Protein western array analysis in human pituitary tumours ...Protein western array analysis in human...

Endocrine-Related Cancer (2008) 15 1099–1114

Protein western array analysis in humanpituitary tumours: insights and limitations

Antonio Ribeiro-Oliveira Jr1,2, Giulia Franchi1,3, Blerina Kola1,Paolo Dalino1,4, Sergio Veloso Brant Pinheiro 2, Nabila Salahuddin1,Madalina Musat1, Miklos I Goth5, Sandor Czirjak 6, Zoltan Hanzely 6,Deivid Augusto da Silva 2, Eduardo Paulino Jr 2, Ashley B Grossman1

and Marta Korbonits1

1Department of Endocrinology, Barts and the London School of Medicine and Dentistry, London EC1M 6BQ, UK2Department of Internal Medicine, Pediatrics and Pathology of Federal University of Minas Gerais, 30330-120 Belo Horizonte/Minas

Gerais, Brazil3Endocrinology Unit, ‘Vita-Salute’ San Raffaele University Hospital, via Olgettina 48, 20132 Milan, Italy4Endocrinology Unit, Department of Endocrinology, Niguarda Ca’ Granda Hospital, Piazza Ospedale Maggiore, 3, 20162 Milan, Italy5Endocrinology Unit, Internal Medicine, National Medical Center, 1135 Budapest, Hungary6Endocrinology Unit, National Institute of Neurosurgery, 1143 Budapest, Hungary

(Correspondence should be addressed to A Ribeiro-Oliveira Jr who is now at Laboratorio de Pesquisas em Endocrinologia, Federal

University of Minas Gerais-Brasil, Avenida Alfredo Balena, 190, sala 151, Belo Horizonte, Brazil; Email: [email protected])

Abstract

The molecular analysis of pituitary tumours has received a great deal of attention, although themajority of studies have concentrated on the genome and the transcriptome. We aimed to study theproteome of human pituitary adenomas. A protein array using 1005 monoclonal antibodies was usedto study GH-, corticotrophin- and prolactin-secreting as well as non-functioning pituitary adenomas(NFPAs). Individual protein expression levels in the tumours were compared with the expressionprofile of normal pituitary tissue. Out of 316 proteins that were detected in the pituitary tissue samples,116 proteins had not previously been described inhumanpituitary tissue.Four prominent differentiallyexpressed proteins with potential importance to tumorigenesis were chosen for validation byimmunohistochemistry and western blotting. In the protein array analysis heat shock protein 110(HSP110), a chaperone associated with protein folding, and B2 bradykinin receptor, a potentialregulator of prolactin secretion, were significantly overexpressed in all adenoma subtypes, whileC-terminal Src kinase (CSK), an inhibitor of proto-oncogenic enzymes, and annexin II, a calcium-dependent binding protein, were significantly underexpressed in all adenoma subtypes. Theimmunohistochemical analysis confirmed the overexpression of HSP110 and B2 bradykinin receptorand underexpression of CSK and annexin II in pituitary adenoma cells when compared with theircorresponding normal pituitary cells. Western blotting only partially confirmed the proteomics data:HSP110 was significantly overexpressed in prolactinomas and NFPAs, the B2 bradykinin receptorwas significantly overexpressed in prolactinomas, annexin II was significantly underexpressedin somatotrophinomas, while CSK did not show significant underexpression in any tumour. Proteinexpression analysis of pituitary samples disclosed both novel proteins and putative proteincandidates for pituitary tumorigenesis, though validation using conventional techniques arenecessary to confirm the protein array data.


Introduction

Pituitary tumours are very common neoplasms, with a

reported prevalence of 17% in the general population,

and they constitute 10% of intracranial neoplasms (Asa

&Ezzat 2002, Ezzat et al. 2004).Although these tumours


1351–0088/08/015–001099 q 2008 Society for Endocrinology Printed in Gr

cause considerable morbidity and mortality due to their

hormonal and local space-occupying effects, they only

very rarely transform into true metastatic cancers

(Kaltsas et al. 2005). Pituitary adenomas therefore offer

an important model to study how cell proliferation is

eat Britain

DOI: 10.1677/ERC-08-0003

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A Ribeiro-Oliveira Jr et al.: Proteomics of pituitary tumours

regulated, and how dysregulation may be compatible

with adenoma transformation.

Studies over recent years have established that

sporadic pituitary tumours rarely show somatic

mutational changes in genes implicated in genetic

pituitary tumour syndromes: menin (in multiple

endocrine neoplasia-1 (MEN 1) syndrome (Chandra-

sekharappa et al. 1997)), CDKN1B (causing extremely

rarely the MEN1 syndrome (Pellegata et al. 2006,

Georgitsi et al. 2007)), protein kinase A regulatory

subunit-1A (PRKAR1A, in a proportion of Carney

complex cases (Kirschner et al. 2000)) and AIP (aryl

hydocarbon receptor-interacting protein, causing a

proportion of familial isolated pituitary adenoma

cases (Vierimaa et al. 2006, Daly et al. 2007, Leontiou

et al. 2008)). In terms of mRNA expression, MEN1,

CDKN1B and PRKAR1A mRNA expression is not

changed in sporadic pituitary adenomas (Dahia et al.

1998, Satta et al. 1999, Kaltsas et al. 2002) while AIP

mRNA is up-regulated rather than down-regulated in

sporadic pituitary adenomas (Leontiou et al. 2008). In

terms of protein expression, MEN1 and PRKAR1A

show no change while CDKN1B protein is down-

regulated in pituitary adenomas, especially in cortico-

trophinomas (Lidhar et al. 1999, Lloyd et al. 2002)

and, again contrary to expectation, AIP protein is

up-regulated in sporadic pituitary adenomas (Leontiou

et al. 2008). Transgenic animal models suggest that

changes in the regulation of the cell cycle are likely to

be important in pituitary oncogenesis (Fero et al.

1996), although it is probable that many of these

changes are secondary to more proximal abnormalities

in cell signalling pathways. Therefore, it has been of

benefit to explore the phenotypic and genotypic

changes seen in pituitary adenomas as compared with

normal human pituitary tissue in order to identify the

dysregulated pathway(s).

Several dysregulated genes and proteins are

expected to occur in different pituitary tumours, and

we and others have recently published analyses of

pituitary tumour mRNA using microarray technology

(Zhan et al. 2003, Moreno et al. 2005, Morris et al.

2005). However, it is well known that a number of cell

cycle proteins in particular are regulated at the protein

level, and it is probable that mRNA analysis would not

detect such changes. We have chosen to investigate the

level of protein expression in pituitary tumours with a

protein array using pituitary adenomas, and compared

them with normal pituitary protein expression patterns,

in order to identify novel expressed proteins in human

pituitary tissue and to search for putative proteins

involved in pituitary tumorigenesis. Furthermore, we

used both immunohistochemistry and immunoblotting

1100

to quantify protein expression individually on a subset

of these proteins in order to attempt validation of the

findings. Our results confirm that protein array may be

a useful approach to identify novel candidate proteins,

but the results obtained must be confirmed by more

established techniques. This in turn suggests that the

results of proteomic analysis must always be

considered in the light of the other data.

Subjects and methods

Tissue specimens

Samples from pituitary adenomas were removed at

transsphenoidal surgery and frozen immediately.

