowering cancer risk in patients
through natural immunoenhancement. A phase I/II trial in women with metastatic
breast cancer using the anti-oestrogen, tamoxifen, plus fenretinide showed no significant
adverse effects and improvement or stabilisation of disease in 80% of cases (Cobleigh,
et al, 1993). Recently, a phase I/II trial was reported in the use of atRA in combination
27
Chapter 1 Literature Review
with tamoxifen in patients with advanced breast cancer (Budd, et al, 1998). However,
improvement or disease stability for more than 6 months was only found in 9 of 25
patients.
In addition to the additive anti-proliferative effects of atRA and anti-oestrogens
as shown in breast cancer cell lines (Wetherall and Taylor, 1986), the combination of
retinoids and interferons (IFNs) a or y produces synergistic anti-proliferative effects on
cultured breast cancer cells (Marth, et al, 1993). Furthermore, both types of IFNs were
shown to increase the expression of RARy mRNA in breast cancer cells, but only in
combination with a retinoid (Widschwendter, et al, 1995, 1996; Fanjul, et al, 1996).
The growth inhibition observed by synergism of retinoids and IFNs may be due to an
anti-angiogenic effect, as shown in T47D breast cancer cells (Majewski, et al, 1995).
It has long been known that oestrogens are important for the growth and
differentiation of normal mammary tissue, and carefully regulates these events (Topper
and Freedman, 1980). Conversely, in breast cancers which are oestrogen-dependent
(ER+), the fine-tuning of oestrogen-stimulated growth and differentiation is lost,
resulting in uncontrolled proliferation. It has been shown that atRA and other retinoids
can inhibit the growth of ER+ breast cancer cell lines, such as MCF-7, T47D and ZR-
75-1, but affects only some ER- cell lines, such as BT-20, SKBR-3 and Hs578T (van
der Burg, et al, 1993; Rubin, et al, 1994; Rishi, et al, 1996; Kazmi, et al, 1996). This
suggests that there is a correlation between retinoid growth inhibition of mammary
carcinoma and oestrogen receptor status. Indeed, the growth inhibition in breast cancer
cells by retinoids has been attributed to an anti-oestrogenic effect, whereby retinoids can
directly or indirectly inhibit binding of ER to its response element (Demirpence, et al,
1992, 1994). Furthermore, transforming growth factor a (TGFa), a potent inducer of
28
Chapter 1 Literature Review
proliferation, is up-regulated by oestradiol but its synthesis is inhibited by atRA in
MCF-7 cells (Fontana, etal, 1992).
Analysis of RARs in mammary carcinoma cell lines has been extensive. Trends
in RAR expression in these cell cultures have been found in addition to the effects
observed between retinoid growth inhibition and oestrogen receptor positivity. The
levels of RARa mRNA are significantly higher in ER+ breast cancer cell lines than in
ER- cells (Roman, et al, 1992; van der Burg, et al, 1993). This was also observed in
ER+ breast cells grown in steroid-depleted media (Roman, et al, 1993; Sheikh, et al,
1993). The levels of RARP expression appears to be low in tumour cell lines compared
to normal breast cell cultures, and are predominantly found in ER- cells (Roman, et al,
1992; van der Burg, et al, 1993; Swisshelm, et al, 1994; Zhang, et al, 1996).
Interestingly, induction of RARp expression by atRA in ER+ cells correlated with
growth inhibition as well as induction of apoptosis (Liu, et al, 1996). Furthermore,
exposure of the DNA methylation inhibitor 5-aza-2'-deoxycytidine resulted in
significant anti-neoplastic activity in the ER- cell line, MDA-MB-231, and increased
expression of RARp and ERa (Bovenzi and Momparler, 2001). Expression levels of
RARy are variable in normal and breast tumour cells and are independent of ER status
(Roman, etal, 1992; Swisshelm, etal, 1994).
Oestrogen was found to up-regulate mRNA levels of RARa in a time- and
concentration-dependent manner and analysis of the RARa promoter revealed an
imperfect, half-palindromic oestrogen response element (Rishi, et al, 1995; van der
Leede, et al, 1995). However, oestradiol could not up-regulate RARp or y gene
expression (Roman, et al, 1993). Furthermore, when the ER- cell line MDA-MB-231,
a retinoid unresponsive cell, was transfected with ER cDNA, it expressed higher levels
Chapter 1 Literature Review
of R A R a m R N A and its growth was inhibited by atRA (Sheikh, et al, 1993). These
authors later found that transfection of RARa cDNA into two retinoid-resistant, ER-
breast cancer cell lines (MDA-MB-231 and MDA-MB-468) led to growth inhibition by
retinoids (Sheikh, et al, 199A). Thus, the association of RARa with oestrogen action
has led authors to implicate this receptor to be predominantly involved in retinoid
inhibition of mammary carcinogenic growth.
Much of the in vitro data described above has provided many insights as to how
RARs are involved in retinoid growth inhibition of breast cancer cells. However, it is
obvious that the effects observed in this system do not necessarily reflect the in vivo
events. Therefore, studies in mammary carcinoma biopsies are required to gain a better
understanding of the involvement of RARs in human breast cancer. Unfortunately,
there is little information published in this area. Furthermore, the studies in some cases
are relatively small, which has resulted in several conflicting conclusions. For example,
in 9 ER+ and 9 ER- breast carcinoma biopsy specimens, Northern blotting revealed that
RARa mRNA was significantly higher in ER+ samples that in ER- samples (Shao, et
al, 199A). This finding was supported by another study which analysed RARa mRNA
levels in a modest sample size consisting of 94 ER+ and 22 ER- primary mammary
carcinoma biopsies, also by Northern blotting (Roman, et al, 1993). A third group,
however, analysed RARa mRNA levels, using in situ hybridisation, in 43 ER+
mammary carcinomas and 19 ER- mammary carcinomas and found no correlation
between RARa and ER status (Xu, et al, 1997). Interestingly, two independent
laboratories analysed the protein levels of RARa in breast carcinoma specimens and
arrived at different results. One group analysed 10 ER- and 9 ER+ breast cancer
biopsies using immunohistochemistry and found approximately 2-fold higher levels of
Chapter 1 Literature Review
R A R a protein in E R + breast cancers than in ER- specimens (Han, et al, 1997). This
correlated with previous findings that RARa mRNA levels are higher in ER+ than ER-
mammary carcinomas. However, in another study consisting of 33 breast carcinoma
specimens, RARa protein levels were higher in tumours with greater proliferative
activity, but were independent of oestrogen status, as determined by
immunohistochemistry (van der Leede, et al, 1996). This implies that the RARa
mRNA levels may not be reflected in protein levels in breast cancer, although the data
are conflicting.
The expression pattern of RARp in breast cancer specimens indicates a loss of
its mRNA and supports the previous in vitro work. By Northern analysis in one study,
only 1 of 18 breast carcinoma samples expressed RARp mRNA (Shao, et al, 199A),
while another group found RARP mRNA in 1 of 14 mammary carcinomas using in situ
hybridisation (Widschwendter, et al, 1997). Others using a semi-quantitative RT-PCR
method (Pasquali, et al, 1997) have detected RARp mRNA at higher levels in breast
cancer samples with low ER and progesterone receptor levels. They also showed that
RARa expression was not related to ER status. However, their method involved the use
of a housekeeping gene as a reference and such genes may not always be constitutively
expressed in malignant tissue (Ostrowski, et al, 1989). This effect may be further
exacerbated when semi-quantitation is performed on RT-PCR samples. Furthermore,
only 18 breast cancer specimens were analysed. Although administration of the DNA
methylation inhibitor 5-aza-2'-deoxycytidine increased RARp expression in vitro
(Bovenzi and Momparler, 2001), hypermethlyation does not account for frequent loss of
RARp in breast tumour tissues (Yang, et al, 2001). However, Yang et al. (2001)
examined the promoter region of the RARp2 gene for the presence of methylation,
Chapter 1 Literature Review
while others have detected methylation in the first exon of RAJRJ32 (Widschwendter, et
al, 2000). This suggests that RARp gene silencing by methylation may occur at
multiple sites.
The expression level of RARy mRNA appears to be high in breast biopsy
specimens from patients with mammary carcinoma and is independent of ER status
(Shao, et al, 199A; Xu, et al, 1997). A slight decrease of RARy expression was
observed in mammary carcinomas compared to normal breast tissues, but was
insignificant, probably due to the low sample size used in the study (Widschwendter, et
al, 1997). Surprisingly, only one study has looked at whether mutations in RARs exist
in human malignant breast cancer specimens (Widschwendter, et al, 1997). However,
the study analysed only the RARp promoter region and found no mutations in this area
of the gene in 14 breast cancer biopsies. Thus, clinical information of RAR expression
patterns in breast cancer is scant and most of the available data may be unreliable given
the low sample sizes used in such studies. Clearly, more work is needed in this area of
research.
1.7 RESEARCH PROPOSAL, AIMS AND HYPOTHESIS OF THIS STUDY
There is extensive information in the literature gathered from in vitro
experiments on retinoic acid receptors and their involvement in breast cancer.
However, there is little information of in vivo perturbations of RARs in human breast
cancer, and most of what is available is statistically unreliable due to the low number of
samples studied. To date, only two papers have been published that investigated the
status of RARs in normal breast tissue (Widschwendter, et al, 1997; Xu, et al, 1997)
using the semi-quantitative technique of in situ hybridisation. There has been very little
Chapter 1 Literature Review
work done in analysing R A R protein levels in normal and malignant breast tissue (van
der Leede, et al, 1996; Han, et al, 1997), and the results obtained are contradictory.
Moreover, there is no information on RARp and y protein levels in human breast
tissues. Analysis of RAR mutations in mammary carcinoma is virtually non-existent,
yet they are present in other neoplastic diseases, such as acute promyelocytic leukaemia
(Larson, et al, 1984).
The hypothesis of this thesis is that decreased expression of RARs and the
presence of polymorphisms in their mRNA are associated with the development
and/or progression of mammary carcinoma.
Therefore the aims of this study are:
1. To determine the expression patterns and levels of mRNA for RARa, p and y in
a large number of malignant breast tissue samples obtained from patients with
mammary carcinoma, and to compare these findings in normal breast specimens.
2. To analyse protein levels of RARa, P and y in a group of patient-matched
normal breast and mammary carcinoma specimens.
3. To screen for any polymorphisms in the coding sequences of RARa, P and y.
To gain a better understanding of the role of RARs in human breast cancer, breast tissue
specimens were used rather than focusing on in vitro studies of established mammary
carcinoma cell lines. Studies on mRNA expression patterns were performed using
reverse transcription-polymerase chain reaction (RT-PCR), and mRNA expression
33
Chapter 1 Literature Review
levels quantitated by competitive RT-PCR (Becker-Andre and Hahlbrock, 1989). In
this technique, a synthetic internal RNA standard was used as a reference, in that its
sequence is recognised by the same PCR primers used to amplify the wild-type mRNA,
thereby "competing" for all reagents used in the RT-PCR process (Figure 1.7.1). This
in effect controls for variations in the reaction efficiencies in both the reverse
transcription and PCR amplification processes that occur across reaction samples, thus
providing a highly sensitive and accurate method of RNA quantitation. Data obtained
from these experiments were correlated with classical phenotype markers of mammary
carcinoma, including oestrogen receptor (ER) status and tumour grade, to determine if
any associations exist.
Analysis of the RAR protein levels was performed by Western immunoblotting
and results were compared with the mRNA levels. As proteins are the ultimate
mediators of RAR signalling, it is important to determine if RAR protein levels
correlate with trends observed in RAR mRNA levels between normal and mammary
carcinoma tissues. Screening for polymorphisms in RARs in mammary carcinoma was
achieved using the single-strand conformation polymorphism technique followed by
sequencing of polymorphic bands. Polymorphisms were also assessed in a normal
population and their frequencies compared with those found in mammary carcinoma.
The data presented in this thesis provides further understanding of RARs and
their involvement in mammary carcinoma by assessing changes in their expression
level, correlations with other disease indicators, and associations with polymorphisms in
retinoic acid receptors. This information may be a useful springboard for further studies
on the aetiology and treatment of breast cancer.
34
Chapter 1 Literature Review
wild-type c D N A 5 '- •3'
5'-wild-type R N A (target)
•3'
deletion
5- • 3' competitor D N A
m v.7ro transcription
! •
5- • 3' competitor R N A
reverse transcription
3'-target c D N A
•5'
3-competitor c D N A
•5'
I P C R amplification 5'H
primer 1
3'
1-3'
primer 2 5'
primer 1
primer 2
•• agarose gel electrophoresis
target
competitor
CHAPTER TWO
MATERIALS
AND
METHODS
Chapter 2 Materials And Methods
2.1 SPECIMENS
2.1.1 Specimens used for PCR Analysis
Fresh breast tissue specimens were obtained from PathCentre, Sir Charles
Gairdner Hospital and from the Mount Hospital, West Perth, Western Australia, which
included eight uninvolved breast tissues taken from mastectomies that were
macroscopically and microscopically regarded as normal. A total of 140 primary
mammary carcinomas were examined, with oestrogen receptor status determined by
routine screening in the Department of Clinical Biochemistry, PathCentre. Screening
for oestrogen receptors was performed by the ABBOTT ER-EIA monoclonal antibody
kit, according to the maufacturer's instructions (ABBOTT Laboratories, IL, USA). All
tissues were frozen as soon as possible following surgical excision and stored at -70°C.
For competitive PCR analysis, 59 primary mammary carcinomas were obtained
from the Mount Hospital, West Perth, Western Australia. These samples also had
oestrogen receptor levels assayed by routine screening in the Department of Clinical
Biochemistry, PathCentre. Twelve normal breast samples obtained from PathCentre
were also used in this study.
2.1.2 Specimens used for Protein Analysis
Frozen breast tissue specimens were obtained from the tumour bank provided by
the University of Western Australia Department of Pathology, Sir Charles Gairdner
Hospital. Eight patients provided matched histologically normal and mammary
carcinoma tissue. Oestrogen receptor levels were also obtained from tumour tissue in
these specimens, performed by routine screening in the Department of Clinical
Biochemistry, PathCentre as noted in 2.1.1.
38
Chapter 2 Materials And Methods
2.1.3 Specimens used for SSCP Analysis
Fresh breast tissue specimens were obtained from PathCentre, Sir Charles Gairdner
Hospital, including eleven from mastectomies that were macroscopicaliy and
microscopically regarded as normal. Seventy-two primary mammary carcinomas were
also examined. All tissues were frozen immediately following surgical excision and
stored at -70°C prior to RNA processing. Whole blood was taken from 21 human
females who had no clinical breast cancer at the time of collection and were regarded as
normal. A further 40 samples of serum taken from another group of normal females,
which were stored frozen for 6 years, were used to match the number of tumour tissues
studied. To determine if polymorphisms were hereditary, a volunteer family was
selected consisting of a father and mother, three daughters (one married) and two
granddaughters and a grandson (see Figure 6.4.1a). No members of this family were
diagnosed with disease at the time of obtaining blood and were regarded as normal.
2.2 EXTRACTION OF TOTAL RNA FROM HUMAN BREAST TISSUE
2.2.1 Reagents
Solution D:
4 M guanidinium isothiocyanate
25 mM sodium citrate, pH 7
0.5% 7V-lauroylscarcosine (Sarcosyl)
0.1 M P-mercaptoethanol
2 M Sodium acetate, pH 4
DEPC-treated water-saturated phenol
49:1 chloroform:iso-amyl alcohol
Isopropanol
Chapter 2 Materials And Methods
2.2.2 Procedure
Tissue homogenates were prepared by pulverising snap-frozen slices of specimens in a
microdismembrator. Approximately 250 mg of frozen breast tissue was cut into thin
slices with a sterile scalpel blade, with care taken to avoid fatty tissue, which was
yellow in contrast with white breast tissue. The slices were placed into a clean metal
chamber, frozen under liquid nitrogen, and then treated for 25 s in a "Mikro-
Dismembrator U" apparatus (B. Braun Biotech International). The powdered tissue was
placed into a sterile lOmL polypropylene tube and kept in ice. Total RNA was
extracted by the guanidinium isothiocyanate method as described by Chomzsynski and
Sacchi (1987). Basically, 2 mL solution D was added to the pulverised tissue, gently
vortexed, then 0.2 mL 2 M sodium acetate, pH 4, was added, followed by 2 mL DEPC-
treated water-saturated phenol and 0.4 mL 49:1 chloroform:iso-amyl alcohol. The tube
was briefly vortexed and chilled on ice for 15 min. Tubes were centrifuged at 10, 500 *
g for 30 min at 4°C. The upper aqueous phase was removed and placed into another
sterile centrifuge tube. An equal volume of isopropanol (~2 mL) was added to the tube
and RNA was precipitated at -20°C for 1 hr. Tubes were again centrifuged at 10, 500 x
g for 30 min at 4°C. The supernatant was carefully removed and the pellet was
dissolved in 0.3mL solution D, which was then transferred into an Eppendorf tube. An
equal volume of isopropanol was added and RNA was re-precipitated at -20°C for 1 hr.