Autopsy normal pituitary samples were collected

within 24 h of death from patients with no evidence

of any endocrine abnormality. For the BD PowerBlot

Western array (BD Biosciences Pharmingen, La Jolla,

CA, USA), a total of 20 human pituitary adenomas

were selected, representative of each of the main

adenoma subtypes (growth hormone-(GH), prolactin-

(PRL) and corticotrophin (ACTH)-secreting tumours

and non-functioning pituitary adenomas (NFPAs)),

whilst 5 normal pituitaries were used as controls. For

the immunohistochemistry, three GH-, three PRL-,

three ACTH-secreting tumours, three NFPA and four

normal pituitary samples were studied. For the western

blotting, 10 GH-, 7 PRL-, 7 ACTH-secreting tumours,

10 NFPAs and 12 normal pituitary samples were

studied. The tumour type was determined on the basis

of clinical and biochemical findings before surgery,

and by morphological and immunohistochemical

analysis of the removed tissue samples, as described

previously (Lidhar et al. 1999). Informed consent was

obtained from all patients, and the protocol was

approved by the institutional Research Ethics

Committee.

Protein extraction and quantification

For the BD PowerBlot Western array, five samples for

each tumour subtype and five samples of normal

pituitary were combined in order to obtain a single

sample of 150 mg tissue for each diagnosis. Samples

were lysed and the protein concentration measured as

previously described (Musat et al. 2005).

BD powerblot western array

The BD PowerBlot was performed by BD Biosciences

Pharmingen utilising a high-resolution two-dimen-

sional electrophoresis as previously described (Phelan

& Nock 2003). Protein concentrations were quantified

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and samples were run on six high-resolution 4–15%

gradient SDS–polyacrylamide gels in triplicate. Each

of the four subtypes of pituitary adenomas and the

normal pituitary tissue was studied using a single array.

After electrophoresis, the gels were transferred to

PVDF membranes. Each membrane was divided into

41 lanes by applying a chamber-forming grid and

probed with 1005 high-quality monoclonal antibodies

per run. After 1 h of incubation at room temperature,

the chambers were rinsed and incubated with single

fluorescent secondary antibody under the same

conditions (Alexa 680-labelled goat anti-mouse IgGs;

Molecular Probes, Eugene, OR, USA). Image data

were captured using the Odyssey Infrared Imaging

System (LI-COR). Molecular masses were determined

using molecular weight standards (BD Biosciences).

Samples were run in triplicate and analysed using a

3!3 matrix comparison method. For example, run 1 of

the control was compared with runs 1, 2 and 3 of the

experimental groups. Run 2 of the control was compared

with runs 1, 2 and 3 of the experimental groups. Run 3 of

the control was compared with runs 1, 2 and 3 of the

experimental groups – altogether 9 comparisons.

Comparative analysis (i.e. fold change in expression

level in treated versus control) followed, and protein

expression level changes were determined. Changes

were divided into 10 categories based on reproducibility

and signal intensity, with ‘level 10’ (the highest)

representing changes greater than twofold in all 9

comparisons that were from good quality signals and

passed a visual inspection. Only the proteins which were

expressed in pituitary samples and reached at least ‘level

1’ of confidence are reported (316 altogether from the

1005 tested antibodies).

We then filtered the data in order to identify the most

overexpressed or underexpressed proteins, correspond-

ing to thosewith good replicates (coefficient of variation

!10%) and a confidence level of 10 for each pituitary

adenoma subtype. We then selected four proteins with

prominent changes seen in all pituitary adenoma

subtypes, and with potential relevance to human

tumorigenesis, as putative protein candidates for further

study and confirmation using immunohistochemistry

and western blotting.

Immunohistochemistry

In order to study the distribution of the four selected

proteins (heat shock protein (HSP110), B2 bradykinin

receptor, CSK (C-terminal Src kinase) and annexin II),

and also to compare their presence in neoplastic cells with

their respective non-neoplastic cells, we utilised confocal

immunofluorescent analysis of the GH-, PRL- and


ACTH-secreting adenomas and normal pituitary tissue,

or the streptavidin–biotin method (DakoCytomation Inc.,

Carpinteria, CA, USA) for the NFPAs. The clinical-

pathological diagnosis of all pituitary adenomas had been

confirmed using immunohistochemical determination of

the expressed pituitary hormones: for the adenoma

samples, all slides utilised showed more than 90% of

the specific tumour, as confirmed by haematoxylin and

eosin. Normal pituitary architecture was evaluated by

reticulin staining. For confocal immunofluorescent

analysis of GH-, PRL- and ACTH-secreting adenomas,

after deparaffinisation and hydrating procedures, the

sections were incubated in blocking solution (1% BSA

and 0.1%Tween 20) at room temperature for 1 h and then

incubated overnight at 4 8C with one of the primary

antibodies (purified mouse anti-HSP110 antibody

(1:200), anti-mouse B2 bradykinin receptor antibody

(1:200), goat polyclonal to CSK antibody (1:200) and

goat polyclonal to annexin II antibody (1:400)). In order

to colocalise the selected proteins in different non-

neoplastic cells, rabbit anti-GH (1:200), rabbit anti-

prolactin (1:200) or rabbit anti-ACTH (1:200), supplied

by Dako (Oxford, UK), were also added to the incubation

buffer used in normal pituitary samples. After an

overnight incubation, slides were rinsed five times in

PBS and then incubated for 1h in the dark at room

temperature with anti-mouse IgG-conjugated with FITC

(1:400, Jackson Immunoresearch,WestGrove,PA,USA)

or anti-goat conjugated with Cy5 (1:400, Jackson),

according to the primary antibody previously used. An

anti-rabbit conjugatedwithCy3 (1:400, Jackson)was also

added to the incubation buffer used on normal pituitary

samples. Following washes with PBS, sections were

mounted in 90%glycerol/10%TRIS1Mand imageswere

captured through confocal microscope (Zeiss LSM 510),

40! objective, and 400! original magnification. All

confocal settings were determined at the beginning of

imaging session and remained unchanged. For quan-

titative analysis, images were captured at eight bits and

analysed in grey scale. Three to four images were

captured from each sample and three measurements

were obtained for each image. ImageJ (NIH, Bethesda,

USA) software was used to quantify fluorescence

intensity and area. Background fluorescence was then

subtracted from the region of interest. The intensity of

fluorescence corresponded to the unit ‘grey level’,

varying from zero (black) to 255 (white), as an average

of the area (sum of grey value of all pixels divided by the

number of pixels/area).

For the non-functional pituitary adenomas, while it is

accepted that the majority of them are of gonadotrophic

origin, many do not express gonadotrophins uniformly

or may express only one subunit (follicle-stimulating

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hormone, FSH, luteinizing hormone and/or human

chorionic gonadotrophin, HCG) rendering the double-

labelling technique difficult. The NFPA tumours were

therefore selected on the basis of their immunohisto-

chemical expression of FSH using monoclonal anti-

FSH (Novocastra, Newcastle-upon-Tyne, UK).

Conventional immunohistochemistry using consecu-

tive cuts was performed, as described previously

(Lidhar et al. 1999), and FSH staining positive areas

were compared between NFPA and normals. Sections

were assessed by a single observer blinded to the

diagnosis. In each section, at least 200 cells were

analysed. The percentage of positive cells was then

expressed as a ratio of positive cells to the total number

of cells counted. Photographs were taken with a Canon

PowerShot A530 colour video camera and processed in

Corel photo-Paint, version 12.0.0.458, 2003, Corel

Corporation.

Western blotting

Human pituitary adenoma tissue and normal pituitary

tissuewere studied for the assessment of the four selected

proteins: HSP110, B2 bradykinin receptor, CSK and

annexin II. In each case, 10 mg protein samples were

subjected to 10% SDS-PAGE separation, with protein

transfer to a PVDF membrane. The membrane was

blocked with 5% non-fat milk for 90 min and incubated

overnight at 4 8C with the primary antibody. The

membrane was washed six times with PBS containing

0.01–0.05% Tween and subsequently incubated for

90min at room temperature. A chemiluminescent

peroxidase substrate, ECL (Amersham-Pharmacia),

was applied according to themanufacturer’s instructions,

and the membranes were exposed briefly to X-ray film.

The PVDF membrane was then stripped with 0.985 g

Tris–Hydrochloride, 2 g SDS, 781 ml 2-mercaptoethanol

(pH 4), and reprobed. The antibodies and the respective

dilutions thatwere used are the following: purifiedmouse

anti-HSP110 antibody at 1:1000 (BD Biosciences

Pharmingen), mouse anti-B2 Bradykinin Receptor

antibody at 1:1000 (Fitzgerald, Concord, MA, USA),

polyclonal goat anti-CSK (1:500) and anti-annexin II

(1:200) antibody (Abcam,Cambridge,UK) and for equal

loading rabbit polyclonal anti-GAPDH antibody at

1:10 000 (Santa Cruz Biotechnology, Santa Cruz, CA,

USA). An anti-goat antibody at 1:5000, an anti-rabbit

antibody at 1:2000, and an anti-mouse antibody at 1:2000

Dakowere the secondary antibodies used. Densitometric

readings of the resulting bands were evaluated using the

ImageJ program, and the ratio toGAPDHwas calculated

and compared with controls.

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Statistical analysis

The data were tested for statistical significance with the

non-parametric Mann–Whitney test or multivariate

analysis using the Kruskal–Wallis test followed by

Conover–Inman analysis (StatsDirect, Ian Buchan,

Cambridge, UK). Significance was taken as P!0.05.

All values given are meansGS.E.M).

Results

The 1005 antibodies utilised in the BD PowerBlot

Western array detected a total of 316 proteins from the

human pituitary tissues. These proteins were cate-

gorised into different functional groups, according to

their areas of activity, and they are summarised in Fig. 1

and listed with their functional categories in Supple-

mentary Table 1, which can be viewed online at http://

erc.endocrinology-journals.org/supplemental/. Out of

these 316 proteins, we found through a literature survey

and interrogation of a human pituitary database (Zhan&

Desiderio 2003, Zhao et al. 2005a) that 116 proteins had

not previously been described as being expressed in

human pituitary tissue, and 81 of these have also not

been studied in the pituitary of any other species.

Expression profile of adenomas

The expression profiles for GH-, PRL-, ACTH-secreting

and non-functioning adenomas and NFPAs, and the

expression profile comparisons to normal autopsy

pituitary tissues, using the BD PowerBlot Western array,

were analysed. The main criteria used to select potential

candidate proteins were ‘level 10’ of confidence for all

pituitary adenoma subtypes. We also considered in this

selection process the consistency within the replicates in

the array, our previous microarray data (Morris et al.

2005), and thepossible relevanceof theproteins for human

tumorigenesis. Themost significant and consistently over-

and underexpressed ‘level 10’ proteins occurring in all

adenoma types studied are shown in Table 1. ‘Level 10’

proteins that were specific to only one or two adenoma

subtypes are shown in Table 2. Interestingly, there were

some ‘level 10’ proteins that were present only in certain

tumours and not present in normal pituitary tissue, while

others were present in normal pituitary tissue but not

present in certain tumours (Table 3).

In all pituitary adenomas subtypes, the number of

‘level 10’ overexpressed proteins was higher than

the number of ‘level 10’ underexpressed ones

(Table 4). However, the fold-change of underexpression

was higher for the underexpressed proteins when

compared with the fold change for the overexpressed

ones (Table 4).



http://erc.endocrinology-journals.org/supplemental/

http://erc.endocrinology-journals.org/supplemental/

Figure 1 Functional categories of 316 characterised proteins covered by the protein microarray. Some proteins were categorised tomore than one functional group. The proteins with areas of research reaching less than 2% in the array were grouped to others(Calcium Signalling, Stats, Immunology, Nitric Oxide, Protein Phosphatase, Protein Sorting and Modification). See Supplementalmaterial Table 1, which can be viewed online at http://erc.endocrinology.org/supplemental/, for each individual protein.


Identification of potential candidate proteins

We selected four proteins which showed significant

changes in all four adenoma subtypes, two being

overexpressed (HSP110 and B2 bradykinin receptor)

and two underexpressed (CSK and annexin II) for

further study . HSP 110 and C-terminal Src kinase had

not specifically been described before as part of the

human pituitary proteome database (Zhan & Desiderio

2003, Zhao et al. 2005a).

HSP110 showed an average of 17-fold overexpres-

sion in all adenoma subtypes as compared with normal

pituitary tissues (33-, 9-, 10- and 14-fold overexpres-

sion for GH-, PRL-, ACTH-secreting adenomas and

NFPAs respectively) in the protein array analysis. In

our previous pituitary microarray study HSP110 gene

expression showed underexpression although this was

not significant (Morris et al. 2005).

The B2 bradykinin receptor showed an average of

11-fold overexpression in all adenomas subtypes as

compared with normal pituitary tissues (16-, 20-, 5- and

4-fold overexpression for GH-, PRL-, ACTH-secreting


adenomas and NFPAs respectively). No data from our

previousmicroarraywere available for the B2 bradykinin

receptor. Other studies have suggested a role for this

receptor in anteriorpituitary regulation (Sharif et al. 1988,

Kuan et al. 1990), and the importance of its up-regulation

in other human cancers has been emphasised (Ikeda et al.

2004, Dlamini & Bhoola 2005, Zhao et al. 2005b).

The CSK protein showed an average of 117-fold

underexpression in all adenoma subtypes as compared

with normal pituitary tissues (25-, 168-, 134- and 140-

fold underexpression for GH-, PRL-, ACTH-secreting

adenomas and NFPA respectively). Our previous

microarray data also showed reduced mRNA

expression in pituitary adenomas (Morris et al. 2005).

Finally, annexin II showed an average of 27-fold

underexpression in all adenoma subtypes as compared

with normal pituitary tissues (25-, 20-, 32- and 31-fold

underexpression for GH-, PRL-, ACTH-secreting ade-

nomas and NFPAs respectively). A significant level of

underexpression had also been shown in our previous

microarray data, where an average fourfold under-

expression was found (Morris et al. 2005).

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http://erc.endocrinology.org/supplemental/

Table 1 Most consistently changing proteins in all adenoma subtypes

Average fold change for each adenoma

subtype

Protein Swiss-Prot ID GH PRL ACTH NFPA

Direction

of change

ARF3 (ADP-ribosylation factor 3) P16587 14 26 N/A 28 CAnnexin I P46193 12 13 31 24 K

Annexin II P07355 25 20 32 31 K

B2 bradykinin receptor P30411 16 20 05 04 C

CSK tyrosine-protein kinase CSK (C-Src Kinase) P32577 25 168 134 140 KHeat shock-110 kDa Q60446 33 09 10 14 C

MEF 2D (myocyte-specific

enhancer factor 2D)

Q63943 20 15 06 21 C

P54nrb (54 kDa nuclear RNA-

and DNA-binding protein)

Q15233 39 50 29 68 C

Rab3 (Rab-protein 3, member

RAS oncogene)

04 N/A 14 11 C

RBP (retinol-binding protein I, cellular) P09455 06 177 N/A 26 K

SCAMP1 (secretory carrier membrane protein 1) O15126 12 10 N/A 06 C

Average fold change for each adenoma subtype of the most consistent over- (C) or underexpressed (K) ‘level 10’ proteins from thearray in alphabetical order. N/A, not available.