Tubes were centrifuged at 7000 x g for 10 min at 4°C. The supernatant was removed
and the pellet was freeze-dried for 8 min before storage at -70°C.
40
Chapter 2 Materials And Methods
2.3 ESTIMATION AND INTEGRITY CHECK OF TOTAL RNA
2.3.1 Reagents
10x MOPS:
0.2 M 3-(AT-morpholino)propanesulfonic acid (MOPS)
0.05 M sodium acetate (anhydrous)
0.01MNa2EDTA
Solution is made up with DEPC-treated water and then autoclaved before use.
Sample buffer:
lxMOPS
20% Formaldehyde, pH >4.0
50% Deionised formamide
Loading dye:
1 mM EDTA, pH 8.0
0.25% (w/v) bromphenol blue
50% (w/v) glycerol
(this buffer is made up in DEPC-treated water)
1 mg/mL ethidium bromide, made up in DEPC-treated water
2.3.2 Procedure
Estimation of RNA was performed by dissolving the RNA pellet in sterile, DEPC-
treated water, and a 1/100 dilution used for quantitation. Using standard
spectrophotometric methods, lOOuL of diluted RNA was placed into a quartz micro-
cuvette and its absorbance read from a Carey UV-Vis spectrophotometer (Varian,
Victoria, Australia) to derive its concentration. Total RNA was quantitated assuming
41
Chapter 2 Materials And Methods
that 1 O D equals 40 |ig/mL (Sambrook, et al, 1989). All R N A samples used in this
study had an A260/A280 ratio of at least 1.75.
The integrity of each RNA sample was checked by denaturing formaldehyde-agarose
gel electrophoresis on 1.2% agarose gels containing 1 x MOPS, 6% formaldehyde and
0.5 uL 1 mg/mL ethidium bromide. Samples were mixed in a 1:4 ratio of RNA: sample
buffer and incubated at 55°C for 15 min. Loading dye was added in a 16.6% v/v, and
samples were electrophoresed at 60V for 90 min in a 1 x MOPS running buffer.
Samples were used in the study if both the 28S and 18.9 rRNA bands were observed.
2.4 SYNTHESIS OF cDNA FROM RNA SAMPLES
2.4.1 Reagents
5* first strand synthesis buffer
100 mM dithiothreitol (DTT)
200 U/uL Moloney Murine Leukaemia Virus (M-MLV) reverse transcriptase
500 ug/mL oligo(dT)i5
40 U/uL recombinant RNasin® (Promega Corp) Ribonuclease Inhibitor
25 mM dNTPs
2.4.2 Procedure
The cDNA synthesis reaction was broadly based on the manufacturer's instructions
(GIBCO BRL, Melbourne, Australia). Each reaction contained 200 ng total RNA, 10
ng oligo(dT)i5, lx first strand buffer, 10 mM DTT, 0.5 mM dNTPs, 8 U RNasin and 40
U M-MLV reverse transcriptase. For the competitive PCR study, an additional amount
of competitor RNA was added and the total volume of sterile water was adjusted.
42
Chapter 2 Materials And Methods
Tubes were incubated at 37°C for 1 hr, followed by an enzyme denaturation step at
90°C for 5 min, and tubes were then stored at 4°C prior to PCR analysis.
2.5 EXTRACTION OF DNA FROM WHOLE BLOOD
Fresh whole blood was centrifuged at 1200 x g for 10 min. Buffy coats were
removed for proteinase K digestion followed by phenol/chloroform/iso-amyl alcohol
treatment to extract DNA (Sambrook, et al, 1989). Following ethanol precipitation,
DNA was re-dissolved in sterile nuclease-free water and stored at 4°C.
2.6 EXTRACTION OF DNA FROM HUMAN SERUM
Extraction of DNA from serum was performed by heating five lots of 20 uL aliquots of
patient serum at 100°C in 0.5 mL microcentrifuge tubes for 10 min. Tubes were then
centrifuged at 13 500 x g for 3 min. The supernatant was removed from each tube and
pooled into a single 0.5 mL microcentrifuge tube. Ten microlitres of supernatant was
used for PCR analysis.
2.7 EXTRACTION OF PROTEIN FROM HUMAN BREAST TISSUES
2.7.1 Reagents
Lysis buffer:
50 mM Tris-HCl, pH 7.6
150 mM NaCl
2mMNa2EDTA
1% Nonidet P-40
10% bacitracin
43
Chapter 2 Materials And Methods
10 mg/mL aprotinin
1 mg/mL leupeptin
2.7.2 Procedure
Tissue homogenates were prepared by microdismembration of frozen breast tissue as
described in Section 2.2.2. The pulverised tissues were placed in sterile 2 mL screw-
capped tubes, kept on ice and 1 mL of lysis buffer was added per tube containing 0.05%
bacitracin, 10 u.g aprotinin and 10 ug leupeptin. Tubes were left on ice for 30 min with
occasional vortexing, followed by centrifugation at 13, 500 x g for 10 min at 4°C. The
supernatant was carefully removed and placed into a sterile 1 mL Eppendorf tube and
stored at -20°C.
2.8 ESTIMATION OF TOTAL CELL PROTEIN FROM BREAST TISSUES
2.8.1 Reagents
Coomassie Brilliant Blue solution.
per litre:
100 mg Coomassie Brilliant Blue G-250
50 mL 95% ethanol
100 mL 85% phosphoric acid
-filter through Whatman No. 1 filter paper
10 mg/mL Bovine serum albumin (BSA) standard (prepared in lysis buffer, see Section
2.7.1)
2.8.2 Procedure
Estimation of protein was performed by the Bradford assay (Bradford, 1976).
The range of BSA used in quantitation of breast tissue protein was 1.25 mg, 3.75 mg,
44
Chapter 2 Materials And Methods
5.00 mg and 10.00 mg. Each protein standard was prepared in lysis buffer as outlined
in Section 2.7.1 and 1 uL was added to 99 uL double-deionised water for the assay. For
each tissue sample protein extract, 1 uL was used and added to 99 uL double-deionised
water. For all tubes, 1 mL Coomassie stain was added and allowed to stand at room
temperature for 5 min before quantitation. Protein was quantitated at A595 by the Carey
UV-Vis spectrophotometer (Varian, Victoria, Australia) in glass cuvettes. This
spectrophotometer automatically creates a standard curve based on the absorbance of
the BSA standards and automatically calculates the protein concentration of each tissue
sample based on this standard curve.
2.9 PREPARATION OF RARa DNA COMPETITOR FOR SEMI
QUANTITATIVE COMPETITIVE POLYMERASE CHAIN REACTION
(PCR)
Complementary DNA (cDNA) was reverse-transcribed (refer to Section 2.4) from RNA
extracted from a culture of MCF-7 cells, provided by co-workers in the laboratory. The
cDNA was used as a template for RARa PCR using the primers shown in Table
2.11.2.1 to yield a 827 bp fragment, with amplification conditions described in Section
2.11.2. The PCR product was then digested with Pvu II (Promega, Sydney, Australia)
resulting in four fragments, as shown on Figure 2.9.1. Following separation of the
fragments by gel electrophoresis, the 5' and 3' fragments of the original 827 bp PCR
product were isolated from the gel. This was performed by cutting the desired bands
out of the gel, elution of DNA through sterile glass wool and centrifugation at 13 500 x
g, followed by ethanol precipitation (Sambrook, et al, 1989). These two fragments
were blunt-end ligated together with T4 DNA ligase (Promega, Sydney, Australia) to
Chapter 2 Materials And Methods
Pvu II Pvu II Pvu II 715 836 1046
_ J I I 607 1436
Figure 2.9.1 Pvu II restriction map of the R A R a c D N A region amplified as described
in Section 2.10.2. The nucleotide positions are in reference to that given in Giguere, et
al. (1987).
Chapter 2 Materials And Methods
give a 496 bp fragment. After P C R using the primers for R A R a as shown on Table
2.11.2.1, this competitor DNA fragment would yield a 174 bp product. The competitor
was further purified by the Wizard® Purification System (Promega, Sydney, Australia)
for future use in semi-quantitative competitive PCR reactions.
2.10 PREPARATION OF COMPETITOR RNA FOR COMPETITIVE RT-
PCR
2.10.1 Generation of Deletion RAR DNA Competitor Fragments
Before synthesising competitor RNA molecules, it was necessary to construct
deletion DNA fragments for each receptor. For RARs a and y, the DNA template was
prepared by synthesising cDNA (refer to Section 2.4) from RNA extracted from the
MCF-7 cell line, while cDNA was synthesised from RNA extracted from the Hs058T
cell line to obtain the RARP DNA template. All RNA from cell lines was acquired
from co-workers in the laboratory. The PCR conditions used to obtain the RAR DNA
templates are described in Section 2.11.2. For RARa, the deletion fragment was
prepared by digesting the PCR product with Pst I and Sma I (both from Promega,
Sydney, Australia), which gave rise to 3 fragments, as shown on Figure 2.10.1.1a. This
was followed by 5' end-filling of the Sma I sticky-end fragment using Klenow fragment
DNA polymerase (Promega, Sydney, Australia). The 5' and 3' fragments were isolated,
purified and ligated as described in Section 2.9. For RARs p and y, the deletion
47
Chapter 2 Materials And Methods
a) RARa
Pst I Sma I 1156 1309 I I
607 1436
b) RARp
Pvu II Pvu II 844 912 I I
800 1571
c) RARy
Pvu II Pvu II 1032 1077
930 1517
Figure 2.10.1.1 Restriction maps of the RAR cDNA regions amplified as described in
Section 2.11.2. a) Cut sites used in generation of RARa RNA competitor, b) Pvu II
sites used in generation of RARP RNA competitor, c) Pvu II sites used in generation of
RARy RNA competitor. The nucleotide positions are in reference to that given in
Giguere, et al. (1987), de The, et al. (1987) and Krust, et al. (1989) for RARs a, p and
y, respectively.
Chapter 2 Materials And Methods
fragment was prepared by digesting the P C R product with Pvu II (Promega, Sydney,
Australia), each of which gave rise to 3 fragments as shown on Figures 2.10.1.1b and c.
Similar to RARa, the 5' and 3' fragments were isolated, purified and ligated as
described in Section 2.8. Using the PCR conditions described in Section 2.11.4, the
deletion DNA fragments yielded the following sizes: RARa: 674 bp; RARp: 255 bp
and; RAR: 482 bp. The competitors were further purified by the Wizard® Purification
System (Promega, Sydney, Australia) prior to cloning.
2.10.2 Cloning of Deletion RAR DNA Competitor Fragments
Each purified RAR competitor DNA fragment was inserted into the nMOSBlue
T-vector (Amersham Pharmacia Biotech, Sydney, Australia) according to the
manufacturer's instructions. These vectors were transformed into JM101 E. coli
competent cells and successful transformants were selected by blue/white colony
screening (Sambrook, et al, 1989). One colony for each receptor was picked and
directly amplified under PCR conditions described in Section 2.10.4 to confirm the
presence of the deletion competitor fragment. These colonies were then cultured
overnight at 37°C in LB broth containing ampicillin and tetracycline. Plasmid DNA
was extracted from culture by the Wizard® Plasmid Purification System (Promega,
Sydney, Australia) and then linearized with^va I restriction enzyme (Promega, Sydney,
Australia). The linearized plasmid was then purified by phenol/chloroform extraction
(Sambrook, etal, 1989).
2.10.3 Construction of RAR RNA Competitors
The pM0S5/we T-vector contains a T7 promoter site 5' from the multiple
cloning site where the RAR competitor DNA fragments were inserted. Using the TNT®
Coupled Reticulocyte Lysate System (Promega, Sydney, Australia), RNA was
49
Chapter 2 Materials And Methods
transcribed from the linearized plasmid according to the manufacturer's instructions.
Following treatment with DNase I, RNA was extracted as described in Section 2.2 and
quantitated as described in Section 2.3. Specificity of the RNA was confirmed by
reverse transcribing the RNA under PCR conditions described in Section 2.11.4 before
it was used for competitive PCR.
2.11 PCR DETECTION OF RARs IN HUMAN BREAST TISSUE cDNA
2.11.1 Reagents and Equipment
10 x reaction buffer (supplied with Tth Plus DNA Polymerase):
670 mM Tris-HCl, pH 8.8
166 mM (NH4)S04
4.5% Triton X-100
2mg/mL gelatin
25 mM magnesium chloride (supplied with Tth Plus DNA Polymerase)
5.5 U/uL Tth Plus DNA polymerase (Fischer-Biotech, Perth, Australia)
10 x reaction buffer (supplied with AmpliTaq Gold DNA Polymerase):
100 mM Tris-HCl, pH 8.3
500 mM KC1
AmpliTaq Gold™ DNA polymerase 5 U/uL (Perkin-Elmer, Melbourne, Australia)
1 U/uL Perfect Match® PCR Enhancer (Stratagene, California, USA)
5 mM dNTPs (Promega, Sydney, Australia)
10 pmol/uL PCR primers - refer to each section
MJ-Research PTC-60 Thermal Cycler with hot bonnet (Geneworks, Sydney, Australia)
Sterile wax beads:
50
Chapter 2 Materials And Methods
prepared personally using autoclaved paraffin wax and an electronic automatic step-
pipettor to pipette 20 uL volume beads.
Nuclease-free sterile water
2.11.2 PCR Detection of RARs in Human Breast Tissues
Specific oligonucleotide regions used to detect RARs a, P and y are shown on
Table 2.11.2.1. These primers spanned large introns and therefore would not amplify
genomic DNA. A reaction mix of 30 uL was used to amplify cDNA, which consisted
of: 10 uL cDNA, 0.2 mM dNTP, 1.25 mM MgCl2, 1 x reaction buffer, 1.65 U Tth plus
DNA polymerase (Fischer-Biotech, Perth, Australia) and 5 pmol of primers. The PCR
cycles began with 4 min at 94°C, 1 min at 70°C (63°C for RARs p and y) and 2 min at
72°C. This was followed with 35 cycles of 1 min at 94°C, 1 min at 70°C (63°C for
RARs p and y) and 2 min at 72°C, and a final extension step at 72°C for 5 min. As a
positive control, cDNA synthesised from total RNA extract of the MCF-7 cell line was
used for RARs a and y, whereas cDNA synthesised from total RNA extract of the
Hs578T cell line was used for RARp. The RNA from these mammary carcinoma cells
lines were provided by co-workers in the laboratory. A tube containing the PCR
cocktail plus nuclease-free water and no sample was used in each run as a blank control.
2.11.3 Semi-quantitative Competitive PCR Detection of RARa in Human Breast
Cancer Tissue
Estimation of RARa expression levels was done using a semi-quantitative
competitive PCR technique. In this approach, the PCR cocktail was spiked with 7uL of
1:1000 RARa competitor DNA as described in Section 2.9. The PCR mix was identical
to that as described in Section 2.11.2 for RARa, except that 1 mM MgCl2 and the
51
Chapter 2 Materials And Methods
Table 2.11.2.1. Sequences of Oligonucleotide Primers
c D N A Transcript* Sequence (5' -» 3') Genomic Location 609-626 1436-1419
800-817 1571-1552
930-947 1517-1500
Fragment Size (bp)
827
771
587
RARa sense GCC CAA GCC CGA GTG CTC anti-sense CTA CAG CTG CCT GGC GGG RARP sense AGG AGA CTT CGA AGC AAG anti-sense GTC AAG GGT TCA TGT CCT TC RARy sense GGA AGA AGG GTC ACC TGA anti-sense CGG CGC CGG GCG TAC AGC
* R A R a sequences were derived from Giguere, et al. (1987) R A R p sequences were derived from de The, et al. (1987) RARy sequences were derived from Krust, et al. (1989)
Chapter 2 Materials And Methods
following primers were used per reaction; sense: 5'- G G A A G A A G G G T C A C C T G A
-3' and anti-sense: 5'- CTC CGC AGA TGA GGC AGA TGG -3'. The amplification
cycle was as described in Section 2.11.2. A tube containing the PCR cocktail plus
nuclease-free water and no sample or competitor was used in each run as a blank
control. For quantitation, lOuL of PCR sample was loaded on a 12% polyacrylamide
gel and stained with ethidium bromide following electrophoresis at 100V for 2 hr. The
polyacrylamide gel was then used for image analysis, with the sample RARa fragment
migrating 505 bp and the competitor DNA fragment at 174 bp. The amount of RARa
in each sample was expressed as a ratio and calculated by measuring the intensity of the
sample fragment relative to the intensity of the competitor DNA PCR product (refer to
Section 2.18).
2.11.4 Competitive PCR Detection of RARs in Human Breast Tissue
Using this quantitative PCR method for detection of RAR levels in breast tissue,
known amounts of competitor RNA are added into tubes containing a constant amount
of sample RNA for cDNA synthesis. During the cDNA synthesis step, 100 ng sample
RNA was placed into 4 separate cDNA reaction tubes. For RARa and p quantitation,
600 fg, 800 fg, 5 pg or 10 pg competitor RNA was added into those tubes, whereas for
RARy, 10 pg, 30 pg, 50 pg, or 70 pg was used. Each PCR reaction mix consisted of the
following components: 0.2 mM dNTP, 2.0, 2.7 or 3.2 mM MgCl2 for RARa, p or y,
respectively, 1 x reaction buffer and 10 pmol of primers. The primers used for
competitive PCR are shown on Table 2.11.4.1. For RARa and p, 1.65 U Tth plus DNA
polymerase (Fischer-Biotech, Perth, Australia) was used, whereas for RARy, 1 U
AmpliTaq Gold DNA polymerase was used with the appropriate reaction buffer and
underwent a 10 min hot-start as instructed by the company. Also, 0.3 U Perfect Match®
Chapter 2 Materials And Methods
Table 2.11.4.1. Sequences of Oligonucleotide Primers for Competitive RT-PCR
cDNA Transcript*
Sequence (5' —> 3') Genomic Fragment Competitor Location Size (bp) Size (bp)
RARa sense GCC CAA GCC CGA GTG CTC anti-sense CTA CAG CTG CCT GGC GGG RARp sense AGG AGA CTT CGA AGC AAG anti-sense TGC AAA TTC TAA GAA TCA GGA TG RARy sense GGA AGA AGG GTC ACC TGA anti-sense CGG CGC CGG GCG TAC AGC
609-626 1436-1419
800-817 1123-1101
930-947 1517-1500
827
323
587
674
255
482
* RARa sequences were derived from Giguere, et al. (1987)
RARp sequences were derived from de The, et al. (1987)
RARy sequences were derived from Krust, et al. (1989)
Chapter 2 Materials And Methods
was used for R A R a to increase specificity and incorporated the use of wax beads for the
hot-start procedure. As a result of this, all reagents and sample minus the polymerase
and sterile water were added in the RARa PCR mix before placing a wax bead into
each tube. Tubes were then heated at 94°C for 1 min in the thermal cycler followed by
a ramp-down to 18°C for 2 min to re-solidify the paraffin wax. The remaining volume
of sterile water plus thermostable DNA polymerase was then added on top of the wax
layer. The PCR reaction consisted of the following cycles: 4 min at 94°C, 1 min at 66°C
for RARa (63°C for RARs p and y) and 2 min at 72°C. This was followed by 35 cycles
(34 for RARp) of 1 min at 94°C, 1 min at 66°C for RARa (63°C for RARs p and y) and
2 min at 72°C, and a final extension step at 72°C for 5 min. In each run, a blank control
was used containing nuclease-free water and PCR reaction mix.
2.11.5 PCR Detection of RAR Polymorphisms in Human Breast Tissue
Specific oligonucleotide primers were designed to detect the entire coding
regions of RARs a, P and y, which are shown on Tables 2.11.5.1a, 2.11.5.2a and
2.11.5.3a, respectively. A reaction mix of 25uL was used for PCR amplification, which
consisted of lOuL cDNA, 1 x reaction buffer, 0.2 mM dNTPs, 1.1 U Tth plus DNA
polymerase (Fischer-Biotech, Perth, Australia) and 5 pmol of primers. The
concentration of MgCl2 varied depending on the primer pair and is detailed in Tables
2.11.5.lb, 2.11.5.2b and 2.11.5.3b. The PCR reaction began with an initial hot start at
94°C for 5 min prior to adding Tth plus DNA polymerase (except for RARy). This was
followed by 35 cycles at 94°C for 1 min, with variable annealing temperature (see
Tables 2.11.5.1b, 2.11.5.2b and 2.11.5.3b) for 1 min and 2 min at 72°C. As a positive
control, cDNA synthesised from total RNA extract of the MCF-7 cell line was used for
RARs a and y, whereas cDNA synthesised from total RNA extract of the Hs578T cell
Chapter 2 Materials And Methods
Table 2.11.5.1a. Sequences of Oligonucleotide Primers for R A R a Primer Sequence (5' -»3') Coding
position* Fragment size (bp)
AlphalF AlphalR Alpha2F Alpha2R Alpha3F Alpha3R Alpha4F Alpha4R Alpha5F Alpha5R
5'-ACT GTG GCC GCT TGG CAT GGC C-3' 5'-CTT GTA GAT GCG GGG TAG AGG GG-3' 5'-GCC TTG CTT TGT CTG TCA GGA CAA G-3' 5'-GCG TGT AGC TCT CAG AGC ACT CGG-3' 5'-GGA GCT CAT TGA GAA GGT GCG-3' 5'-GCG TGT ACC GCG TGC AGA TCC-3' 5 '-CCG AGC AGG ACA CCA TGA CCT T-3' 5'-CCG CAC GTA GAC CTT TAG CGC C-3' 5'AAG CGG AGG CCC AGC CGC CCC CAC A-3' 5'-CGG TCA CGG GGA GTG GGT GGC CGG GCT-3'
87->108 338->360 360->384 617->640 657->677 917->937 938->959 1173->1194 1195->1219 1468-^1494
274
281
281
257
300
Coding position corresponds with the D N A position number published in Giguere, et al. (1987).
Table 2.11.5.1b. P C R Conditions for Amplification of R A R a c D N A Primer — — LMgCl2J used
in PCR (mM) Annealing temperature
Alphal Alpha2 Alpha3 Alpha4 Alpha5
0.5 0.8 1.5 0.8 0.5
68°C 68°C 66°C 68°C 68°C
Chapter 2 Materials And Methods
Table 2.11.5.2a. Sequences of Oligonucleotide Primers for R A R P Primer Sequence (5' ->3') Coding
position* Fragment Size (bp)
BetalF BetalR Beta2F Beta2R Beta3F Beta3R Beta4F Beta4R Beta5F Beta5R
5'-GAT C A T GTT T G A CTG TAT G G A TGT T-3' 5'- TGT CCT GGC AGA CGA AGC AGG GT-3' 5 '-ATC ATC AGG GTA CCA CTA TGG-3' 5 '-CAA GTC AGC TGT CAT TTC ATA-3' 5'-GAC GAT CTC ACA GAG AAG AT-3' 5 '-TGC AAA TTC TAA GAA TCA GGA TG-3' 5 '-CCA GGT ATA CCC CAG AAC AAC A-3' 5 '-GTG AGG CTT GCT GGG TCG TCT T-3' 5'-ATG TTT CCA AAG ATC TTA ATG AA-3' 5'-ATG TCT TAT TGC ACG AGT GGT GA-3'
318->342 558->581 582->602 832-»852 853->872 1101̂ .1123 1124-̂ 1145 1395->1416 1417̂ -1439 1651->1673
264
271
271
293
257
* Coding position corresponds with the D N A position number published in de The, et al. (1987).
Table 2.11.5.2b. Primer
Betal Beta2 Beta3 Beta4 Beta5
PCR Conditions for Amplification [MgCl2] used inPCR(mM)
1.4 2.0 2.7 2.0 1.4
of RARP cDNA Annealing temperature
64°C 64°C 64°C 64°C 56°C
Chapter 2 Materials And Methods
Table 2.11.5.3a. Sequences of Oligonucleotide Primers for RARy Primer Sequence (5'-> 3') Coding
position* Fragment Size (bp)
Gamma IF GammalR Gamma2F Gamma2R Gamma3F Gamma3R Gamma4F Gamma4R Gamma5F Gamma5R
5'-TGC CAT G G C CAC CAA TAA G G A GCG-3' 5'-GGC TTG TAG A C C CGA G G A G G C GGA-3' 5'-ATG CTT CGT GTG CAA TGA CAA GTC C-3' 5'-CTG TCA G G T G A C CCT TCT TCC TTC A-3' 5'-CTA TGA GCT G A G CCC TCA GTT A G A A-3' 5 '-TCT AGG CAG GCA GCT TTG AGC AGA GT-3' 5'-TAT CCT GAT GCT GCG TAT CTG CAC A-3' 5 -TCC AGC AGT GGC TCC TGC AGC TT-3' 5'-AGC CCT GAG GCT GTA CGC CCG-3' 5'-CTG GTC AGG CTG GGG ACT TCA-3'
411-̂ 434 657->680 681-V705 926->950 951->975 1195-̂ 1220 1221->1245 1468-̂ 1490 1491-»1511 1763̂ -1783
270
270
270
270
293
Coding position corresponds with the D N A position number published in Krust, et al. (1989).
Table 2.11.5.3b. P C R Conditions for Amplification of RARy c D N A Primer [MgCl2] used Annealing
in P C R (mM) temperature Gamma 1 Gamma2 Gamma3 Gamma4 Gamma5
"OX 0.8 0.8 0.8 0.8
"63T" 65°C 65°C 65°C 65°C
Chapter 2 Materials And Methods
line was used for RARp. A tube containing the PCR cocktail plus nuclease-free water
and no sample was used in each run as a blank control.
2.11.6 PCR Detection of RARy Polymorphism in Serum and Buffy Coats
Ten microlitres of serum supernatant or luL buffy coat DNA was used for PCR
analysis. The primers used to amplify RARy were as follows; sense: 5'-AGA TGG
AGA TTC CAG GCC CGA TGC-3'; antisense: 5'-CTG GTC AGG CTG GGG ACT
TCA-3'. Each 25uL PCR mix consisted of 1 x reaction buffer, 5 pmol primers and 0.2
mM dNTPs. Samples underwent a 5 min hot start at 94°C prior to adding 1.1 U Tth
plus DNA polymerase, followed by a 60 cycle amplification run comprising of 94°C for
1 min, 67°C for 1 min and 72°C for 2 min. A blank containing PCR mix without
sample DNA (substituted with nuclease-free water) was used in each run.
2.12 AGAROSE GEL ELECTROPHORESIS OF PCR PRODUCTS
2.12.1 Reagents
5 mg/mL Ethidium bromide
Bromphenol Blue Loading Dye:
0.25% Bromphenol blue
25% Ficoll Type 400 in H20
10 x Tris/Borate/EDTA Buffer (TBE)
0.89 M Tris-base
0.89 M Boric acid
0.2 M EDTA-Na2, pH 8.0
123 bp DNA Standard ladder (GIBCO-BRL, Melbourne, Australia)
59
Chapter 2 Materials And Methods
2.12.2 Procedure
Electrophorese 5 uL of PCR product plus 2 uL of bromphenol blue loading dye in a
1.2% agarose gel containing 0.15ug ethidium bromide, in 1 x TBE buffer using a
BioRad Wide Mini-Sub Cell GT (BioRad, NSW, Australia) tank. Electrophorese at
100V for 1 hr. For competitive PCR products, 10 uL of sample was loaded plus 2 uL
bromphenol blue loading dye in a 2% agarose gel containing 0.15 ug ethidium bromide,
in the same type of gel tank. Gels were electrophoresed at 100V for 50 min for RARa
and P, and for 1 hr 20 min for RARy. All gels contained lug of 123 bp DNA ladder.
The gel was then visualised on an UV transilluminator in the dark room. The image
was captured by a Kodak DC 120 digital camera (cl- Amersham Pharmacia Biotech,
NSW, Australia), saved on the computer and printed out on an Epson Stylus 800
(Epson, NSW, Australia) inkjet printer using photo-quality paper.
2.13 PREPARATION OF POLYACRYLAMIDE GELS FOR SSCP ANALYSIS
2.13.1 Reagents
35:1 acrylamide:Af, AT-fo's-acrylamide solution
Glycerol
10 x Tris/Borate/EDTA Buffer (TBE) -refer to section 2.12.1
10% ammonium persulphate
N, N, N', AT-tetramethylethylenediamine (TEMED)
2.13.2 Procedure
A 13% polyacrylamide gel with 5% glycerol was prepared as described by
Ausubel, et al. (1994). Gel solutions were poured onto a Mini-Protean II gel system
(BioRad, NSW, Australia) and allowed to set for at least one hour.
60
Chapter 2 Materials And Methods
2.14 SSCP SCREENING AND SEQUENCE ANALYSIS OF POLYMORPHIC
RARS
Denaturation of the RAR PCR products to produce single stranded DNA for SSCP
analysis was performed by diluting 2uL PCR product with 8uL formamide loading
buffer (95% formamide, 10 mM sodium hydroxide, 0.05% bromphenol blue) and
heating at 95°C for 10 min. Denatured samples were then loaded on a 13% non-
denaturing polyacrylamide solution (35:1 acrylamide: N, Ar-to-acrylamide) containing
5% glycerol. Electrophoresis was carried out in cold-room temperature at 4°C under
constant current of 60mA for 3 hr, with a change of cold 1 x TBE running buffer at the
half-way point of the run. Detection of DNA bands was achieved by silver staining
based on a method described by Soong and Iacopetta (1997). This involved soaking
each gel in 10% ethanol for 3 min, 1% nitric acid for 3 min, followed by 3 x 10 sec
washes with Milli-Q water and then soaking in staining solution (0.05 g silver nitrate
and 75 uL formaldehyde in 50 mL Milli-Q water) for 10 min. Following further 3x10
sec washes with Milli-Q water, bands were observed by soaking in developing solution
(1.5 g sodium carbonate, 75 uL formaldehyde and 50 uL sodium thiosulphate (2
mg/mL) in 50 mL Milli-Q water) for 2-3 min and fixed in 10% acetic acid for 5 min.
Gels were finally soaked for 15 min in a 30% methanol/3% glycerol solution before
dried overnight using the GelAir drying system (BioRad, NSW, Australia).
Polymorphic bands seen on the gels were repeated for confirmation by re-synthesising
cDNA from the relevant patient's RNA, followed by PCR amplification using the
appropriate primers. Confirmed polymorphic bands were then excised with a clean
scalpel blade. Gel slices were destained by washing five times in a 0.5mL solution of
2.5% sodium thiosulphate and 1% potassium hexacyanoferrate for 5 min each.
61
Chapter 2 Materials And Methods
Following a final rinse with distilled water, 30uL distilled water was added and D N A
was eluted from the gel by heating at 80°C for 15 min (Hara, et al, 1994). A luL
aliquot was then re-amplified in a PCR reaction mix using primers specific for that RAR
region of interest to enrich the polymorphic sequence. After checking for amplification
by agarose gel electrophoresis, the remaining PCR sample was purified using the
Wizard® PCR Preps DNA purification system (Promega, Sydney, Australia), and then
subjected to direct sequencing in an ABI Prism automated DNA sequencing system
(Perkin-Elmer, Melbourne, Australia).
2.15 PROTEIN SEPARATION ON SDS-POLYACRYLAMIDE GELS (SDS-
PAGE)
2.15.1 Reagents
4 x Tris-HCl/SDS. pH 6.8:
5 mM Tris-base, pH 6.8
0.4 % sodium dodecyl sulphate (SDS) (w/v)
4 x Tris-HCl/SDS. pH 8.8:
0.375 M Tris-base, pH 8.8
0.4% SDS (w/v)
37.5:1 (40%) acrylamide N, AT-Zn's-acrylamide solution (BioRad, NSW, Australia)
10% ammonium persulphate
N, N, N', TV'-tetramethylethylenediamine (TEMED)
5 x SDS electrophoresis buffer:
0.125 M Tris-base
0.96 M glycine
Chapter 2 Materials And Methods
0.5% SDS
2 x SDS sample buffer
lx Tris-HCl/SDS, pH 6.8
20% glycerol
4% SDS
0.001% bromphenol blue
P-mercaptoethanol
Rainbow™ coloured protein molecular weight markers (Amersham Pharmacia Biotech,
Sydney, Australia)
2.15.2 Procedure
Preparation of the SDS/polyacrylamide gel was adopted from the methods
described by Ausubel, et al (1994). An 8% stacking/resolving gel was used over a
3.9% stacking gel. For protein separation, 50 ug of sample was mixed with 2 x SDS
sample buffer and P-mercaptoethanol was added freshly to a final concentration of 5%
v/v. For each gel, 7 uL marker was used and treated with sample buffer and P-
mercaptoethanol as stated previously. In order to compare protein levels across gels on
a semi-quantitative basis, 50 |ig of one protein sample was loaded as a standardising
control onto every gel. This sample was previously checked by Western blotting to
ensure it had expressed all RAR proteins, and aliquots were made to prevent protein
degradation over time. Prior to electrophoresis, the protein mixes (including standard)
were denatured by heating to 100°C for 10 min. After loading all samples onto the gel,
empty lanes were loaded with blank sample buffer to ensure even running. Gels were
electrophoresed at 100V for 2 hr 45 min in cold-room temperature of 4°C, using 1 x
SDS electrophoresis buffer.
63
Chapter 2 Materials And Methods
2.16 ELECTROBLOT TRANSFER OF PROTEINS ONTO MEMBRANES
2.16.1 Reagents
1 x transfer buffer:
25 mM Tris-base
192 mM glycine
20% methanol
0.5% Ponceau S stain (in 1% acetic acid)
2.16.2 Procedure
Preparation of the electroblotting tank using the BioRad (NSW, Australia) mini
blotter was carried out as described by Ausubel, et al. (1994). Protein on the gel was
electroblotted onto Hybond-P (PVDF) membranes (Amersham Pharmacia Biotech,
Sydney, Australia) at either 380 mA for 45 min, or 30 V overnight at 4°C, using cold 1 x
transfer buffer. Control of loading was checked by soaking membranes in 0.5%
Ponceau S stain for 5 min, followed by several washes with Milli-Q water and then
visualised for uniformity of load. The membrane was destained further in Milli-Q water
for 5 min prior to immunoblotting.