Immunohistochemistry for HSP110, B2

bradykinin receptor, CSK and annexin II

In order to compare the expression levels of an individual

protein between specific cell types in the normal pituitary

and the specific cell type-derived tumours, we performed

confocal immunofluorescent analysis for GH-, PRL- and

ACTH-secreting adenomas. For theNFPA, conventional

immunohistochemistry with consecutive cuts was

performed and FSH-positive tumour areas were

compared with FSH-positive areas from the normal

pituitary. To this end, 12 pituitary tumours and 4 normal

pituitaries were selected.

Table 2 Most consistently changing proteins for each adenoma su

Protein Swiss-

Acetylcholine receptor b P25109

ASS (argininosuccinate synthase) P00966

Apoptosis regulator BAX Q07812

Dematin Q08495

EBP 50 (Ezrin–radixin–moesin-binding

phosphoprotein 50)

O14745

ECA39 (branched-chain-amino-acid aminotransferase) P54687

Epithelial-cadherin precursor P12830

HILP/XIAP(inhibitor of apoptosis protein 3/X-linked IAP) P98170

Lamin A/C Q969I8

L-Caldesmon Q05682

MEPHX (microsomal epoxide hydrolase) P07099

The 11 most consistent ‘level 10’ proteins for pituitary adenoma sudirection of change (C/K).

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The confocal immunofluorescent analysis showed

that HSP110was expressed in the normal pituitary, with

mild coexpression in normal GH, PRL and ACTH cells.

It also showed the overexpression of HSP110 in GH-,

PRL- and ACTH-secreting tumour cells when

compared with respective normal cells from normal

pituitary (P!0.01 for all comparisons). HSP110 was

also overexpressed in FSH-staining tumour when

compared with FSH cells from normal pituitary

(P!0.01). These data confirm the overexpression of

this protein in tumorous cells when compared with the

corresponding normal pituitary cells (Fig. 2).

btype

Prot ID Adenoma subtype

Average fold

change

Direction

of change

ACTH 14 CPRL 21 C

NFPA 15 C

ACTH and GH 10 and 10 C

PRL and NFPA 19 and 15 C

NFPA 14 C

PRL 39 C

NFPA and ACTH 18 and 11 CACTH 14 K

ACTH 18 C

GH, PRL and NFPA 14, 17 and 10 K

btypes in alphabetical order, including average fold change and



Table 3 Proteins that were not detected in normal pituitary or in one adenoma type

Protein Swiss Prot ID Adenoma subtype Comment

14-3-3e-23KD 14-3-3 protein epsilon

(mitochondrial import stimulation factor

L subunit)

P42655 Underexpression in GH secreting

adenomas and NFPA

Cannot be detected in GH

secreting tumours and

NFPA

CPG16/CaM kinase VI

(calcium/calmodulin-dependent protein

kinase type I-like)

O08875 Overexpression in ACTH-secreting

adenomas

Cannot be detected in normal

pituitary

GRIM-19 (cell death-regulatory

protein 19)

Underexpression in ACTH-secreting

adenomas

Cannot be detected in

ACTH-secreting tumours

HRF (histamine-releasing factor) P13693 Underexpression in ACTH-secreting

adenomas


ACTH-secreting tumours

L22-18-19 KD (60s ribosomal

protein L22)

P35268 Overexpression in PRL-secreting

adenomas


normal pituitary

MEK5-53-57KD (dual specificity

mitogen-activated protein kinase

kinase 5; MAP kinase kinase 5)

Q13163 Overexpression in PRL-secreting

adenomas


normal pituitary

PP2A catalytic a-34KD (protein

phosphatase 2, catalytic subunit, alpha)

Overexpression in PRL-secreting

adenomas


normal pituitary

Reps1(RalBP1-associated Eps

domain-containing protein 1;

RalBP1-interacting protein 1)

O54916 Overexpression in GH-secreting

adenomas


normal pituitary

RONa (macrophage-stimulating protein

receptor precursor; MSP receptor;

p185-Ron; CD136 antigen; CDw 136)

Q04912 Overexpression in GH-secreting

adenomas


normal pituitary

Consistent level 10 proteins for specific adenoma subtypes that showed gross over or underexpression compared with normalpituitary. NFPA, non-functional pituitary adenoma.



that B2 bradykinin receptor was expressed in the

normal pituitary, with mild coexpression in normal

GH, PRL and ACTH cells. It also showed over-

expression of this protein in GH-, (P!0.05), PRL-,

(P!0.01) and ACTH-secreting tumour cells

(P!0.01) when compared with their respective

pituitary cells (P!0.01). B2 bradykinin receptor was

also overexpressed in FSH-staining tumour when

compared with FSH cells from normal pituitary

(P!0.01). These data confirm the overexpression of

B2 bradykinin receptor in tumorous cells when

Table 4 The number of the most consistently changing proteins in

‘Level 10’ proteins GH

Number of ‘level 10’ overexpressed proteins 29

Number of ‘level 10’ underexpressed proteins 12

MeansGS.E.M. of fold changes in ‘level 10’

overexpressed proteins

8.9G0.8*

MeansGS.E.M. of fold changes in ‘level 10’

underexpressed proteins

10.6G1.0

Number of ‘level 10’ proteins and profile of their over and underexp*P!0.05 for comparisons between fold changes in ‘level 10’ over anfold changes in ‘level 10’ over and underexpressed proteins.


compared with the corresponding normal pituitary

cells (Fig. 3).

For the CSK protein, the confocal immunofluor-

escent analysis showed expression of this protein in

normal pituitary, with coexpression in GH, PRL and

ACTH cells. It also showed underexpression in GH-,

PRL- and ACTH-secreting tumour cells when

compared with their respective normal pituitary cells

(P!0.01). CSK was also underexpressed in FSH-

staining tumour when compared with FSH cells from

normal pituitary (P!0.01). These data confirm the

underexpression of CSK in all adenoma tumour cells

each adenoma subtype

PRL ACTH NFPA

29 36 36

11 22 20

10.9G1.1† 6.7G0.5* 9.0G1.1†

25.7G7.0 13.8G2.0 16.4G2.4

ressions in each adenoma subtype compared with controls.d underexpressed proteins. †P!0.01 for comparisons between

1105


Figure 2 Double immunofluorescent staining using HSP110 (blue staining) and polyclonal GH, PRL and ACTH (yellow staining)antibodies in normal pituitary, and HSP110 (blue staining) in GH-, PRL- and ACTH-secreting adenomas (3A, 3B and 3Crespectively). Colocalisation is shown by green colour (scale bar 50 mm): In figure 2D, conventional immunohistochemistry stainingusing HSP110 (brown) in consecutive cuts of normal pituitary tissue and non-functional pituitary adenomas (FSH positive); scale bar:25 mm. All data are shown as meansGS.E.M.; AU, arbitrary units; *P!0.05, **P!0.01.


when compared with the corresponding normal

pituitary cells (Fig. 4).


expression of annexin II in normal pituitary, with

coexpression in GH, PRL and ACTH cells. It also

showed underexpression of this protein in GH-, PRL-,

and ACTH-secreting tumour cells when compared

with their respective normal pituitary cells (P!0.01

for all comparisons). Annexin II was also under-

expressed in FSH-staining tumour when compared

with FSH cells from normal pituitary (P!0.01). These

data confirm the underexpression of annexin II in all

adenoma tumour cells when compared with the

correspondent normal pituitary cells (Fig. 5).

1106

Western blotting for HSP110, B2 bradykinin

receptor, CSK and annexin II

In order to validate the expression patterns of HSP110,

B2 bradykinin receptor, CSK and annexin II as shown

by protein array, conventional western blotting was

also performed on the proteins from 34 pituitary

tumours and 12 normal pituitaries. Certain samples

were the same as the ones included in the protein array,

to these we added additional tumours and normal

pituitary samples for this validation process.

HSP110 immunoblotting confirmed overexpression

in NFPAs (P!0.01 compared with normal controls)

and prolactin-secreting adenomas (P!0.05), but no



Figure 3 Double immunofluorescent staining using B2 bradykinin receptor (blue staining) and polyclonal GH, PRL and ACTH(yellow staining) antibodies in normal pituitary, and B2 bradykinin receptor (blue staining) in GH-, PRL- and ACTH-secreting adenomas(4A, 4B and 4C respectively). Colocalisation is shown by green colour (scale bar 50 mm). In figure 3D, conventionalimmunohistochemistry staining using B2 bradykinin receptor (brown) in consecutive cuts of normal pituitary tissue and non-functionalpituitary adenomas (FSH positive); scale bar 25 mm. All data are shown as meansGS.E.M.; AU, arbitrary units; *P!0.05, **P!0.01.


significant overexpression was detected in GH- or

ACTH-secreting adenomas (Fig. 6A). These data

therefore confirmed in part, the protein overexpression

shown in the protein array to all adenomas subtypes.

Immunoblotting for the B2 bradykinin receptor

confirmed overexpression in prolactin-secreting ade-

nomas (P!0.05), correlating well with the degree of

overexpression shown for this adenoma subtype in the

protein array; ACTH-secreting adenomas also showed

a trend towards overexpression (PZ0.1), but neither

the NFPAs nor the GH-secreting adenomas showed

significant changes (Fig. 6B). These data thus

confirmed the overexpression of B2 bradykinin

receptor shown in the protein array for the prolactin-

secreting adenomas alone.


For the underexpressedCSKprotein, the results on the

conventional western blotting were not confirmed; none

of the adenoma subtypes showed significantly reduced

expression by immunoblotting (Fig. 6C). Annexin II,

another prominent underexpressed protein in the protein

array, was underexpressed in GH-secreting adenomas

(PZ0.01) and there was also a trend for underexpression

in the NFPAs (PZ0.09). However, this was not seen in

either ACTH- or PRL-secreting adenomas (Fig. 6D).

Discussion

We have utilised a proteomics technique to identify a

series of proteins that are differentially expressed in

pituitary adenomas when compared with the normal

1107


Figure 4 Double immunofluorescent staining using CSK (red staining) and polyclonal GH, PRL and ACTH (green staining)antibodies in normal pituitary, and CSK (red staining) GH-, PRL- and ACTH-secreting adenomas. Colocalisation is shown by orangecolour (scale bar 50 mm). In figure 4D, conventional immunohistochemistry staining using CSK (brown) in consecutive cuts of normalpituitary tissue and non-functional pituitary adenomas (FSH positive); scale bar 25 mm. All data are shown as meansGS.E.M.; AU,arbitrary units; *P!0.05, **P!0.01.


pituitary. Due to the magnitude of the amount of

protein required for protein array, and the relatively

small amount of material available from each tumour,

we had to amalgamate tissue from five separate

tumours for each tumour subtype sample. Following

the analysis of protein array data, we explored the

expression of four grossly over- and underexpressed

proteins seen in all tumour subtypes by immunohis-

tochemistry and conventional western blotting. The

proteomic array data were fully reproduced by the

immunohistochemistry but only partially reproduced

by western blotting.

High-resolution two-dimensional gel electro-

phoresis (2-DE) has been the standard tool for

expression proteomics since its introduction more

than 30 years ago. The protein array that we describe

1108

here covered 1005 proteins with monoclonal

antibodies, out of which 316 were expressed in

pituitary tissue. Excellent previous studies have set

up a database of proteins expressed in pituitary

tumours (Zhan & Desiderio 2003, Zhao et al. 2005a)

and now our data add a large number of novel proteins

into this database. These proteins are shown in

Supplementary Table 1 according to their functional

categories and direction of changes observed in

adenomas. Despite various improvements of the

2-DE technique, one of the main inherent limitations

of this method is the fact that many proteins that are

expressed at low levels may escape detection. On the

other hand, the findings shown in Table 4 of many-fold

changes in underexpression suggest a considerable

sensitivity for this array.



Figure 5 Double immunofluorescent staining using annexin II (red staining) and polyclonal GH, PRL and ACTH (green staining)antibodies in normal pituitary, and annexin II (red staining) in GH-, PRL- and ACTH-secreting adenomas. Colocalisation is shown byorange colour (scale bar 50 mm). In figure 5D, conventional immunohistochemistry staining using annexin II (brown) in consecutivecuts of normal pituitary tissue and non-functional pituitary adenomas (FSH positive); scale bar 25 mm. All data are shown asmeansGS.E.M.; AU, arbitrary units; *P!0.05, **P!0.01.


The molecular analysis of pituitary tumours has

been extensively studied, and a number of factors

involved in pituitary tumorigenesis have been

described. The genome of a given cell or organism is

relatively static, subject to epigenetic modulation,

whereas the transcriptome and proteome are more

dynamic, and change with time and conditions

reflecting the different state of the disease. Indeed,

the proteome is much more complex than the

transcriptome due to post-translational modifications,

translocation, protein–protein interactions and

regulation, but otherwise may provide more direct

information when compared with the transcriptome in

relation to oncogenesis. An important aim of proteo-

mics has been to understand the cellular function at the


protein level by the dynamic proteome, clarifying the

functions of proteins and disclosing new biomarkers

for disease states.

The four proteins chosen from the protein array for

validation by immunoblotting showed consistent pro-

minent over- or underexpression in all adenoma

subtypes in the original array. Furthermore, two of

these proteins (HSP110 and CSK) had not previously

been described as expressed in normal human pituitary

tissue. The data obtained from the immunohistochemi-

cal analysis confirmed these changes for all four

proteins. Immunoblotting showed only partial paralle-

lism with the array data; it confirmed the results

shown in the protein array of overexpression of

HSP110 in NFPAs and prolactin-secreting adenomas,

1109


Figure 6 Expression of (A) HSP110, (B) B2 bradykinin receptor, (C) CSK and (D) annexin II in each adenoma subtype comparedwith normal pituitary tissue by western blotting. Data are shown as meansGS.E.M., *P!0.05, **P!0.01.


and also overexpression of the B2 bradykinin

receptor in prolactin-secreting adenomas. Furthermore,

it confirmed the underexpression of annexin II in

GH-secreting adenomas, although it completely failed

tomirror the results for CSK.These data implicate a role

for HSP110, the B2 bradykinin receptor, CSK and

annexin II in the process of pituitary tumorigenesis in

these tumour subtypes, although whether these changes

are causal or consequential remains unclear.