2.17 IMMUNOBLOTTING (WESTERN BLOT) OF BREAST TISSUE
PROTEIN FOR RARs
2.17.1 Reagents
10 x TBST:
0.1 M Tris-base
1.5 M NaCl
0.2%) Tween 20 (polyoxyethylene sorbitol monolaureate) " " 64
Chapter 2 Materials And Methods
Blocking solution:
5 % non-fat milk powder in 1 x TBST
ECL detection system (Amersham, Sydney, Australia)
2.17.2 Procedure
The PVDF membranes containing separated breast tissue protein were blocked
for 1 hr with blocking solution before incubation with antibody. The RAR polyclonal
antibodies, obtained from Santa Cruz Biotechnology, Inc., Ca, USA, were all derived
from rabbit and the following dilutions were used: RARa: 1:2000 (C-20, sc-551);
RARP: 1:500 (C-19, sc-552); and RARy: 1:500 (C-19, sc-550). Primary antibodies
were incubated with the membranes in blocking solution for 1 hr, followed by 3 x io
min washes with blocking solution. A 1:5000 goat anti-rabbit IgG conjugated with
horseradish peroxidase secondary antibody (Sigma, NSW, Australia) was incubated
with the membrane in blocking solution for 1 hr, followed by 3 x 10 min washes in 1 x
TBST buffer.
Specific binding of primary antibodies was visualised by the enhanced
chemiluminescence (ECL) detection system (Amersham, Sydney, Australia) after
exposure to Hyperfilm (Amersham, Sydney, Australia) for 5 min.
2.18 QUANTITATION OF RAR COMPETITIVE PCR FRAGMENTS AND
PROTEIN BANDS
All competitive PCR gels and Western blot film/membranes were quantitated on
an IBM PC computer using the freeware program Scion Image, version Beta 4.0.1, with
the 'Gel plot 2' macro setting in accordance to the software developer's instruction
manual. For quantitation of competitive PCR, the intensity of the two fragments was
65
Chapter 2 Materials And Methods
measured and expressed as a ratio of sample R A R (target) to competitor R A R intensity.
After plotting the ratio of target: competitor versus amount of competitor RNA, the
amount of RAR in the sample RNA was determined where the ratio equalled one.
Samples that were not in the range of this ratio were appropriately diluted (or, the
amount of competitor used was lowered) and underwent another competitive RT-PCR
run as described in section 2.11.4. The final amount of receptor was adjusted by a
correction factor to account for the difference in size of the competitor/target. The
correction factors are as follows: RARa: 0.815; RARp: 0.789; RARy: 0.821. All results
was statistically analysed by InStat software, v3.01 (GraphPad Software, Inc.).
66
CHAPTER THREE
PCR DETECTION
OF RARs IN
BREAST TISSUE
Chapter 3 P C R Detection of R A R s in Breast Tissue
3.0 Introduction
Considerable in vitro research into the associations between RARs and breast
cancer has been published, but surprisingly, there is little information on breast cancer
biopsies. As a result of this and the use of small sample groups, current clinical
research is conflicting, with some data confirming established in vitro results, while
other have been contradictory. This chapter provides more data on RARs in clinical
breast cancer biopsy samples, by analysing using RT-PCR a large sample of ER+ and
ER- mammary carcinoma biopsies and comparing the RAR status in these tissues with
normal breast tissues. Comparative analyses using normal mammary tissue are also
lacking in the literature. The hypothesis that is being proposed is that RARs are present
in mammary carcinoma, but their mRNA expression levels are lower in neoplastic than
normal tissues, thereby resulting in abrogated retinoid signalling. Furthermore, the
RAR status in mammary carcinoma biopsy samples has been compared with ER status
and tumour grade to determine any associations, as in vitro studies have shown that ER
and RARs, particularly RARa, are correlated (Roman, et al, 1992; Sheikh, et al, 1993;
van der Burg, et al, 1993). The development of a semi- and a fully quantitative RT-
PCR technique are also described and documented thus providing an extremely
sensitive and specific method for determining RAR mRNA expression levels in breast
tissue samples.
3.1 PCR Detection of RARs in Breast Tissue
3.1.1 Detection of RARs in Normal and Malignant Mammary Tissue
The presence of RARs in breast tissues has been detected by RT-PCR (see
Section 2.11.2), producing fragment sizes of 827 bp, 771 bp and 587 bp, for RARs a, p
68
Chapter 3 P C R Detection of R A R s in Breast Tissue
and y, respectively. One hundred and forty primary mammary carcinomas, of which 97
were found to be ER+ and 43 ER-, and 18 normal breast tissue samples were analysed
in this study. The expression pattern of RARs in normal tissues is shown in Table
3.1.1.1, while the patterns observed in primary mammary carcinomas is shown in Table
3.1.1.2. In all the tissue categories, RARa was present in at least 70% of samples, with
15/18 in normal breast, 69/97 in ER+ mammary carcinomas and 37/43 in ER-
mammary carcinomas. There was no significant difference in the presence of RARa in
normal and neoplastic tissue, irrespective of ER status (p=0.3687, Fisher's exact test).
RARP mRNA was detected in 13/18 normal breast tissue samples, 45/97 ER+
mammary carcinomas and 23/43 ER- mammary carcinomas, with no difference
between tumour and normal tissue (p=0.0193, Fisher's exact test). Similarly, there was
no significant difference between the presence of RARa or p and oestrogen receptor
status (p=0.0861 and 0.4680 for RARa and p, respectively, by Fisher's exact test) in
mammary carcinomas. This suggests that the expression of oestrogen receptor is
independent of RARa or p expression. However, there was a significant absence of
RARy mRNA in mammary carcinomas compared to normal tissues (p=0.0018, Fisher's
exact test). Moreover, the prevalence of RARy was significantly less in ER+ tumours
than in ER- tumours (p=0.0016, Fisher's exact test), which indicates an inverse
relationship between oestrogen receptor status and the presence of RARy.
69
Chapter 3 P C R Detection of RARs in Breast Tissue
Table 3.1.1.1. Detection of RARs in Normal Human Breast Tissues
Receptor
RARa RARP RARy
RAR Positive
15/18 13/18 18/18
%
89 72 100
70
Chapter 3 PCR Detection of RARs in Breast Tissue
Table 3.1.1.2. Detection of RARs in Primary Mammary Carcinomas
Receptor
R A R a RARP RARya
R A R Positive (ER+) %
69/97 71 45/97 46 57/97 59
R A R Positive (ER-) %
37/43 86 23/43 53 37/43 86
a Fisher's exact test,/?=0.0016 between ER+ and ER- mammary carcinomas
Chapter 3 P C R Detection of R A R s in Breast Tissue
3.1.2 RAR Status and Tumour Grading in Mammry Carcinoma
Of the 140 primary mammary carcinoma biopsies studied, histopathology
records were available for 63 samples. From these, 6 were classified as Grade I, 23
were Grade II and 34 were Grade III infiltrating ductal carcinomas by the Nottingham
grading system (Elston and Ellis, 1991). The distribution of RARs among these
tumours is shown on Table 3.1.2.1. RARa was detected in 3/6 Grade I, 18/23 Grade II
and 33/34 Grade III mammary carcinomas. RARp mRNA was found in 1/6 Grade I,
12/23 Grade II and 15/34 Grade III mammary carcinomas. There were no significant
relationships between the presence of RARa or RARP and tumour grade. However,
there was a significant difference in RARy expression in Grade II tumours (6/23)
compared to Grade III tumours (24/34) (p=0.0013, Fisher's Exact test). When Grade I
and Grade II tumour categories were combined (i.e., 8/29 Grades I and II compared
with 24/34 Grade III), this was also significant (p=0.001, Fisher's Exact test),
suggesting that there is a low prevalence of RARy in low-grade tumours compared to
high-grade tumours.
72
Chapter 3 P C R Detection of RARs in Breast Tissue
Table 3.1.2.1. R A R Status and Tumour Grading of Mammary Carcinoma Biopsies
RARa
RARp
RARy0
GRADE I
3/6
1/6
2/6
%
50
17
33
G R A D E II
18/23
12/23
6/23
%
78
52
26
G R A D E III
33/34
15/34
24/34
%
97
44
71
a Fisher's Exact Test,p=0.0013 between Grade II and III tumours
Chapter 3 P C R Detection of R A R s in Breast Tissue
3.2 Quantitation of R A R s Using R T - P C R
3.2.1 Evaluation of the Competitive RT-PCR Method
3.2.1.1 Exponential Amplification of RARa, p and y mRNA
In order to quantitate mRNA levels by competitive PCR, the exponential range
of the PCR reaction before the plateau phase must be determined for each receptor, so
that there is a linear relationship between the amount of competitor amplified compared
to the amount of target amplified. For each receptor, 100 ng of total RNA from a
patient known to express each RAR was co-reverse-transcribed with 40 pg, 2 pg or 40
pg of RARa, P or y competitor RNA, respectively. For RARa and y, the cDNA
samples were amplified for 31 to 40 cycles, whereas for RARp, the sample was
amplified for 29 to 40 cycles, with PCR conditions described in Section 2.11.4.
Following separation by agarose gel electrophoresis and measuring the intensity of each
band, these values were plotted against the number of cycles amplified. The graphs
obtained from these values for RARa, P and y, respectively, are shown in Figures
3.2.1.1.1, 3.2.1.1.2 and 3.2.1.1.3. The exponential range of RARa amplification was
found between 32 and 39 cycles and the ratio of target/competitor remained stable
within that range, as shown on Figure 3.2.1.1.4. For RARP, the exponential range fell
between 31 and 36 cycles and the target/competitor ratio remained stable between 31
and 35 cycles, as shown on Figure 3.2.1.1.5. The exponential range for RARy was
determined to be between 31 and 37 cycles. However, the ratio of target/competitor
was only stable between 31 and 36 cycles (Figure 3.2.1.1.6) before the amplification
rate of the competitor decreased relative to the rate of target amplification. From these
results, the number of cycles used for competitive PCR of RARs in breast tissues was
determined to be 35 cycles for RARa and y, and 34 cycles for RARp.
74
Chapter 3 P C R Detection of R A R s in Breast Tissue
31 32 33 34 35 36 37 38 39 40
w 1100
(0
<
200
100
0
» W MTypeRARa
I CompetiDrRAR a
4
31 32 33 34 35 36 37 38 39 40
Number of Cycles
Figure 3.2.1.1.1. Exponential amplification range for RARa using 100 ng total sample
RNA and 40 pg competitor RNA. Both RNA species were co-reverse-transcribed and
then amplified for various numbers of cycles. Following agarose gel electrophoresis,
the bands were scanned and their intensities measured. These values were plotted on
the y-axis and expressed as arbitrary units.
75
Chapter 3 P C R Detection of R A R s in Breast Tissue
29 30 31 32 33 34 35 36 37 38 39 40
Figure 3.2.1.1.2. Exponential amplification range for R A R P using 100 ng total sample
RNA and 2 pg competitor RNA. Both RNA species were co-reverse-transcribed and
then amplified for various numbers of cycles. Following agarose gel electrophoresis,
the bands were scanned and their intensities measured. These values were plotted on
the y-axis and expressed as arbitrary units.
76
Chapter 3 PCR Detection of RARs in Breast Tissue
31 32 33 34 35 36 37 38 39 40
31 32 33 34 35 36 37 38 39 40
Number of Cycles
Figure 3.2.1.1.3. Exponential amplification range for RARy using 100 ng total sample
RNA and 40 pg competitor RNA. Both RNA species were co-reverse-transcribed and
then amplified for various numbers of cycles. Following agarose gel electrophoresis,
the bands were scanned and their intensities measured. These values were plotted on
the y-axis and expressed as arbitrary units.
tn
co
n I. <
Chapter 3 P C R Detection of R A R s in Breast Tissue
1.4 1
1.2
° 1 ;M *3 &0.8 E o O 0.6 Si CU S> 0.4 (0 1-
0.2
o|
^& ' — # — •
J 1 1 1 1 1 1
A •
1
A •
- — K —
4 •
— i
31 32 33 34 35 36 37 N u m b e r of Cycles
38 39 40
Figure 3.2.1.1.4. Stability of P C R amplification over a number of cycles for R A R a
using 100 ng total sample RNA and 40 pg competitor RNA. Both RNA species were
co-reverse-transcribed and then amplified for various numbers of cycles. Following
agarose gel electrophoresis, the bands were scanned and their intensities measured. The
ratio of target/competitor was plotted on the y-axis.
Chapter 3 P C R Detection of RARs in Breast Tissue
29 30 31 32 33 34 35 36 37 38 39 40
Number of Cycles
Figure 3.2.1.1.5. Stability of PCR amplification over a number of cycles for RARp
using 100 ng total sample R N A and 2 pg competitor RNA. Both R N A species were co-
reverse-transcribed and then amplified for various numbers of cycles. Following
agarose gel electrophoresis, the bands were scanned and their intensities measured. The
ratio of target/competitor was plotted on the y-axis.
79
Chapter 3 P C R Detection of R A R s in Breast Tissue
fc.
o CJ) Q.
E o
o 1-•5
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
31 32 33 34 35 36 37 Number of Cycles
Figure 3.2.1.1.6. Stability of P C R amplification over a number of cycles for R A R y
using 100 ng total sample RNA and 40 pg competitor RNA. Both RNA species were
co-reverse-transcribed and then amplified for various numbers of cycles. Following
agarose gel electrophoresis, the bands were scanned and their intensities measured. The
ratio of target/competitor was plotted on the y-axis.
Chapter 3 P C R Detection of R A R s in Breast Tissue
3.2.1.2 Linearity of Competitive RT-PCR
As a result of the size difference between the target and competitor template, the
amplification kinetics of DNA synthesis may be slightly different between these two
cDNA species, especially when the amount of each starting material is different. It was
therefore necessary to assess a range of concentrations of competitor against a constant
amount of target RNA for which there was a linear relationship. For each receptor, 100
ng was co-reverse transcribed with decreasing amounts of competitor RNA. Figure
3.2.1.2.1 shows a linear response for RARa RT-PCR with constant target RNA and 0.5,
1, 3, 5, 7 and 10 pg competitor RNA (^=0.9371). In Figure 3.2.1.2.2, a linear
relationship was observed for RARP amplification over the range of 0.2, 1, 3, 5, 7 and
10 pg competitor RNA (^=0.9491). For RARy, a linear range was observed with 5, 10,
30, 50, 70 and 100 pg competitor RNA (^=0.9461), as shown in Figure 3.2.1.2.3.
These results demonstrate that the competitive PCR reaction for each receptor can
reliably detect the amount of RAR over a wide range of concentrations.
3.2.2 Comparison between Semi-Quantitative and Competitive RT-PCR
A semi-quantitative competitive PCR method was developed for RARa as described in
Section 2.11.3. This method allowed quantitation of RNA relative to a DNA competitor
of known concentration, which was constant for every sample assayed. Analysis was
performed on 59 breast tissue samples and Figure 3.2.2.1 illustrates representative
samples using semi-quantitative competitive RT-PCR, separated out on a 12% non-
denaturing polyacrylamide gel. The amount of RARa determined by this method was
expressed as an arbitrary unit, calculated as a ratio of the intensity of sample RARa
against the intensity of the competitor fragment. To validate this method, the 59 breast
81
Chapter 3 P C R Detection of R A R s in Breast Tissue
0 2 4 6 8 10
Amount of Competitor (pg)
Figure 3.2.1.2.1. Linearity assay for R A R a competitive RT-PCR. A constant 100 ng
total R N A was co-reverse-transcribed with 0.5, 1, 3, 5, 7 and 10 pg competitor RNA.
Following amplification and gel electrophoresis, the bands were quantitated and
recorded and expressed as a ratio of competitor/target. A linear relationship was
observed over this range of competitor concentration (^=0.9371).
Chapter 3 P C R Detection of R A R s in Breast Tissue
1.6 J
1.4
1.2
petitor/Target
p
bo
-»•
E 0.6 o
o 0.4
0.2
0 I •/
•
•
• /
i
/ •
i i i
0 2 4 6 8 10
Amount of Competitor (pg)
12
Figure 3.2.1.2.2. Linearity assay for R A R p competitive RT-PCR. A constant 100 ng
total R N A was co-reverse-transcribed with 0.2, 1, 3, 5, 7 and 10 pg competitor R N A .
Following amplification and gel electrophoresis, the bands were quantitated and
recorded and expressed as a ratio of competitor/target. A linear relationship was
observed over this range of competitor concentration (^=0.9491).