HSP110 is expressed in most tissues, and like other

stress proteins, HSP110 is a chaperone protein

associated with protein folding. It can be induced by

heat shock and its induction is associated with

thermotolerance. HSP’s overexpression has been

recently described as important in a series of human

cancers (Feng et al. 2005, Melle et al. 2006). Our

previous microarray data showed a trend for HSP110 in

the opposite direction (Morris et al. 2005), suggesting

that some form of post-transcriptional modification

accounts for these results. The novel data regarding the

overexpression of molecular chaperone HSP110 in

NFPAs and prolactin-secreting tumours demonstrates

the added value of proteomic analysis subsequent to

mRNA analysis. Recent proteomic studies on breast

cancer cells have shown that HSPs, including HSP110,

showedprotein expression patterns thatmight be altered

by 17b-oestradiol treatment (Lee et al. 2006). Further-

more, APG-2 protein, a member of the HSP110 family,

has shown developmental changes in its expression at

the protein level (Okui et al. 2000). These data

demonstrate the utility of an analysis at the protein

level. As recent publications have characterised

HSP110 and other HSPs as potential anti-tumour agents

(Wang et al. 2001, Casey et al. 2003), it is possible that

HSP110 overexpression in these adenomas may be a

1110

protective mechanism to reduce adenomatous

proliferation, and may relate to the benign nature of

the majority of such tumours. Other heat shock-induced

proteins have also been demonstrated to be differen-

tially induced in tumorous tissue (Feng et al. 2005,

Melle et al. 2006).

The B2 bradykinin receptor is a member of the

kallikrein system, a subgroup of the serine protease

family of enzymes, which also includes the enzyme

known as prostate-specific antigen, an important

clinical marker in human prostate cancers (Diamandis

& Yousef 2002). The B2 bradykinin receptor was

overexpressed in prolactin-secreting tumours; previous

work has shown that bradykinin increases prolactin,

but not GH release, and that this stimulation could be

antagonised by a B2-type bradykinin receptor antagon-

ist and inhibited by dopamine (Kuan et al. 1990). Other

studies have suggested a role for this receptor in

anterior pituitary regulation (Sharif et al. 1988). The

B2 bradykinin receptor belongs to the kallikrein-kinin

system, and several of its components have been

reported to be tumour markers for many malignancies

(Diamandis & Yousef 2002), especially those of

endocrine-related organs (Yousef & Diamandis

2003). The importance of its up-regulation in other

human cancers has also been described (Ikeda et al.

2004, Dlamini & Bhoola 2005, Zhao et al. 2005b).

Furthermore, it has been suggested that some

kallikreins may be part of a novel enzymatic cascade

pathway that is activated in some cancers, with a

particular potential role for kallikreins in central

nervous system (Yousef & Diamandis 2003, Yousef

et al. 2003). While its function in pituitary oncogenesis

remains obscure, it may offer a novel therapeutic target

(Stewart et al. 1997).




CSK is a non-receptor protein tyrosine kinase, its

major function being to specifically phosphorylate a

conserved C-terminal tyrosine of Src family kinases.

As a consequence of down-regulation of the Src kinase

protein expression and presumably its activity, proto-

oncogenic enzymes controlling cell growth and

proliferation would diminish. It is well known that

Src-family protein tyrosine kinases are tightly

regulated in normal cells and aberration in their

regulation can lead to constitutive activation, which

is associated with certain types of cancer (Kim et al.

2004, Chong et al. 2005, Martin 2006).

As a calcium-dependent phospholipids-binding

protein, annexin II has been suggested to play a role

in exocytosis from anterior pituitary secretory cells

(Turgeon et al. 1991, Senda et al. 1994). It has

previously been reported as being down-regulated in

some human cancers (Xin et al. 2003, Gillette et al.

2004), although overexpression of annexin II on the

cell surface has been suggested to contribute to the

development of metastases by enhancing tumour cell

detachment and tissue invasion (Choi et al. 2000, Ma

et al. 2000). Annexin II was described in a previous

human pituitary proteomics analysis (Zhan & Desi-

derio 2003); in this case our finding of underexpression

in GH-secreting adenomas is in accordance with our

previous microarray showing down-regulation of

annexin II mRNA in GH-secreting adenomas (Morris

et al. 2005). The importance of the annexin family as

potential regulatory factor in pituitary tumours has

been described, and expression of annexins I and V

have been demonstrated in pituitary tumours (Traverso

et al. 1999, Mulla et al. 2004). Specifically, annexin II

overexpression has previously been shown in a

GH-secreting carcinoma (Mulla et al. 2004). It is

therefore possible that carcinomatous transformation

per se leads to a specific annexin II increase.

While factors involved in pituitary tumorigenesis

have been subject to intensive study (Asa&Ezzat 2002,

Heaney & Melmed 2004, Kaltsas et al. 2005, Farrell

2006) there have been relatively few studies of the

specific pituitary protein changes using proteomics

(Zhan & Desiderio 2003, Zhan et al. 2003, Asa & Ezzat

2005,Moreno et al. 2005, Zhao et al. 2005a). This study

was performed using a screening commercial protein

array to track many different proteins simultaneously in

order to obtain greater insight into cellular function.

However, there are problems associatedwith the limited

sampling that is often employed, and also some

uncertainty regarding the use of autopsy controls for

comparison with fresh adenomatous tissue. In our study

all tissues were normalised for protein content, and such

tissue is the best currently available for comparison to


tumours. Reassuringly, a considerable proportion of

proteins showed higher expression in the normal

autopsy tissue than in the surgical adenoma samples.

Despite the recent advances in human proteomics

(Garbis et al. 2005, Senzer et al. 2005), we believe

confirmation with additional technique(s) can provide

furtherusefuldata.Ourproteomicsdatawerecorrespond-

ing with the immunohistochemistry but the immuno-

blotting results were more variable. Especially, the fold

changes over- or underexpressions shown by the protein

array were only weakly correlated with the quantitative

changes observed in western blotting.

Indeed, several shortcomings have been described in

proteomic studies, and while there are few reports on

proteomics for pituitary adenomas, the findings may be

even more difficult to interpret with these small tumours

when compared with other human cancers. Furthermore,

this study was performed utilising a commercial

screening array that was not designed specifically to

focus on the pituitary. As a consequence, novel proteins

were shown to be present in pituitary while others were

not assessed. However, our findings of the expression of

the pituitary transcription factor Pit 1 in controls,

up-regulation of this protein in GH- and PRL-secreting

adenomas, and the absence of this protein in ACTH-

secreting adenomas and non-functional pituitary adeno-

mas, corroborates the hypothesis that this is indeed a

reliable protein array. Although whole tissue samples

lead to frank admixture of tumour cellswith various other

cells (stromal cells, macrophages, red blood cells, etc.) it

may be argued that these cells may also be important for

the physiology of the tumour, and that they should

therefore be included. On the other hand, it would have

been ideal that this study hadmatched for age and gender

tumour bearing patients and control pituitaries, although

the small availability of pituitary material for research

generally precludes this selection. It is also known that

the comparisons between adenomatous tissues consisting

of a monoclonal, homogeneous tumour cell population

and normal pituitaries representing various proportions

of endocrine cell types may also carry some bias. This

study, however, overcame this important obstacle,

showing by immunofluorescent and conventional immu-

nohistochemistry the expected over- or underexpression

of the four selected proteins in human pituitary tumour

cells when compared with their normal pituitary cells.

Interestingly, these results were much less obvious in the

immunoblotting study, and in some cases were absent.

The concordance of the proteomic results with the

immunohistochemistry suggests that conventional

immunoblotting in this situation is of much less

sensitivity.

1111



In summary, our data show the limitations of

proteomic profiling, but do reveal novel proteins in

human pituitary tissue and some possible candidates

for pituitary oncogenesis. Our data also encourage

future studies targeting these novel protein candidates

as possible biomarkers to this diagnosis, monitoring of

disease progression, and developing of novel drug

therapeutic approaches to some specific pituitary

adenomas. However, we caution using proteomic

array data without validating its findings using

alternative more conventional techniques.

Declaration of interest

The authors declare that there is no conflict of interest that

could be perceived as prejudicing the impartiality of the

research reported.

Funding

This work was supported by the Cancer Research Committee

of Saint Bartholomew’s Hospital and by the OTKA K68660

grant. A R O Jr was supported by CNPq (Conselho Nacional

de Desenvolvimento Cientıfico e Tecnologico).

Acknowledgements

We are grateful for the support of Cancer Research

Committee of Saint Bartholomew’s Hospital and CNPq

(Conselho Nacional de Desenvolvimento Cientıfico e

Tecnologico).

References

Asa SL & Ezzat S 2002 The pathogenesis of pituitary

tumours. Nature Reviews. Cancer 2 836–849.

Asa SL & Ezzat S 2005 Genetics and proteomics of pituitary

tumors. Endocrine 28 43–47.

Casey DG, Lysaght J, James T, Bateman A, Melcher AA &

Todryk SM 2003 Heat shock protein derived from a non-

autologous tumour can be used as an anti-tumour vaccine.

Immunology 110 105–111.

Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE,

Collins FS, Emmert-Buck MR, Debelenko LV, Zhuang Z,

Lubensky IA, Liotta LA et al. 1997 Positional cloning of

the gene for multiple endocrine neoplasia-type 1. Science

276 404–407.

Choi S, Kobayashi M, Wang JX, Habelhah H, Okada F,

Hamada J, Moriuchi T, Totsuka Y & Hosokawa M 2000

Activated leukocyte cell adhesion molecule (ALCAM)

and annexin II are involved in the metastatic progression

of tumor cells after chemotherapy with Adriamycin.

Clinical & Experimental Metastasis 18 45–50.

1112

Chong YP, Mulhern TD & Cheng HC 2005 C-terminal Src

kinase (CSK) and CSK-homologous kinase (CHK)-

endogenous negative regulators of Src-family protein

kinases. Growth Factors 23 233–244.

Dahia PL, Aguiar RC, Honegger J, Fahlbush R, Jordan S,

Lowe DG, Lu X, Clayton RN, Besser GM & Grossman

AB 1998 Mutation and expression analysis of the

p27/kip1 gene in corticotrophin-secreting tumours.

Oncogene 16 69–76.

Daly AF, Vanbellinghen JF, Khoo SK, Jaffrain-Rea ML,

Naves LA, Guitelman MA, Murat A, Emy P, Gimenez-

Roqueplo AP, Tamburrano G et al. 2007 Aryl hydro-

carbon receptor–interacting protein gene mutations in

familial isolated pituitary adenomas: analysis in 73

families. Journal of Clinical Endocrinology and Metab-

olism 92 1891–1896.

Diamandis EP & Yousef GM 2002 Human tissue kallikreins:

a family of new cancer biomarkers. Clinical Chemistry 48

1198–1205.

DlaminiZ&BhoolaKD2005Upregulationof tissuekallikrein,

kinin B1 receptor, and kinin B2 receptor in mast and giant

cells infiltrating oesophageal squamous cell carcinoma.

Journal of Clinical Pathology 58 915–922.

Ezzat S, Asa SL, Couldwell WT, Barr CE, DodgeWE, Vance

ML & McCutcheon IE 2004 The prevalence of pituitary

adenomas: a systematic review. Cancer 101 613–619.

Farrell WE 2006 Pituitary tumours: findings from whole

genome analyses. Endocrine-Related Cancer 13 707–716.

Feng JT, Liu YK, Song HY, Dai Z, Qin LX, Almofti MR,

Fang CY, Lu HJ, Yang PY & Tang ZY 2005 Heat-shock

protein 27: a potential biomarker for hepatocellular

carcinoma identified by serum proteome analysis.

Proteomics 5 4581–4588.

Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E,

Polyak K, Tsai LH, Broudy V, Perlmutter RM et al. 1996

A syndrome of multiorgan hyperplasia with features of

gigantism, tumorigenesis, and female sterility in

p27(Kip1)-deficient mice. Cell 85 733–744.

Garbis S, Lubec G & Fountoulakis M 2005 Limitations of

current proteomics technologies. Journal of Chromatog-

raphy. A 1077 1–18.

Georgitsi M, Raitila A, Karhu A, van der Luijt RB, Aalfs CM,

Sane T, Vierimaa O, Makinen MJ, Tuppurainen K,

Paschke R et al. 2007 Germline CDKN1B/p27Kip1

mutation in multiple endocrine neoplasia. Journal of

Clinical Endocrinology and Metabolism 92 3321–3325.

Gillette JM, Chan DC & Nielsen-Preiss SM 2004 Annexin 2

expression is reduced in human osteosarcoma metastases.

Journal of Cellular Biochemistry 92 820–832.

Heaney AP & Melmed S 2004 Molecular targets in pituitary

tumours. Nature Reviews. Cancer 4 285–295.

Ikeda Y, Hayashi I, Kamoshita E, Yamazaki A, Endo H,

Ishihara K, Yamashina S, Tsutsumi Y, Matsubara H &

Majima M 2004 Host stromal bradykinin B2 receptor

signaling facilitates tumor-associated angiogenesis and

tumor growth. Cancer Research 64 5178–5185.




Kaltsas GA, Kola B, Borboli N, Morris DG, Gueorguiev M,

Swords FM, Czirjak S, Kirschner LS, Stratakis CA,

Korbonits M et al. 2002 Sequence analysis of the

PRKAR1A gene in sporadic somatotroph and other

pituitary tumours. Clinical Endocrinology 57 443–448.

Kaltsas GA, Nomikos P, Kontogeorgos G, Buchfelder M &

Grossman AB 2005 Clinical review: diagnosis and

management of pituitary carcinomas. Journal of Clinical

Endocrinology and Metabolism 90 3089–3099.

Kim SO, Avraham S, Jiang SX, Zagozdzon R, Fu YG &

Avraham HK 2004 Differential expression of Csk

homologous kinase (CHK) in normal brain and brain

tumors. Cancer 101 1018–1027.

Kirschner LS, Sandrini F, Monbo J, Lin JP, Carney JA &

Stratakis CA 2000 Genetic heterogeneity and spectrum of

mutations of the PRKAR1A gene in patients with the

Carney complex.HumanMolecular Genetics 9 3037–3046.

Kuan SI, Judd AM, Jarvis WD, Login IS & MacLeod RM

1990 Physiological and biochemical effects of bradykinin

and lys-bradykinin in pituitary cells. Molecular and

Cellular Endocrinology 72 239–246.