Chapter 3 PCR Detection of RARs in Breast Tissue
0 20 40 60 80 100
Amount of Competitor (pg)
120
Figure 3.2.1.2.3. Linearity assay for RARy competitive RT-PCR. A constant 100 ng
total R N A was co-reverse-transcribed with 5, 10, 30, 50, 70 and 100 pg competitor
RNA. Following amplification and gel electrophoresis, the bands were quantitated and
recorded and expressed as a ratio of competitor/target. A linear relationship was
observed over this range of competitor concentration (r2=0.9461).
Chapter 3 P C R Detection of R A R s in Breast Tissue
Figure 3.2.2.1. Semi-quantitative competitive RT-PCR of R A R a using a D N A
competitor. Samples were taken from human mammary carcinoma biopsies and
underwent PCR using a constant amount of DNA competitor as described in Section
2.11.3. Samples were run on a non-denaturing 12% polyacrylamide gel. Top band
represents the target (sample) fragment and the lower band the competitor fragment.
85
Chapter 3 P C R Detection of R A R s in Breast Tissue
tissue samples also underwent competitive RT-PCR for RARa by the methods
described in Section 2.11.4. Figure 3.2.2.2 shows a plot of the values determined by
both competitive RT-PCR methods. There was no correlation between the two methods
(^=0.0051, p=0.592A, Pearson's correlation), which showed that this semi-quantitative
method, although requiring fewer reaction steps, gave highly variable results compared
to the more stringent competitive RT-PCR method.
3.2.3 Quantitation of RARs in Normal and Malignant Breast Tissues using
Competitive RT-PCR
The expression of RAR mRNA was examined in 59 mammary carcinoma
biopsy samples by competitive RT-PCR as described in Section 2.11.4. In addition, 12
normal breast tissues were analysed in this study and all tissues demonstrated
expression of all three RARs as determined in Section 3.1.1. A representative
competitive RT-PCR can be seen on the gel photographs shown in Figures 3.2.3.1,
3.2.3.2 and 3.2.3.3 for RARa, P and y, respectively, using constant amount of target
RNA co-reverse-transcribed with increasing amount of competitor RNA. Analysis of
the amount of RARs found in normal breast tissue, compared to primary mammary
carcinomas, is summarised as a semi-logarithmic plot in Figure 3.2.3.4. For RARa, the
mean value (± S.E.M.) found in normal tissues was 2.27 ± 0.55 pg, while in tumour
tissues, the mean value was 3.66 ± 0.50 pg. No significant difference was found for
RARa expression in normal and tumour breast tissues using the 2-tailed Mann-Whitney
test (p=0.AA16). The average level of RARp mRNA in normal tissues was 4.72 ± 1.05
pg and was significantly higher compared to the levels in tumour tissues, which was
0.72 ± 0.08 pg (pO.0001, Mann-Whitney test). For RARy, the mean level of
86
Chapter 3 P C R Detection of R A R s in Breast Tissue
20000
18000
E
3 6000
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Seml-Quantltatlve Values (Arbitrary Unit)
7.00 8.00 aoo
Figure 3.2.2.2. Comparison between semi-quantitative and competitive RT-PCR.
Human mammary carcinoma biopsies taken from 59 patients underwent both PCR
methods as described in Sections 2.11.3 and 2.11.4 for RARa. Following quantitation,
values were plotted on a scattergram to assess the correlation between the two methods.
No such correlation was found, as ^=0.0051 and there was no significance by Pearson's
test (p=0.592A).
Chapter 3 P C R Detection of R A R s in Breast Tissue
Figure 3.2.3.1. Detection of R A R a m R N A levels by competitive RT-PCR.
Representative sample separated on a 2% agarose gel containing ethidium bromide.
Top band in each lane represents the sample (target) fragment, whereas the lower band
represents the competitor fragment with increasing amount from left to right of the gel,
as described in Section 2.11.4.
88
Chapter 3 P C R Detection of R A R s in Breast Tissue
Figure 3.2.3.2. Detection of R A R p m R N A levels by competitive RT-PCR.
Representative sample separated on a 2% agarose gel containing ethidium bromide.
Top band in each lane represents the sample (target) fragment, whereas the lower band
represents the competitor fragment with increasing amount from left to right of the gel,
as described in Section 2.11.4.
89
Chapter 3 P C R Detection of R A R s in Breast Tissue
Figure 3.2.3.3. Detection of R A R y m R N A levels by competitive RT-PCR.
Representative sample separated on a 2% agarose gel containing ethidium bromide.
Top band in each lane represents the sample (target) fragment, whereas the lower band
represents the competitor fragment with increasing amount from left to right of the gel,
as described in Section 2.11.4.
Chapter 3 P C R Detection of R A R s in Breast Tissue
R A R a R A R p RARy
Figure 3.2.3.4. Quantitation of RAR mRNA levels by competitive RT-PCR in normal
breast and primary mammary carcinoma tissue. Bar graphs correspond to mean mRNA
levels and error bars representing standard error of the mean. No significant change
was observed for RARa (p=0.AA16, Mann-Whitney test) between normal and tumour
tissue. There were significant decreases in expression of RARp and y (pO.0001 and
/?=0.001 for RARp and y, respectively, Mann-Whitney test) in tumour tissue compared
to normal breast tissue.
Chapter 3 P C R Detection of R A R s in Breast Tissue
expression in normal breast tissue was 110.93 ±31.27 pg, whereas in tumour tissue, the
mean level was 30.02 ± 2.05 pg. There was also a significant difference between the
levels of RARy expression in normal and tumour breast tissues (p=0.00l, Mann-
Whitney test). Thus, it appears that there is lowered expression of RARp and y in
mammary carcinoma.
3.2.4 Comparison between RAR mRNA Levels and ER Status in Mammary
Carcinoma
Oestrogen receptor status had been determined on the 59 mammary carcinoma
specimens used for competitive RT-PCR analysis of RARs and comparisons were made
between these two receptors. Scatter diagrams comparing ER status and RARa, p and y
mRNA levels are shown in Figures 3.2.4.1, 3.2.4.2 and 3.2.4.3, respectively.
Statistically significant positive correlations were observed with ER status and RARa
(r2=0.2647, /?<0.0001) and y (r2=0.1325, p=0.005A) mRNA levels, by Pearson's
correlation. There was no relationship between RARp mRNA levels and ER status by
Pearson's correlation (^=0.0127,^=0.4042).
92
Chapter 3 P C R Detection of R A R s in Breast Tissue
0 50 100 150 200 250 300 350 400 450
Amount of ER (fmol/mg protein)
Figure 3.2.4.1. Relationship between E R status and R A R a m R N A levels in mammary
carcinoma tissues. The amount of RARa in each tissue was determined using
competitive RT-PCR and the ER levels assayed routinely as described in Section 2.1.1.
A significant correlation was found between ER status and RARa mRNA levels using
Pearson's correlation test (r2=0.2647,/?<0.0001).
Chapter 3 P C R Detection of R A R s in Breast Tissue
3000
2500
< z DC
•a 2000
I e o gl 1500
5 1000 o E <
500
50 100 150 200 250 300
Amount of ER (fmol/mg protein)
350 400 450
Figure 3.2.4.2. Relationship between E R status and R A R p m R N A levels in mammary
carcinoma tissues. The amount of RARp in each tissue was determined using
competitive RT-PCR and the ER levels assayed routinely as described in Section 2.1.1.
No significant correlation was observed between ER status and RARp mRNA levels
using Pearson's correlation test (r^O.0127,^0.4042).
Chapter 3 P C R Detection of R A R s in Breast Tissue
0 50 100 150 200 250 300 350 400 450
Amount of ER (fmol/mg protein)
Figure 3.2.4.3. Relationship between E R status and RARy m R N A levels in mammary
carcinoma tissues. The amount of RARy in each tissue was determined using
competitive RT-PCR and the ER levels assayed routinely as described in Section 2.1.1.
A significant correlation was observed between ER status and RARy mRNA levels
using Pearson's correlation test (r =0.1325,/?=0.0054).
Chapter 3 P C R Detection of R A R s in Breast Tissue
3.3 Discussion
Although there has been considerable research on the expression of RARs in
human breast tissue, progress in this area has been limited, primarily because tumour
biopsies are small in size and the extraction of RNA may sometimes be difficult.
Furthermore, assays such as Northern blotting require a substantial amount of RNA and
may not be as sensitive as RT-PCR, which was the method of choice in this study. One
can rule out the possibility that the PCR results presented here were obtained from
contaminating genomic DNA, as all PCR primers were designed to span introns. These
primers were also tested for amplification of human genomic DNA, which either
produced a faint fragment of larger size, or failed to amplify (data not shown). Normal
breast tissues excised distant from the site of the tumour were also used in this study
instead of "normal" tissue located at the immediate vicinity of the neoplasm. This was
done to avoid the possibility of any neoplastic cells contaminating the samples used for
the RNA extraction process, which may have influenced results of the PCR analyses.
The use of different molecular biology techniques in the literature may have contributed
to the conflicting data over the status of RARs in mammary carcinoma, although this
may also be due in part to low sample size used in some studies.
The results presented here show no significant change in RARa expression in
mammary carcinoma compared to normal breast tissue. There also appears to be no
significant relationship between ER status and the occurrence of RARa expression,
which is in agreement with a previous study using in situ hybridisation (Xu, et al,
1997). Using Northern analysis, it was shown by others that there was a significant
relationship between ER and RARa co-expression in human breast cancers, by %2
contingency analysis (/?=0.0052) (Roman, et al, 1993). This discrepancy may be due to
96
Chapter 3 P C R Detection of R A R s in Breast Tissue
the use of RT-PCR, which is more sensitive than Northern hybridisation. The sample
size used in this study comprised a large cohort of ER+ and ER- human mammary
carcinoma biopsies (97 and 43, respectively), and was comparable to the sample size
used by Roman et al. (1993). However, approximately twice as many ER- mammary
carcinoma tissues were used in this study, which provides more statistically reliable
results. Previous studies in vitro have shown that atRA inhibits the growth of ER+
mammary carcinoma cells, whereas ER- cells are refractory to this effect (van der Burg
et al, 1993). The present data, however, suggest mechanisms not involving RARa may
contribute to atRA sensitivity in ER+ mammary carcinomas.
The frequency of RARp in mammary carcinoma appeared to be lower when
compared to normal breast tissue. This is, however, not significant, although others
have found a significant loss of RARp expression in cultured mammary carcinoma cells
(Swisshelm, et al, 1994). It has been demonstrated by in situ hybridisation that RARp
expression was absent in 13 of 14 breast tumour tissues, but not in adjacent normal
breast tissue, in formalin-fixed, paraffin-embedded specimens (Widschwendter, et al,
1997). This information, together with the data shown in this study, suggests a possible
role for RARp in mammary carcinogenesis via its absence of expression.
A significant loss of RARy expression was observed in mammary carcinoma
compared to normal tissue, using PCR detection. Furthermore, the frequency of RARy
expression in these neoplasms was significantly less in ER+ tumours than in ER-
tumours. Other studies have found no correlation between ER status and RARy
expression in breast cancer cells (Roman, et al, 1992, 1993; Shao, et al, 199A; Xu et
al, 1997) and in another study, there was a slight loss of RARy expression in breast
tumour tissue compared to normal breast tissue (Widschwendter, et al, 1997).
Chapter 3 P C R Detection of R A R s in Breast Tissue
However, in the latter study, the researchers only analysed 14 formalin-fixed, paraffin-
embedded tissue samples. The results presented here show for the first time a
significant loss of RARy expression in mammary carcinoma and suggest an
involvement in the initiation or progression of the malignancy. Indeed, in BE(2)-C
neuroblastoma cells, overexpression of RARy resulted in a reduced growth rate
(Marshall, et al, 1995). Moreover, overexpression of RARy in NT2/D1 embryonal
carcinoma cells led to terminal differentiation (Moasser, et al, 1995).
It has been reported that the expression of RARp was progressively lost in
human mammary carcinoma as the neoplasm became increasingly aggressive (Xu et
al, 1997). Their data found a significant drop in RARp expression from normal breast,
to ductal carcinoma in situ (DCIS) and finally to low- and high-grade invasive tumours.
The results obtained here show no apparent relationship between RARa and p
expression and tumour grade. However, a significant decrease in the prevalence of
RARy expression was observed in grade I and II tumours compared to normal breast
tissue. This was followed by a significant increase in presence of RARy expression in
poorly differentiated grade III neoplasms, compared to the low-grade mammary
carcinomas. This also relates to the findings in this chapter that RARy expression was
present more significantly in ER- tumours, of which are usually classified with high
grade neoplasms. Thus, the data suggest that loss of RARy expression may be involved
in the early stages of mammary carcinogenesis and once the neoplasm has become
highly aggressive, this loss of expression may not be of importance for any further
tumour progression. A study of RARy expression in pre-malignant DCIS tissues and its
comparison with normal mammary tissue and invasive breast may substantiate this
theory.
Chapter 3 P C R Detection of R A R s in Breast Tissue
The results described in Section 3.1 reveal relevant trends in the expression
patterns of RARs in mammary carcinoma and normal breast tissue simply through their
presence or absence. However, more meaningful results could be obtained from a
quantitative viewpoint rather than qualitative assessment. Thus, methods were designed
to estimate mRNA levels of RARs using a PCR approach. This has obvious advantages
over Northern and other blot analyses, such as higher sensitivity and avoiding the use of
radioactive material. Furthermore, many breast tumours collected in this study were
small in size, therefore using Northern blotting on these samples was not achievable, as
these samples contained very low quantities of total RNA. The PCR method was
further warranted in this instance, as it does not require RNA in microgram quantities
for observable amplification. There have been numerous reports on methods of
quantitating RNA using RT-PCR by incorporating an internal standard cDNA of known
amount in the PCR reaction (Zhao, et al, 1995; Corry, et al, 1996; Schieldrop, et al,
1996; Vidal, et al, 1996). Some studies even use housekeeping genes, such as
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or p-actin, as internal standards
for quantitation (Zhao, et al, 1995; Pasquali, et al, 1997). However, in tumour cells,
constant expression of such genes may not necessarily be the case (Ostrowski, et al,
1989). Competitive PCR (Becker-Andre and Hahlbrock, 1989) is a more reliable
approach, where sample DNA (target) competes with a synthetic standard (competitor),
of known amount, for components in the same PCR reaction mix, including the same
primer pair. This method, however, has the disadvantage in that quantitation of each
sample requires titration of decreasing amounts of competitor that is co-amplified with a
constant amount of target, thus making the procedure laborious. Therefore, a semi
quantitative competitive PCR protocol was devised where each sample is quantitated in
Chapter 3 P C R Detection of R A R s in Breast Tissue
a single tube containing a known amount of sample cDNA and a known amount of
competitor DNA, both of which are constant for every sample. A relative amount of
expression is obtained by analysing the ratio of target intensity against the intensity of
the competitor fragment. This procedure has been published by others (Jiang et al,
1996) and has the advantage over conventional competitive RT-PCR in that it requires
fewer tubes and therefore less sample RNA and laboratory preparation. This method
was tested among 59 mammary carcinoma samples for RARa and validation attempted
by conventional competitive RT-PCR on the same samples. The result of each method
provided disparate values when compared to one another, for two possible reasons.
Firstly, the semi-quantitative approach does not control for the efficiency of the cDNA
synthesis reaction, which is likely to be variable for different samples. Secondly, the
large difference in size between target and competitor in the semi-quantitative method
may affect the efficiency of PCR amplification (McCulloch, et al, 1995). However,
others dispute this latter point, claiming that amplification efficiency primarily depends
on the primer sequences (which are identical for target and competitor in this study) and
not template size (Siebert and Larrick, 1992). Since the semi-quantitative approach
could not be validated, the conventional but laborious competitive RT-PCR method was
chosen for quantitation of RAR mRNA.
To effectively calculate levels of RAR mRNA using competitive RT-PCR, the
amplification conditions must be optimised. Quantitation was performed during the
exponential phase of the amplification procedure. Some authors have reported that this
is unnecessary and that it is possible to quantitate during the plateau phase (Siebert and
Larrick, 1992). However, as there may be some variation in amplification efficiency
between different sized templates (i.e., target and competitor), one template may plateau
Chapter 3 P C R Detection of R A R s in Breast Tissue
before the other, which may result in a false indication of the amount of target. The
linearity assays showed that competitive RT-PCR could detect very low quantities of
RNA, which can be extrapolated further by linear regression. Thus, although
competitive RT-PCR provides a powerful tool in detecting and quantitating very small
amounts of specific mRNA, other disadvantages were found in this technique besides its
laborious nature. As described in Section 2.11.4, competitive RT-PCR protocols varied
for each type of RAR to be analysed. This was done to ensure that the two PCR
fragments amplified for each sample (i.e., target and competitor), as observed following
agarose gel electrophoresis, were absolutely clean and did not contain feint smears or
non-specific fragments, which would affect the quantitation procedure. Hence,
competitive RT-PCR can be a tedious procedure, especially during the optimisation
stage where clean PCR fragments are a necessity, and this can be seen in the
competitive PCR protocol for RARa, which required extra components to optimise the
procedure.