Lee SU, Kim BT, Min YK & Kim SH 2006 Protein profiling

and transcript expression levels of heat shock proteins in

17beta-estradiol-treated human MCF-7 breast cancer

cells. Cell Biology International 30 983–991.

Leontiou CA, Gueorguiev M, van der Spuy J, Quinton R,

Lolli F, Hassan S, Chahal HS, Igreja SC, Jordan S, Rowe J

et al. 2008 The role of the aryl hydrocarbon receptor-

interacting protein gene in familial and sporadic pituitary

adenomas. Journal of Clinical Endocrinology and

Metabolism 93 2390–2401.

Lidhar K, Korbonits M, Jordan S, Khalimova Z, Kaltsas G,

Lu X, Clayton RN, Jenkins PJ, Monson JP, Besser GM

et al. 1999 Low expression of the cell cycle inhibitor

p27Kip1 in normal corticotroph cells, corticotroph

tumors, and malignant pituitary tumors. Journal of

Clinical Endocrinology and Metabolism 84 3823–3830.

Lloyd RV, Ruebel KH, Zhang SY & Jin L 2002 Pituitary

hyperplasia in glycoprotein hormone alpha subunit-,

p18(INK4C)-, and p27(kip-1)-null mice – analysis of

proteins influencing p27(kip-1) ubiquitin degradation.

American Journal of Pathology 160 1171–1179.

Ma JX, Finley RL, Waisman DM & Sloane BF 2000 Human

procathepsin B interacts with the annexin II tetramer on

the surface of tumor cells. Journal of Biological

Chemistry 275 12806–12812.

Martin GS 2006 Fly Src: the Yin and Yang of tumor invasion

and tumor suppression. Cancer Cell 9 4–6.

Melle C, Bogumil R, Ernst G, Schimmel B, Bleul A & von

Eggeling F 2006 Detection and identification of heat

shock protein 10 as a biomarker in colorectal cancer by

protein profiling. Proteomics 6 2600–2608.

Moreno CS, Evans CO, Zhan X, Okor M, Desiderio DM &

Oyesiku NM 2005 Novel molecular signaling and

classification of human clinically nonfunctional pituitary

adenomas identified by gene expression profiling and

proteomic analyses. Cancer Research 65 10214–10222.


Morris DG, Musat M, Czirjak S, Hanzely Z, Lillington D,

Korbonits M & Grossman AB 2005 Differential gene

expression in pituitary adenomas by oligonucleotide array

analysis. European Journal of Endocrinology 153 1–10.

Mulla A, Christian HC, Solito E, Mendoza N, Morris JF &

Buckingham JC 2004 Expression, subcellular localization

and phosphorylation status of annexins 1 and 5 in human

pituitary adenomas and a growth hormone-secreting

carcinoma. Clinical Endocrinology 60 107–119.

MusatM,KorbonitsM,KolaB,BorboliN,HansonMR,Nanzer

AM, Grigson J, Jordan S, Morris DG, Gueorguiev M et al.

2005 Enhanced protein kinase B/Akt signalling in pituitary

tumours. Endocrine-Related Cancer 12 423–433.

Okui M, Ito F, Ogita K, Kuramoto N, Kudoh J, Shimizu N &

Ide T 2000 Expression of APG-2 protein, a member of the

heat shock protein 110 family, in developing rat brain.

Neurochemistry International 36 35–43.

Pellegata NS, Quintanilla-Martinez L, Siggelkow H, Samson

E, Bink K, Hofler H, Fend F, Graw J & Atkinson MJ 2006

Germ-line mutations in p27Kip1 cause a multiple

endocrine neoplasia syndrome in rats and humans. PNAS

103 15558–15563.

Phelan ML & Nock S 2003 Generation of bioreagents for

protein chips. Proteomics 3 2123–2134.

Satta MA, Korbonits M, Jacobs RA, Bolden-Dwinfour DA,

Kaltsas GA, Vangeli V, Adams E, Fahlbusch R &

Grossman AB 1999 Expression of menin gene mRNA in

pituitary tumours. European Journal of Endocrinology

140 358–361.

Senda T, Okabe T, Matsuda M & Fujita H 1994 Quick-

freeze, deep-etch visualization of exocytosis in anterior–

pituitary secretory-cells – localization and possible roles

of actin and annexin-Ii. Cell and Tissue Research 277

51–60.

Senzer N, Shen YQ, Hill C & Nemunaitis J 2005

Individualised cancer therapeutics: dream or reality?

Expert Opinion on Therapeutic Targets 9 1189–1201.

Sharif NA, Hunter JC, Hill RG & Hughes J 1988 Bradykinin-

induced accumulation of [3H]inositol-1-phosphate in

human embryonic pituitary tumour cells by activation of a

B2-receptor. Neuroscience Letters 86 279–283.

Stewart JM, Gera L, Chan DC, Whalley ET, Hanson WL &

Zuzack JS 1997 Long-acting, orally-active bradykinin

antagonists for a wide range of applications. Immuno-

pharmacology 36 167–172.

Traverso V, Christian HC, Morris JF & Buckingham JC 1999

Lipocortin 1 (annexin 1): a candidate paracrine agent

localized in pituitary folliculo-stellate cells. Endo-

crinology 140 4311–4319.

Turgeon JL, Cooper RH & Waring DW 1991 Membrane-

specific association of annexin-I and annexin-Ii in

anterior–pituitary-cells. Endocrinology 128 96–102.

Vierimaa O, Georgitsi M, Lehtonen R, Vahteristo P, Kokko

A, Raitila A, Tuppurainen K, Ebeling TM, Salmela PI,

Paschke R et al. 2006 Pituitary adenoma predisposition

caused by germline mutations in the AIP gene. Science

312 1228–1230.

1113



Wang XY, Kazim L, Repasky EA & Subjeck JR 2001

Characterization of heat shock protein 110 and glucose-

regulated protein 170 as cancer vaccines and the effect of

fever-range hyperthermia on vaccine activity. Journal of

Immunology 166 490–497.

Xin W, Rhodes DR, Ingold C, Chinnaiyan AM & Rubin MA

2003 Dysregulation of the annexin family protein family

is associated with prostate cancer progression. American

Journal of Pathology 162 255–261.

Yousef GM & Diamandis EP 2003 An overview of the

kallikrein gene families in humans and other species:

emerging candidate tumour markers. Clinical Biochem-

istry 36 443–452.

Yousef GM, Kishi T & Diamandis EP 2003 Role of kallikrein

enzymes in the central nervous system. Clinica Chimica

Acta 329 1–8.

1114

Zhan X & Desiderio DM 2003 A reference map of a

human pituitary adenoma proteome. Proteomics 3

699–713.

Zhan X, Evans CO, Oyesiku NM & Desiderio DM 2003

Proteomics and transcriptomics analyses of secretagogin

down-regulation in human non-functional pituitary ade-

nomas. Pituitary 6 189–202.

Zhao Y, Giorgianni F, Desiderio DM, Fang B & Beranova-

Giorgianni S 2005a Toward a global analysis of the

human pituitary proteome by multiple gel-based tech-

nology. Analytical Chemistry 77 5324–5331.

Zhao Y, Xue Y, Liu Y, FuW, Jiang N, An P, Wang P, Yang Z

& Wang Y 2005b Study of correlation between

expression of bradykinin B2 receptor and pathological

grade in human gliomas. British Journal of Neurosurgery

19 322–326.



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