The results of competitive RT-PCR of RARs in breast tissues indeed provide
further information than the qualitative data discussed earlier. Variations in levels of
expression of the RARs, as represented by the standard error of the mean, were
expected and this may be due to differences found between females with respect to age,
time of menstruation (or lack of) and lifestyles, which may affect hormonal status.
There was no significant difference in the mean expression levels of RARa between
normal breast and mammary carcinoma tissues, which implies that this receptor alone
may not have a strong involvement in the development or progression of breast cancer.
Interestingly, although a trend in loss of RARp expression was observed in mammary
carcinoma compared to normal breast tissue, competitive RT-PCR analysis of RARp
101
Chapter 3 P C R Detection of R A R s in Breast Tissue
expression levels reveal that it is 6.5 times lower in breast tumours compared to normal
tissue. This highly significant decrease in R A R p expression may suggest a causal role
in mammary carcinogenesis. Indeed, it has been shown in vitro that R A R p induction by
atRA correlates with growth inhibition in breast cancer cells, and this anti-proliferative
effect was attributed to promotion of apoptosis by R A R p (Liu, et al, 1996). A low
concentration of R A R p m R N A in breast tumour tissue may prevent such growth
inhibitory and apoptotic events to occur using atRA treatment.
Quantitation of R A R y in breast tissue showed that its expression levels were
much higher compared to R A R a and R A R p . This was also observed by another group
studying 18 breast carcinoma biopsy specimens using Northern analysis (Shao, et al,
1994). In addition, the mean level of expression in normal tissues was significantly
higher compared to mammary carcinoma tissues, which correlates well with the
qualitative analysis discussed previously, as the prevalence of R A R y expression is also
significantly lower in mammary carcinoma compared to normal breast tissue. It has
been shown that the combination of retinoids and IFNa or y produces a synergistic anti
proliferative effect in breast cancer cells (Marth, et al, 1993). This combination
treatment also increased the expression of R A R y m R N A in breast cancer cells
(Widschwendter, et al, 1995, 1996; Fanjul, et al, 1996) and more recently in mouse
renal carcinoma cells (Hara, et al, 2001). As the data here show decreased expression
levels of R A R y m R N A in breast tumour tissue, combination therapy using retinoids and
IFNs could possibly bring R A R y m R N A back to levels usually found in normal breast
tissue. As a result, this may allow responsiveness of mammary carcinoma tissue to the
growth-inhibitory effects of atRA. Thus, a combination treatment of retinoids and IFNs
could potentially offer a new approach to treatment of breast cancer.
~ 102
Chapter 3 P C R Detection of R A R s in Breast Tissue
The use of the highly sensitive competitive RT-PCR technique has also
confirmed an existing trend as well as a new insight in the relationship between ER
status and RAR mRNA levels. As discussed earlier, no relationship was observed
between ER status and the occurrence of RARa expression. However, a highly
significant positive correlation was found between ER status and RARa mRNA levels.
This verifies previous studies using Northern blotting in the detection of RARs in
malignant breast tumours, which also found a significant relationship between ER status
and RARa mRNA levels (Roman, et al, 1993; Shao, et al, 1994). It also has been
shown that oestrogen up-regulates RARa expression in a time- and concentration-
dependent manner (Roman, et al, 1993; van der Leede, et al, 1995). However, it was
also observed that the growth-inhibitory effects of atRA in cultured mammary
carcinoma cell lines correlated with RARa status, irrespective of the ER status
(Fitzgerald, et al, 1997). Thus, although RARa is present in ER- and ER+ mammary
carcinomas, adequate levels of its expression are probably required to acquire the
growth-inhibitory effects of atRA, as shown in ER+ mammary carcinoma cell culture
studies (van der Burg, et al, 1993). However, studies at the protein level need to be
performed to validate these results, as they are the ultimate mediators of RAR
signalling.
Determining the type of RAR that exerts the effects of atRA in mammary
carcinoma is further complicated by findings in this study that RARy mRNA expression
significantly correlates with ER status. This significance has not been previously
reported. Studies in vitro have shown that RARy mRNA expression levels were similar
in both ER+ and ER- mammary carcinoma cell lines, and a previous study examining
18 mammary carcinoma biopsy specimens found no significant relationship (Roman, et
Chapter 3 P C R Detection of R A R s in Breast Tissue
al, 1992; Shao, et al, 1994). In normal mouse cervical and vaginal tissues, oestrogen
was found to induce RARy expression and was suggested to be involved in the growth
and development of these tissues (Celli, et al, 1996). It was shown in this study that
RARy expression occurs significantly less frequently in ER+ mammary carcinomas than
in their ER- counterparts. Thus, analysis of the RARy status and expression levels in
mammary carcinomas may provide an indication of the effectiveness of treatment using
retinoid therapy in these patients.
Unfortunately, data on tumour grade was not available for the 59 mammary
carcinoma specimens used for competitive RT-PCR, thereby preventing any
conclusions to be drawn about trends in RAR expression levels and progression of
mammary carcinoma. In summary, the data presented here may clarify the conflict of
existing information found in the literature. The data have been determined on a large
sample size, through the use of a highly sensitive and specific technique. It is more than
likely that combinatory functional losses of at least RARp and y are involved in
mammary carcinogenesis instead of a singular type of receptor.
3.4 Conclusions
From the studies using PCR techniques described in this chapter, the following
conclusions can be made with respect to RARs and mammary carcinoma:
• RARa, p and y mRNA are found in normal breast and mammary carcinoma tissues,
and although there was a decrease in trend in RARp expression in mammary
carcinoma, this was not significant. Furthermore, there was a significant loss of
RARy expression in mammary carcinomas.
104
Chapter 3 P C R Detection of R A R s in Breast Tissue
• The prevalence of RARa and P expression are not associated with ER status,
whereas RARy is present more significantly in ER- mammary carcinomas than in
ER+ mammary carcinomas. Furthermore, although there were no associations
between RARa and P expression and tumour grade, RARy expression was
significantly lower in low-grade mammary carcinomas compared with high-grade
mammary carcinomas. As ER- status correlates with high-grade tumours, the data
suggests that loss of RARy expression may be involved in the early stages of
mammary carcinogenesis.
• By competitive RT-PCR analysis, mRNA levels of RARp and y, but not a, are
significantly lower in mammary carcinomas than in normal breast tissues. This
suggests that low concentrations of RARs may be involved in mammary
carcinogenesis as a result of insufficient signalling of the normal retinoid response.
• There is a positive correlation between RARa mRNA and ER levels in mammary
carcinoma, which has been reported previously in the literature. Surprisingly, a
positive correlation was also observed with RARy mRNA and ER levels in
mammary carcinoma, suggesting that oestrogen may up-regulate levels of RARa
and y expression.
• It is more than likely that a combined loss of expression of at least RARp and y,
rather than only one type, is involved in mammary carcinogenesis.
105
CHAPTER FOUR
ANALYSIS OF RETINOIC ACID
RECEPTOR PROTEIN LEVELS IN
BREAST TISSUES
"106
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
4.0 Introduction
In Chapter 3, RT-PCR methods were described which analysed the mRNA
expression patterns of RARs in breast tissue specimens. Receptor proteins are the
eventual molecules involved in RAR signalling and since these levels do not necessarily
reflect the mRNA levels, it is additionally important to analyse RAR protein patterns.
At the beginning of this study, human RAR antibodies were not commercially available
and thus the RT-PCR approach was chosen initially to analyse RAR expression patterns
in breast tissue. Only RARa protein levels in mammary carcinoma specimens have
been analysed in the literature, however no comparisons with normal breast tissue were
reported (van der Leede, et al, 1996; Han, et al, 1997). The hypothesis in this chapter
is that the RAR protein levels in breast tissue correlates with the mRNA expression
levels. This chapter investigates the protein expression patterns of RARa, p and y in
mammary carcinoma biopsy specimens, as well as in normal breast tissue from the same
patient, and will be compared against the RAR mRNA expression patterns as described
in Chapter 3. Due to refinements in the early diagnosis of breast cancer, it has been
difficult to obtain sufficient biopsy material of normal and tumour tissue to undertake
this work, because often the lesions that are being sampled are only a few mm in
diameter.
4.1 Distribution of RAR Protein Levels in Breast Tissues
Eight females were examined for RAR protein levels, using both mammary
carcinoma biopsies as well as normal breast tissue from the same patient, which was
obtained distant from the site of the tumour. Using Western immunoblotting, all tissue
extracts analysed contained RARa, P and y protein and each receptor detected was «51
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
tn* Nl Tl
97.4-1
66-1
46-
30-
N 2 T2 N3 T3 N 4 T4
kDa N 5 T5 N 6 T6 N 7 T7 N 8 T8 N 4
Figure 4.1.1. Detection of R A R a protein in normal breast and mammary carcinoma
tissue. The RARa protein was detected as a «51 kDa fragment following Western
immunoblotting and chemiluminescent detection (see arrow). The numbers in each lane
represent matched normal breast (N) and mammary carcinoma (T) tissue taken from the
same patient. Sample N4 was loaded onto every gel as a standardising control for semi
quantitative analysis.
108
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
Nl Tl N 2 T2 N3 T3 N 4 T4
kDa N 5 T5 N 6 T6 N 7 T7 N 8 T8 N 4
Figure 4.1.2. Detection of R A R P protein in normal breast and mammary carcinoma
tissue. The RARp protein was detected as a «51 kDa fragment following Western
irnmunoblotting and chemiluminescent detection (see arrow). The numbers in each lane
represent matched normal breast (N) and mammary carcinoma (T) tissue taken from the
same patient. Sample N4 was loaded onto every gel as a standardising control for semi
quantitative analysis.
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
Nl Tl N 2 T2 N3 T3 N 4 T4
N 5 T5 N 6 T6 N 7 T7 N 8 T8 N 4
Figure 4.1.3. Detection of RARy protein in normal breast and mammary carcinoma
tissue. The RARy protein was detected as a «51 kDa fragment following Western
immunoblotting and chemiluminescent detection (see arrow). The numbers in each lane
represent matched normal breast (N) and mammary carcinoma (T) tissue taken from the
same patient. Sample N4 was loaded onto every gel as a standardising control for semi
quantitative analysis.
110
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
kDa in size (Gaub, et al, 1989; Rochette-Egly, et al, 1991). Representative blots of
RARa, P and y probing of normal and corresponding tumour breast tissue are shown in
Figures 4.1.1, 4.1.2 and 4.1.3, respectively, as detected by chemiluminescence. Due to
the polyclonal nature of each RAR antibody, some samples exhibited several non
specific protein bands. The expected 51 kDa fragment of the RAR protein was detected
in all samples and its size was determined relative to pre-stained protein markers. RAR
protein levels in normal and neoplastic breast tissue are summarised graphically in
Figure 4.1.4. Protein values were determined relative to a standardising control sample
that was loaded onto each gel (refer to Section 2.15.2). The mean level ± S.E.M. (in
arbitrary units) of RARa protein in normal tissue was 10.46 ± 0.77, while in tumour
tissue the mean level was 11.69 ± 1.26. The mean RARp protein levels in normal tissue
were 10.37 ± 1.34, and 6.14 ± 1.06 in neoplastic tissue. The mean levels of RARy
protein were 9.19 ± 1.09 in normal tissue and 6.10 ± 0.64 in mammary carcinoma.
Using the Wilcoxon matched-pairs, signed ranks test, no significant difference was
observed for RARa protein levels between normal and matched breast tumour tissues
(p=0.23). However, RARp and RARy protein levels in normal breast tissue were
significantly greater than in breast tumour tissue (p=0.0A, p=0.02 for RARp and RARy,
respectively).
4.2 Comparison of RAR and Oestrogen Receptor Protein Levels
All breast samples had oestrogen receptor levels routinely determined in their
tumour tissue and these results were compared with their corresponding RAR levels in
tumour tissue. Figures 4.2.1, 4.2.2 and 4.2.3 show scatter diagrams of ER levels and
111
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
R A R a R A R P RARy
Figure 4.1.4. Comparison of RAR protein levels in normal breast and mammary
carcinoma tissues matched for the same patient. Bar graphs correspond to mean protein
levels and error bars representing standard error of the mean. Significantly higher levels
of RARp and y protein were detected in normal tissues compared to mammary
carcinoma tissues (p=0.0A, p=0.02 for RARp and y, respectively, as shown by an
asterisk), but not for RARa (p=0.23), using Wilcoxon matched-pairs, signed ranks
testing.
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
a> 3 (0 to
o> E o E >*-
a> > 4) _l C CD *H
2 Q. DC UJ
1000
900
800
7on
«()()
500
400
300
200
100
0
Figure 4.2.1. Relationship between R A R a protein levels and E R protein levels in
mammary carcinoma tissues. N o significant relationship was found between these two
receptors (r2=0.11 l,p=0.A2, Pearson's correlation).
2 4 6 8 10 12 14 16 18 20
RARa Protein Level (Arbitrary Unit)
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
IV 3 (0 (A "P O) E o
1 a> > OJ _i c o HH»
E 0. 0. UJ
1000
900
800
/OO
600
500
400
300
200
100
0 4 6 8 10
RARp Protein Level (Arbitrary Unit) 12
Figure 4.2.2. Relationship between R A R p protein levels and E R protein levels in
mammary carcinoma tissues. N o significant relationship was found between these two
receptors (r2=0.006,/>=0.86, Pearson's correlation).
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
1000 i
900
--. 800 -Cv 3 (0 .2 700 -
o> E 1 is 600-o 1 E >*-- 500l » > a> -i 400 -c '3 «w g 300-Q.
oc LU 200
100
0
4+/
, , •/•» •
•
2 4 6 8
RARy Protein Level (Arbitrary Unit)
10
Figure 4.2.3. Relationship between R A R y protein levels and E R protein levels in
m a m m a r y carcinoma tissues. A significant relationship was observed between these
two receptors (r2=0.540,/?=0.04, Pearson's correlation).
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
levels of RARa, p and y, respectively. There was no significant relationship between
ER status and RARa (r2=0.111,/?=0.42) or RARp (r2=0.006, p=0.86) protein levels by
Pearson's correlation test. However, a significant correlation was observed between ER
status and RARy (r2=0.540, p=Q.0A) protein levels. As shown on the scatter diagrams,
one patient had a relatively high concentration of ER protein (900 fmol/mg tissue)
compared with the other patients. Although this may be considered as an outlier,
removal of this data value in the statistical analysis did not alter the relationship
observed between ER and RAR protein levels.
4.3 Comparison of RAR Protein Levels and Grading of Mammary Carcinoma
Tissue
Histopathology records were available for all samples and were graded
according to the criteria of Elston and Ellis (1991). Malignant tumours included 7
infiltrating ductal carcinomas and one infiltrating tubulo-lobular carcinoma, of which 2
were classified as grade I, 3 were grade II and 3 were grade III neoplasms. The average
level of RAR protein ± standard error of the mean detected among the 3 tumour grades
is summarised in Table 4.3.1. Using one-way analysis of variance (ANOVA), no
significant trends were observed between each RAR isotype protein levels and tumour
grading (p=0.01, p=0.53 and;?=0.91 for RARa, P and y, respectively). However, there
appears to be an increasing trend in RAR protein levels (for all 3 types) as the tumour
progresses from grade I to III, although this may reflect in the increasing tumour cell
load.
116
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
Table 4.3.1. Analysis of R A R Protein Levels and Tumour Grading of Mammary
Carcinoma Biopsies0
RARa6
RARpc
RAR/
GRADE I
9.41 ± 2.07
4.00 ± 0.22
5.53 ± 0.29
GRADE II
9.73 ±1.36
6.29 ±1.32
6.26 ±1.43
GRADE III
15.18± 1.34
7.44± 2.53
6.32 ±1.26
a Protein levels are expressed as average arbitrary units ± standard error of the mean.
b One-way ANOVA, p=0.01, across all three tumour grades
c One-way ANOVA, p=0.53, across all three tumour grades
d One-way ANOVA, ^=0.91, across all three tumour grades
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
4.4 Discussion
Using Western immunoblotting, RARp and y protein levels, but not RARa,
were significantly lower in mammary carcinoma than normal breast tissue. These
results confirm the competitive RT-PCR studies detailed in Chapter 3, where it was
demonstrated that RARp and y mRNA levels were significantly lower in mammary
carcinoma compared with normal breast tissue. However, there were no significant
differences in RARa mRNA expression levels between normal breast and mammary
carcinoma tissue. This finding is also reflected in the protein levels reported in this
chapter. It is important to note that different patient samples were used in the protein
analysis to those used in Chapter 3. This was necessary because of the small amounts
of biopsy samples available for analysis. It was demonstrated in Chapter 3 that RARy
mRNA levels were much higher than RARa and p, as has been shown by others (Shao,
et al., 1994). Due to the use of different antibodies in the Western immunoblots, which
may have variable affinity for tissue protein, comparisons between protein levels of
each type of RAR cannot be made. However, as the protein levels bear a similar trend
to the mRNA data presented in Chapter 3, it appears that decreased expression at the
mRNA level for RARp and y, but not RARa, are associated with mammary carcinoma.
This connection may arise from impairment of normal retinoid signalling through
insufficient levels of these receptors, which would have adverse effects on
differentiation and proliferation of normal breast cells.
Analysis of a relationship between ER status and RAR mRNA expression in
Chapter 3 showed a significantly positive correlation between ER levels and RARa and
y mRNA levels, but not with RARp. However, when comparing RAR protein levels, a
significant correlation was observed only with ER status and RARy. Although the
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
RARa mRNA levels do not correlate with protein levels, others have shown by
immunohistochemistry that there was no relationship between ER status and RARa
protein levels (van der Leede, et al., 1996). It has been postulated that discrepant results
observed between ER status and RARa mRNA and protein levels may arise because of
the use of whole breast tumour tissues, which include connective tissue adjacent to
tumour cells (van der Leede, et al, 1996). This could give rise to false positive results
for RARa status in mammary carcinoma cells. However, in this study, normal breast
tissues were analysed and compared with mammary carcinoma tissues. Furthermore, all
breast tissues used either in the mRNA or protein analysis of RARs underwent identical
procedures in sample pulverisation for extraction of either total RNA or protein. The
data in Chapter 3 suggest that oestrogen may regulate RARa and y expression at the
mRNA level, but at the protein level, RARa may be degraded at an enhanced rate,
thereby nullifying the up-regulatory effects of oestrogen. Recently, it was shown that
addition of atRA in the ER+ MCF-7 cell line resulted in a rapid breakdown of RARa
and y protein levels through a process that involved ubiquitination. Conversely, there
was an accumulation of RARa and y mRNA levels (Tanaka, et al, 2001). Similar to
the correlation found between ER status and RARy mRNA levels in Chapter 3, there is
no indication in the literature of a correlation between ER and RARy protein levels.
The discovery of this relationship reported in this chapter may have important
ramifications on the use of RARy-selective retinoids in the treatment of breast cancer.
Although the design and use of such selective retinoids may prove beneficial in
minimising side effects often observed with non-selective retinoids, the measurement of
the level of RARy expression in each patient would be required prior to treatment,
particularly in ER- breast tumours where RARy expression may be low. However,
" " 119
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
more research is needed to determine if a single RAR, or a combination of RARs, is
required for a response to retinoid treatment, and whether a threshold in RAR protein
levels is necessary to obtain the anti-cancer effects of retinoids.
The data reported here demonstrated that there was no statistically significant
relationship between RAR protein levels and tumour grading. Identical observations
were found in Chapter 3 for RARa and p mRNA expression, although there was a
significant increase in the prevalence of RARy mRNA from low- to high-grade
mammary carcinomas. Unfortunately, no information on tumour grade could be
obtained in mammary carcinoma specimens used in the competitive RT-PCR study.
Therefore, no comparisons can be made concerning trends in RAR mRNA, protein
levels and tumour grading in mammary carcinoma. However, the qualitative PCR
results, together with observations of significantly lower RARP and y protein levels in
mammary carcinomas compared to normal breast tissue, indicate that a loss or reduced
expression of RARs may be an early event in mammary carcinogenesis. It is possible
that investigations on RAR protein levels in pre-malignant breast lesions may provide
some clarification.
In summary, the work presented here provides evidence of reduced
concentrations of RAR protein levels in mammary carcinoma. The findings agree with
most of the mRNA studies discussed in Chapter 3. However, there is not a strong
association between ER status and RARa as previously reported (Roman, et al, 1992,
1993; Sheikh, et al, 1993; van der Burg et al, 1993). The studies detailed by these
authors analysed mRNA levels and not protein levels as has been described in this
chapter. The discrepancy in these results indicate that it is important to determine the
protein levels and not simply the mRNA levels, because mRNA processing can vary
Chapter 4 Analysis of Retinoic Acid Receptor Protein Levels in Breast Tissues
and may not accurately reflect the protein concentrations in mammary carcinoma.
Since RARs are critical in the control of cellular growth, determining their protein
levels would be more advantageous than merely analysis of their mRNA levels. As
there may be clinical ramifications for treatment of breast cancer patients using retinoid
therapy, it is important the results described in this chapter are replicated in other
laboratories.
4.5 Conclusions
The following conclusions can be made in relation to the protein levels of RARs in
breast tissues of normal and neoplastic origin:
• RARp and y protein levels, but not RARa, are significantly lower in mammary
carcinoma than normal breast tissue. This is in agreement with the RAR mRNA
levels described in Chapter 3, and suggests that lowered expression of RARs may be
associated with the development of mammary carcinoma.
• There is no relationship between RARa or p protein levels and ER status in
mammary carcinoma. However, a significant correlation exists with ER and RARy
protein levels, which was also observed at the mRNA level described in Chapter 3.
This finding has not been previously reported.
• There is no relationship between RAR protein levels and grading of mammary
carcinoma, suggesting that decreased RAR expression may be an early event in
mammary carcinogenesis.
• Routine assessment of RAR protein levels, for example by immunohistopathology,
should be considered in patients with mammary carcinoma to determine if therapy
with retinoids is warranted. The use of RARy-selective retinoids may prove useful
in treating ER+ breast cancer patients.
' 121
CHAPTER FIVE
IDENTIFICATION OF
RETINOIC ACID RECEPTOR
POLYMORPHISMS
122
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
5.0 Introduction
Retinoids are involved in many signalling processes in the body and anomalies in these
pathways may lead to the development and progression of neoplasia. Indeed, it is well
known that a specific translocation of the RARa gene (t(15;17)) is a characteristic of
acute promyelocytic leukemia (Larson, et al, 1984; Kastner, et al, 1992). Moreover,
down-regulation of RARp has been observed in several types of human malignancy of
epithelial and neuroectodermal origin (Torma, et al, 1993; Ferrari, et al, 1994; Lotan,
et al, 1995; Xu, et al, 1996), including several breast cancer cell lines (Swisshelm, et
al., 1994). Although it has been shown that retinoids inhibit the growth of human breast
cancer cells and their receptors detected in human breast cancer tissue (Roman, et al,
1993; van der Leede, et al, 1996), whether these receptors are present as fully
functional proteins in malignant cells is yet unknown.
To investigate the role that RARs may play, the presence of any polymorphism or
mutation in RARs a, P and y was determined in human breast tissue, both of normal and
neoplastic origin, and in serum and buffy coats from normal females. The hypothesis to
be tested in this chapter is that polymorphisms in the coding sequence of RARs are
associated with mammary carcinoma. Through the use of the polymerase chain reaction
(PCR), reverse transcription PCR and the single strand conformation polymorphism
(SSCP) technique, the occurrence of an RARy polymorphism was demonstrated in
serum and buffy coat DNA from normal females. Furthermore, this polymorphism as
well as another type was identified in mRNA from primary mammary carcinoma as
well as in DNA from serum of the same patients.
123
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
5.1 Absence of Polymorphisms in RARa and p mRNA in Normal and
Malignant Breast Tissue
Analysis was performed on 27 specimens, which comprised of samples from six
normal breast and 21 primary breast carcinomas and evidence for polymorphisms in the
entire coding regions of R A R s a and P assessed. Figures 5.1.1, 5.1.2, 5.1.3, 5.1.4 and
5.1.5 demonstrate typical band patterns seen for each of the primer sets used for R A R a
(refer to Table 2.11.5.1 a). For R A R p , representative gels for each of the P C R primer
pairs (refer to Table 2.11.5.2a) are in Figures 5.1.6, 5.1.7, 5.1.8, 5.1.9 and 5.1.10,
following SSCP and silver staining. Band patterns seen for each region of the R A R a
receptor amplicons appeared identical when both tissue types were compared with one
another. Likewise, identical band patterns were seen in the R A R p receptor when
normal samples were compared against primary tumour samples. The absence of
polymorphisms in the R A R a or p receptor suggests that mutations in these receptors are
unlikely to be involved in the development and progression of mammary carcinoma.
5.2 Detection of Polymorphisms in RARy mRNA in Normal and Malignant
Breast Tissue
The SSCP band patterns for the R A R y receptor derived from each of the primer
sets listed in Table 2.11.5.3a are represented in Figures 5.2.1, 5.2.2, 5.2.3, 5.2.4 and
5.2.5. Screening of eleven normal breast tissues failed to identify any band shifts.
However, in samples of primary mammary carcinoma, two types of polymorphisms
were found in R A R y m R N A . Representative bands of each polymorphism are shown in
Figure 5.2.6a. These polymorphic bands were detected with the Gamma5 primer set
(Table 2.11.5.3a). Confirmation of these polymorphisms by repeat c D N A synthesis and
~~ 124
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.1.1. Representative SSCP band patterns observed for R A R a breast tumour
cDNA samples using Alphal primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.1.2. Representative SSCP band patterns observed for R A R a breast tumour
cDNA samples using Alpha2 primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.1.3. Representative SSCP band patterns observed for R A R a breast tumour
cDNA samples using Alpha3 primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
127
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.1.4. Representative SSCP band patterns observed for R A R a breast tumour
cDNA samples using Alpha4 primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
Chapter 5 Identification of Retinoic Acid Receptor Polymorphi
Figure 5.1.5. Representative SSCP band patterns observed for R A R a breast tumour
cDNA samples using Alpha5 primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
129
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.1.6. Representative SSCP band patterns observed for R A R p breast tumour
cDNA samples using Betal primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
130
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.1.7. Representative SSCP band patterns observed for R A R p breast tumour
cDNA samples using Beta2 primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
131
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.1.8. Representative SSCP band patterns observed for R A R p breast tumour
cDNA samples using Beta3 primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
132
Chapter 5 Identification of Retinoic Acid Receptor Polymorphi
Figure 5.1.9. Representative SSCP band patterns observed for R A R p breast tumour
cDNA samples using Beta4 primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
133
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.1.10. Representative SSCP band patterns observed for R A R p breast tumour
cDNA samples using Beta5 primer set (refer to Section 2.11.5). Band patterns for this
region of the receptor appeared identical in all tissues studied.
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.2.1. Representative SSCP band patterns observed for R A R y breast tumour
cDNA samples using Gammal primer set (refer to Section 2.11.5). Band patterns for
this region of the receptor appeared identical in all tissues studied.
135
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.2.2. Representative SSCP band patterns observed for R A R y breast tumour
cDNA samples using Gamma2 primer set (refer to Section 2.11.5). Band patterns for
this region of the receptor appeared identical in all tissues studied.
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.2.3. Representative SSCP band patterns observed for R A R y breast tumour
cDNA samples using Gamma3 primer set (refer to Section 2.11.5). Band patterns for
this region of the receptor appeared identical in all tissues studied.
137
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.2.4. Representative SSCP band patterns observed for R A R y breast tumour
cDNA samples using Gamma4 primer set (refer to Section 2.11.5). Band patterns for
this region of the receptor appeared identical in all tissues studied.
138
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.2.5. Representative SSCP band patterns observed for R A R y breast tumour
cDNA samples using Gamma5 primer set (refer to Section 2.11.5). These band patterns
represent the 'normal' profile and do not include polymorphic band shifts.
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.2.6. Band shift patterns seen for RARy in mammary tumour tissues. A: Lane 1
represents the normal phenotype. Lane 2 shows the S427L polymorphism band profile
(shift marked by arrow). Lane 3 shows the G443G polymorphism band profile (marked
by arrow). B: Sequencing data showing point mutations for each polymorphism. 1:
TCG->TTG base change at amino acid 427 (triangle); 2: GGG->GGA base change at
amino acid 443 (triangle).
140
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
PCR of the relevant sample showed the identical band shift as seen in the previous
sample run. Upon sequencing the PCR samples in which a polymorphism was detected
(Figure 5.2.6b), one type was found to be a silent mutation at codon 443, involving a
GGG-»GGA base change (glycine-»glycine). The other polymorphism was shown to
be a serine—^leucine amino acid change at codon 427, which involved a TCG-»TTG
point mutation. A total of 72 primary mammary carcinoma cases were screened for
polymorphisms within this region of RARy, of which 17/72 (23.6%) cases expressed the
G443G polymorphism, while 7/72 (9.7%) were found to express the S427L
polymorphism. These polymorphisms were mutually exclusive; none of the cases
studied expressed both.
5.3 Detection of RARy Polymorphism in Serum from Females with Mammary
Carcinoma
Upon finding the S427L polymorphism in seven mammary carcinoma biopsies,
DNA was extracted from these patients to determine whether the polymorphism was
genetic. Serum was the only available source of DNA from these patients and RARy
was successfully amplified by PCR. Analysis by SSCP of these serum samples also
detected the S427L polymorphism in all seven patients, thus confirming that the
polymorphism is genetic and not acquired through oncogenesis.
5.4 Detection of RARy Polymorphism in Serum and Whole Blood from Normal
Females
Forty serum samples from normal females were analysed for RARy
polymorphisms after it was revealed that the S427L polymorphism could be detected in
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
DNA in the serum from the seven patients with mammary carcinoma. It was found that
1/40 of these normal serum samples showed the S427L polymorphism. An additional
21 samples of whole blood taken from normal females were used to match the number
of tumour cases studied. It was found that 2/21 of these DNA samples showed the
S427L polymorphism. Upon pooling these results with the 11 normal breast tissue
RNA studied above, 3/72 (4.2%) of normal females, compared with 9.7% in females
with mammary carcinoma, had the S427L polymorphism. However, Fischer's exact
test showed no significant differences in these two groups (p=0.3260).
5.5 Heritability of RARy S427L Polymorphism
Analysis of DNA taken from whole blood was done on a three-generation family
(Figure 5.5.1a), using PCR/SSCP analysis (Figure 5.5.1b). The grandson's DNA
(sample 8, Figure 5.5.1a) failed to amplify, and so that result was excluded from this
study. It was found that the mother and two of her daughters exhibited the S427L
genotype, following sequencing of re-confirmed samples that were affected (samples 2,
4 and 5). Since the married daughter (sample 6) did not have this genotype (hence her
offspring did not have the S427L polymorphism) it was not possible to follow it through
three generations. However, the data suggest that this polymorphism is heritable from
parent to offspring.
142
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
B
Figure 5.5.1. Detection of R A R y S427L polymorphism in a three-generation family. A:
family tree of persons analysed in this study, all of whom were regarded as normal at
the time of bleeding. B: SSCP gel showing band patterns for family members.
Numbers in A correspond to lane numbers seen in B. Lane 8 could not be shown as the
sample failed to amplify. Following confirmation of band shifts and sequencing, lanes
2, 4 and 5 showed the S427L polymorphism, suggesting that it is inherited (see arrow in
B).
143
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
5.6 Discussion
To our knowledge, the results reported here indicate for the first time that RARy
polymorphisms exist in human tissues. We have identified two polymorphisms, a silent
base change and an amino acid change at codon 427. Using mammary carcinoma as a
disease model, we were able to show the S427L polymorphism was present in 9.7% of
mammary tumours studied and less than half of that incidence in normal samples
(4.2%). The S427L polymorphism was identified at the genomic level and was not
acquired spontaneously through oncogenesis. It was found that the prevalence of the
S427L polymorphism in normal compared to tumour samples were not statistically
significant. However, those normal females with this polymorphism could not be
followed up to determine if they had developed breast cancer. Furthermore, analysis of
a family possessing the S427L polymorphism showed that it could be inherited.
Although polymorphisms were not identified in the coding sequences of RARa and p,
the possibility that such polymorphisms occur cannot be ruled out, particularly since a
low sample size of 27 specimens was used in this study.
We were able to detect human DNA in serum samples using PCR. Since these
samples were 6 years old, it is interesting that all 47 samples studied had detectable
human DNA. This offers an alternative source of DNA for retrospective studies. The
number of amplification cycles was important for detection of the genomic RARy PCR
product. When 37 cycles were used, only some samples amplified, while at 49 cycles, a
majority but not all samples amplified. This optimisation procedure involved the use of
the seven samples that were identified with the S427L RARy polymorphism through
RT-PCR of their respective RNA extracts (data not shown). Using 60 cycles, all
samples amplified. In all PCR runs performed using this method, only twice did the
144
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
blank appear positive for RARy. However, when new reagents were used, the blank did
not contain any PCR products. Amplification of DNA has been achieved for serum
samples with between 35 and 45 cycles (Chen, et al, 1996; Nawroz, et al, 1996; De
Kok, et al, 1997; Hibi, et al, 1998; Esteller, et al, 1999). The number of cycles we
used may reflect on the age of the serum specimens. To test this, DNA was extracted
from 12 freshly collected serum samples and amplified for RARy for 40 cycles without
an initial hot start (Figure 5.6.1a). All samples amplified, which indicated that
degradation of DNA in the older frozen serum samples could be the reason why more
PCR cycles were required to amplify the RARy product. Interestingly, none of the 12
matching plasma samples that were amplified for 40-cycles for the RARy PCR reaction
contained any detectable DNA (Figure 5.6.1b). This indicated that in serum samples
DNA is released from cells during the clotting process. Plasma samples were collected
in heparinised Vacutainer tubes and the effect of heparin has been previously reported
to inhibit the PCR process (Beutler, et al, 1990). However, DNA has been successfully
analysed from whole blood collected in heparinsed tubes in the detection of markers for
Huntington's disease (Beilby, et al, 199A). The PCR method reported here used a very
small amount of serum (2-3 uL) as a source of DNA. It is important to note that the
supernatant must be removed from the serum precipitate, as this inhibited the PCR
reaction when it was amplified directly (Figure 5.6.2). An alternative method of
extracting DNA from serum has been proposed using a microwave oven (Sandford and
Pare, 1997). Based on the optimal times relative to the power of the microwave oven
(in our case, 700W for 3 min), we were unable to extract DNA from serum with this
method. As shown in Figure 5.6.2, DNA could only be detected by PCR in serum that
145
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
369bp -246bp -
123bp -
369bp 246bp
123bp
Figure 5.6.1. Agarose gel showing P C R products of RARy. (A) Amplification in serum
(lanes 1-12) and (B) matching plasma (lanes 13-24) samples. Lane 25 is the blank
control.
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
Figure 5.6.2. Agarose gel of R A R y P C R amplification comparing extraction methods.
Each run was performed in duplicate, using the same serum sample for all runs. Lanes
1 and 2: micro waved serum with supernatant removed. Lanes 3 and 4: micro waved
serum without removal of supernatant. Lanes 5 and 6: boiled serum with supernatant
removed. Lanes 7 and 8: boiled serum without removal of supernatant. Lane 9: blank
control.
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
was boiled and the supernatant removed. The differences in age and quality of the
serum used may contribute to the discrepancy seen between these two methods.
Thus, we have shown that the human RARy gene can be successfully amplified from
serum stored frozen at -70°C for 6 years, and that RARy can be amplified from serum
but not plasma. This method has the advantages that it requires very low sample
volumes, is simple, cheap and efficient. As a result of this, we propose that this method
could be used as an initial approach to extracting human DNA from serum. It will be of
great value in the field of genotyping markers for human disease and where cells have
not been collected and only serum is available.
Alterations in the RARy gene may affect the normal cell cycle. Indeed, in F9
embryonal carcinoma cells, a study has shown that disruptions in the RARa and y genes
altered regulation of the expression of differentiation-specific genes (Boylan, et al,
1995). Point mutations in the E domain of the RARy receptor have resulted in impaired
ligand binding and transactivation (Renaud, et al, 1995). The polymorphisms
identified here are located in the F domain, a region of the receptor that is poorly
understood. Deletion studies have shown that in RARa, the F domain was not required
for high-affinity ligand binding (Lefebvre, et al, 1995). However, in the oestrogen
receptor, the F domain was found to modulate gene transcription in its liganded form, as
well as in ascertaining the efficacy of antiestrogens (Montano, et al, 1995). These data
led to the conclusion that the conformation of the ligand-receptor complex differs
between the wild type form and a truncated receptor lacking the F domain. This could
subsequently affect the ability of the oestrogen receptor to interact with co-repressors or
co-activators. It is possible that the S427L amino acid change in RARy we have
detected could have a similar effect in altering the ligand-receptor complex, or perhaps
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
other functional regions of the receptor, such as the AF-2 region or the D N A binding
domain. This could possibly arise as a result of impaired binding of coregulator
molecules. W e were unable to perform a ligand-binding assay with the S427L protein
in tissue samples. This was primarily due to the amount of tissue being a limiting factor
and each tissue sample also contained normal R A R y m R N A (Fig. 5.6.2a, c.f. lane 1 and
lane 2), which would interfere with the binding assay.
Polymorphisms have been identified in the R A R a receptor (Larson, et al,
1984). Such mutations were found in patients with acute promyelocytic leukemia
(APL), where a translocation occurs in the R A R a gene (t(15;17)) that results in a
P M L / R A R a fusion protein. T w o missense mutations were found in the R A R a IE
domain of the chimeric gene in A P L patients showing clinical resistance to atRA
therapy. This suggested that mutations in the P M L / R A R a chimeric gene may give rise
to atRA resistance (Imaizumi M, et al, 1998). However, the authors reported that they
only detected 2/23 patients with mutated P M L / R A R a m R N A . Other investigators did
not find any mutations in the P M L / R A R a gene using SSCP (Morosetti, et al, 1996),
which implies that there is a low prevalence of alterations in this chimeric gene. Our
results showed an absence of mutations in the R A R a and p transcripts, suggesting that
other mechanisms must be involved with the role of R A R s in the development and
progression of mammary carcinoma.
Although mutations in the receptor can affect the retinoid signaling process, the
mere absence of these R A R s in the cell may also give rise to faulty cellular function. It
has been shown that reduced expression of endogenous R A R y in neuroblastoma cells
can lead to the malignant phenotype (Marshall, et al, 1995). Conversely, over-
expression of RARy, and not R A R a or R A R P , induces terminal differentiation in the
" " 149
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
DI human embryonal carcinoma cell line (Moasser, et al, 1995). We conclude that in
mammary tumours, aberrant expression of RARy is present and may be involved in its
carcinogenesis. As we could not follow up the health status of those normal females
with the S427L polymorphism, further work needs to be done to determine its
usefulness as a prognostic marker for hereditary breast cancer. Whether these mutations
do affect the functionality of RARy also remains to be answered.
150
Chapter 5 Identification of Retinoic Acid Receptor Polymorphisms
5.6 Conclusions
The following conclusions can be made about RAR polymorphisms in human breast
tissue and blood:
• Polymorphisms could not be found in RARa or p mRNA in breast tissues by SSCP
analysis. However, two polymorphisms were identified in RARy mRNA: G443G
and S427L. Both of these polymorphisms are located in the F domain of the
receptor.
• The S427L polymorphism occurred at the genomic level and was found to be
genetic and not acquired through oncogenesis. Furthermore, this polymorphism was
found to be heritable in a family study.
• The frequency of the S427L polymorphism in normal breast tissue and blood was
not statistically different from that observed in mammary carcinoma.
• Follow-up studies could not be done on those normal specimens that contained the
S427L polymorphism. Thus, it is not clear as to whether this polymorphism can
predispose to disease.
• The functional status of the S427L RARy protein is unclear.
151
CHAPTER SIX
GENERAL
DISCUSSION
152
Chapter 6 General Discussion
6.0 General Discussion
In this thesis it was hypothesised that expression of retinoic acid receptors may
be decreased in mammary carcinoma, and that polymorphisms may exist in their coding
sequences, and together these perturbations contribute to the development and/or
progression of breast cancer. As there has been considerable in vitro work performed
on RAR abnormalities, human breast tissue biopsies were used in experiments to test
this hypothesis because few such reports are available in the scientific literature despite
the obvious relevance. Thus, the aims focused on the determination of the mRNA and
protein expression patterns of RARa, p and y in a large cohort of mammary carcinoma
biopsies and compare with similar estimations in normal breast tissue samples.
Oestrogen status and tumour grading, which are commonly assessed in biopsies of
mammary carcinoma, were also analysed in conjunction with RAR expression to
determine if correlations exist. Finally, breast tissue and leucocyte DNA were screened
for polymorphisms in the coding sequences of RARa, P and y to elucidate if an
association existed with a predisposition to mammary neoplasia.
Breast cancer is a leading cause of morbidity and mortality in women. In
Australia, approximately 1 in 11 women are diagnosed with the disease, irrespective of
age1". Studies investigating the causes of mammary carcinoma have been extensive and
advancements have been made in the earlier diagnosis of the disease. However, despite
this progress, the mortality rate of patients with breast cancer remains high, with
approximately 2,600 Australian women dying from breast cancer every yearf. It is clear
that new approaches are required that will assist with prevention, early diagnosis,
prognosis and treatment of this disease. In addition, markers that may
f Data obtained from Cancer in Australia, 1991-1994 with projections to 1999 (1998) published
by the Australian Institute of Health and Welfare
Chapter 6 General Discussion
indicate those w o m e n w h o are at greater risk of developing breast cancer would be of
great significance.
Natural or synthetic retinoids are an appropriate candidate for chemoprevention
of malignancy as they possess anti-proliferative, differentiative and apoptotic
characteristics. Clinical trials using retinoids in treatment of breast cancer are on-going.
Promising in vitro results have been shown for the synergistic effects of retinoid
treatment in combination with other agents, such as the anti-oestrogen tamoxifen, or the
interferons (Wetherall and Taylor, 1986; Marth, et al, 1993). Therefore it would be
beneficial for women if current clinical trials prove effective using a combined
treatment modality (Budd, et al, 1998; Decensi and Costa, 2000). Indeed, retinoid
treatment has been effectively used in skin cancers and in other malignancies, such as
acute promyelocytic leukaemia (Huang et al, 1988; Lippman and Meyskens, 1989;
Castaigne, et al, 1990). However, the information present in the literature on clinical
trials using retinoids in breast cancer treatment show that they are not always effective
and may only be beneficial to certain subgroups, such as premenopausal women
(Cassidy, et al, 1982; Veronesi, et al, 1999). Thus, further understanding of the nature
of the retinoic acid receptors, which are the mediators of retinoid action, is required in
both malignant and normal breast tissues. It may be, for example, that RARs are
present at levels too low to mediate the retinoid-triggered responses in mammary
carcinoma.
The RARs are considered to be regulators of the homeostatic state of cells in
view of their profound effects in promoting cellular differentiation and inhibition of cell
growth, as well as their involvement in regulating transcription of a myriad of genes as
discussed in Section 1.3.3. The work presented in this thesis demonstrates that there is
abnormal expression of the RARs in mammary carcinoma. Alteration of retinoid
Chapter 6 General Discussion
signalling pathways, which are required for normal cellular growth and differentiation,
may arise as a result of abnormal expression and thus may be implicated in the
causation of mammary neoplasia. Very few studies have analysed expression patterns
of all 3 RAR types in tissues derived from normal breast and mammary carcinoma
(Shao, etal, 1994; Pasquali, etal, 1997; Widschwendter, etal, 1997; Xu, etal, 1997).
It was shown in Chapter 3 that there was an absence of RARy expression in mammary
carcinoma compared to normal breast. Moreover, analysis of the mRNA levels of
RARs in breast tissue using competitive RT-PCR demonstrated there was significantly
reduced expression of RARp and y in mammary carcinoma tissue compared to normal
breast tissue. This was further supported by the data in Chapter 4 by the findings that
protein levels of RARp and y were significantly decreased in mammary carcinoma
compared with patient-matched normal breast tissue. Thus, the decreased expression of
RARs, particularly p and y, in mammary carcinoma provide support for the hypothesis
described in this thesis. As RARs are able to autoregulate their expression due to
inherent RAREs in the promoter sequences (de The, et al, 1990b; Leroy, et al, 1991b;
Lehmann, et al, 1992), it is likely that functional losses of all three RARs may be
involved in mammary carcinogenesis instead of a single type of RAR. Consequently,
these neoplastic cells may not have adequate levels of RARs for normal retinoid
signalling, thus favouring the evolution and progression of mammary carcinoma. It was
also shown in Chapter 4 that there were no significant differences between tumour
grading and RAR protein levels. This suggests that decreased RAR expression may be
an early event in mammary carcinogenesis and could possibly be involved in its
initiation; further work on this aspect, however, is required.
155
Chapter 6 General Discussion
M a m m a r y carcinogenesis is associated with decreased R A R expression and this
may be an important early event in the development of breast cancer as these receptors
modulate the expression of many other genes. Studies in vitro have shown that the
addition of atRA in MCF-7 cells resulted in greater than 70% inhibition of oestrogen-
stimulated synthesis and secretion of transforming growth factor a (TGFa), which is
known to be a potent inducer of proliferation (Fontana, et al, 1992). Thus, decreased
expression of RARs may not be sufficient to antagonize oestrogen stimulation of growth
in mammary cells. Furthermore, an imbalance of the cross-coupling between RARs and
AP-1 can lead to tumourigenesis. Human skin studies in vivo have shown that UV
irradiation activates AP-1 and concomitantly results in reduced expression of RARy
mRNA and protein, which gave rise to photo-aging and development of skin cancer
(Fisher, et al, 1996; Wang et al, 1999). However, pre-treatment of skin with atRA
followed by UV exposure resulted in amelioration of loss of RARy compared with non-
treated, UV-exposed skin (Wang et al, 1999). Thus, the protective effect of atRA pre-
treatment in skin may prevent an imbalance of the cross-talk between AP-1 and RARs,
which would therefore diminish the involvement of AP-1 in the development of
neoplasia in skin.
Another inducer of proliferation, TGFpi, is known to contain three AP-1
binding sites in two of the promoter regions of this gene. Repression of this gene has
been demonstrated in HepG2 hepatocellular carcinoma cells upon treatment with atRA,
which was hormone dependent and a function of RAR concentration (Salbert, et al,
1993). Carcinogenesis can also arise through prevention of apoptosis and it has been
shown that caspase-3 can be activated by retinoids through induction of RARp
(Srivastava, et al, 1999). Thus, deficiencies in RAR expression can result in atRA
Chapter 6 General Discussion
unresponsiveness in cells and may promote molecular events that lead to initiation
and/or progression of neoplasia.
The data presented in this thesis demonstrates that a reduction in RAR
expression occurs in mammary carcinoma tissue compared to normal breast tissue,
although it is unknown what causes this lowered expression. An explanation of this
could be related to dietary uptake of retinoids, as it has been demonstrated in our
laboratory that P-carotene intake is significantly less in patients with breast cancer in
contrast with findings obtained from age-matched control patients (Ching, et al, 2002).
This observation, together with the RAR data in this thesis, show that consumption of
foods rich in retinoids may be protective against the formation of mammary carcinoma.
Another possible involvement of RARs in mammary carcinogenesis could be
because of the expression of defective receptors. In Chapter 6, a polymorphism that
altered an amino acid type was identified in the F domain of the human RARy gene.
This S427L polymorphism was observed in both normal females and in mammary
carcinoma tissue. Although there was more than twice the prevalence of the
polymorphism in subjects with breast cancer, the difference was not significant.
However, follow-up studies could not be done on the normal females with the S427L
polymorphism to determine if they had developed mammary carcinoma. Given that
breast cancer has no age onset, it would be highly improbable to perform such a follow-
up task. As this polymorphism is heritable, it may play a role in the development of
familial breast cancer. Unfortunately it was not possible to develop this part of the
project further. Nevertheless, there is some support for the second part of the
hypothesis and it is clear that more research is required to determine if this
polymorphism can serve as a biomarker for disease. A model describing how
Chapter 6 General Discussion
disruptions in R A R processing may be involved in the initiation and/or progression of
mammary carcinoma is shown in Figure 6.0.1 and could provide the basis for future
investigations.
In conclusion, it can be postulated that effective retinoid signalling can be
achieved when levels of RARs are adequate. This theory has been suggested in the
past, but was based on in vitro studies (van der Burg et al, 1993). Therefore, to
provide a suitable approach for chemoprevention of mammary carcinoma by retinoid
treatment, detection of sufficient levels of RARs at the site of the tumour should be
performed, and this would especially be useful if selective retinoids are to be used in
chemoprevention. In essence, this approach would be similar to the routine detection of
oestrogen receptor levels in mammary carcinomas to determine whether hormone
treatment is warranted. The detection of RARs can be achieved at the rnRNA level
using competitive RT-PCR as described in this thesis. However, the threshold level of
each type of RAR, or whether a combination of all three receptors is required for
effective mediation by retinoids, remains to be determined. Whether the S427L
polymorphism of the RARy gene could serve as a marker for hereditary breast cancer
also remains to be elucidated.
158
Chapter 6 General Discussion
6.1 Future W o r k
The research achieved in this thesis opens new avenues of further investigations
into the role of RARs in breast cancer. Some likely possibilities are enumerated below:
1) Development of a protocol to determine cut-off concentrations of RARs required
for the effects of retinoids. Such an approach can be performed at the mRNA level
using competitive RT-PCR, or at the protein level by antibody detection. The latter
approach could possibly be adopted from current methods in determining ER status in
breast tumours using commercially available antibody kits, or by
immunohistopathology.
2) Screening a larger population of normal females and those with mammary
carcinoma for the S427L polymorphism to determine if it is significantly associated
with breast cancer development.
3) Determine if the S427L RARy protein is functionally active.
4) Examination of the promoter region of RARs for polymorphisms that may be
associated with its de - or down-regulation.
159
Chapter 6 General Discussion
?Methylation O"Normal"
Absence of RARs in cells ^RAR mRNA/protein R A R polymorphism
?DCIS
Mammary carcinoma
TAP-I tTGFa, p 4<Apoptosis
?DCIS Mammary carcinoma
IRAR mRNA/protein
Mammary carcinoma
Figure 6.0.1. Model of mammary carcinogenesis due to disruptions in RAR signalling.
This could occur from their absence, inadequate levels, or non-functional receptors due
to polymorphisms.
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161
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