Selective Suicide Gene Therapy of Colon Cancer Exploiting the Urokinase Plasminogen Activator...

Selective Suicide Gene Therapy of Colon CancerExploiting the Urokinase Plasminogen ActivatorReceptor PromoterLadan Teimoori-Toolabi,1 Kayhan Azadmanesh,2,3 Amir Amanzadeh4 and Sirous Zeinali1

1 Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

2 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran

3 Virology Department, Pasteur Institute of Iran, Tehran, Iran

4 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

Abstract Background: Colon cancer is the third and fourth most prevalent cancer among Iranian men and women,

respectively. Suicide gene therapy is one of the alternative therapeutic modalities for cancer. The application

of specific promoters for therapeutic genes should decrease the adverse effects of this modality.

Objectives:The combined aims of this studywere to design a specific suicide gene therapy construct for colon

cancer and study its effect in distinct representatives of transformed and nontransformed cells.

Study Design: The KRAS oncogene signaling pathway is one of the most important signaling pathways

activated in colon cancer; therefore, we inserted the urokinase plasminogen activator receptor (uPAR;

PLAUR gene) promoter as one of the upregulated promoters by this pathway upstream of a suicide gene

(thymidine kinase [TK]) and a reporter gene (b-galactosidase, b-gal [LacZ]). This promoter is a natural

combination of different motifs responsive to the RAS signaling pathway, such as the transcription factors

AP1 (FOS/JUN), SP1, SP3, and AP2a, and nuclear factor kappa B (NFkB).Results:The reporter plasmid under the control of the uPAR promoter (PUCUPARLacZ) had the ability to

express b-gal in colon cancer cells (human colon adenocarcinoma [SW480] and human colorectal carcinoma

[HCT116] cell lines), while it could not express b-gal in nontransformed human umbilical vein endothelial

cells (HUVEC) and normal colon cells. After confirming the ability of pUCUPARTK (suicide plasmid) to

express TK in SW480 and HCT116 cells by real-time PCR, cytotoxicity assays showed that pUCUPARTK

decreased the viability of these cells in the presence of ganciclovir 20 and 40 mg/mL (and higher), respectively.

Although M30 CytoDEATH� antibody could not detect a significant rate of apoptosis induced by gan-

ciclovir in pUCUPARTK-transfected HCT116 cells, the percentage of stained cells was marked in com-

parison with untreated cells. While this antibody could detect apoptosis in HCT116 cell line transfected with

positive control plasmid, it could not detect apoptosis in SW480 cells transfected with the same positive con-

trol. This discrepancy could be attributed to the different mechanisms of TK/ganciclovir-induced apoptosis

in tumor protein p53 (TP53)-expressing (HCT116) and -deficient (SW480) cells. Annexin-propidium iodide

staining could detect apoptosis in treated, pUCUPARTK-transfected SW480 and HCT116 cells.

Conclusion:This study showed that the uPARpromoter can be considered as a suitable candidate for specific

suicide gene therapy of colon cancer and probably other cancers in which the RAS signaling pathway is

involved in their carcinogenesis process.

Background

Coloncancer is the thirdand fourthmostprevalent cancer among

Iranianwomen andmen, respectively;[1] whereas, in theUS, it is

rated the thirdmost prevalent cancer for bothmen andwomen.[2]

Since conventional therapies cannot fully eradicate the

cancerous cells, alternative therapies such as gene therapy have

attracted much attention in recent years.[3] Efficacy of chemo-

therapy in full eradication of a few types of cancers, such as

leukemia, has led to the hypothesis that all types of cancer can

ORIGINAL RESEARCH ARTICLEBiodrugs 2010; 24 (2): 131-146

1173-8804/10/0002-0131/$49.95/0

ª 2010 Adis Data Information BV. All rights reserved.

potentially be eradicated by higher doses of chemotherapeutic

agents,[4] although this is problematic because of the adverse

effects associated with high drug dosages. In suicide gene

therapy, a gene construct that encodes an enzyme is introduced

to the target tissues. The enzyme converts a nontoxic prodrug

to a toxic drug. Therefore, suicide gene therapy opens a new

horizon for local administration of these toxic drugs.

Thymidine kinase (TK) is the first and most commonly

studied suicide gene in human clinical trials.[5,6] Its mechanism

of action is based on the fact that in contrast to normal mam-

malian TK, herpes simplex virus TK (HSV TK) preferentially

monophosphorylates ganciclovir, making it toxic to normal

mammalian cells. Further phosphorylation of ganciclovir-

monophosphate by cellular kinases produces a metabolite,

which, after integration into DNA, terminates DNA-strand

elongation.[7] In addition to the direct killing effect induced by

this suicide gene, bystander effect also plays an important role

in enhancing its efficacy.[8] Clinical trials using this system have

shown controversial results. Although suicide gene therapy of

malignant mesothelioma or localized prostate carcinoma in-

creased the median survival of patients[6] or the doubling time

of prostate-specific antigen (PSA),[9] respectively, experiments

on brain tumors were unsatisfactory.[10]

Several strategies have been designed to overcome the toxic

effects associated with systemic administration of vectors con-

taining suicide genes.[11] These include targeting the construct to

the desired cells by inserting monoclonal antibodies specific for

tumor cells on the surface of the viral vectors,[12] or restricting the

expression of suicide gene by cancer-specific promoters.[13] Dif-

ferent cancer-specific promoters have been used thus far, such as

PSA promoter for prostate adenocarcinoma[14] or carcinoem-

bryogenic antigen (CEA) promoter for colon carcinoma.[15]

Selecting the best cancer-specific promoters for suicide gene

therapy can be accomplished by investigating deregulated signal-

ing pathways in cancerous cells. Investigating these signaling

pathways would lead to identifying abnormally upregulated

promoters downstream of these pathways. RAS signaling is

the most commonly activated signaling pathway in cancers.

Members of the RAS family (Kirsten RAS [KRAS], neuro-

blastoma RAS [NRAS], and Harvey RAS [HRAS]) have the

role of connecting the growth receptors to the intracellular

tyrosine kinases.[16] They have two main states: the activated

form, which is bound to guanine triphosphate (GTP), and the

inactivated form attached to guanine diphosphate. Mutant

KRAS is insensitive to GTPase activating proteins in the cy-

toplasm; therefore, it remains constitutively active.[17]

In 30% of all types of cancers, mutations in one member of

the RAS family occur.[18] Ninety-five percent of pancreatic,[19]

50% of small[20] and large bowel carcinoma,[21] and about

30–50% of lung cancers[22] have mutations in the KRAS gene.

Effectors of RAS signaling (BRAF, mitogen-activated

protein kinase kinase [MAPKK], mitogen-activated protein

extracellular kinase/extracellular signal-regulated protein

kinase [MEK/ERK], and phosphatidylinositol 3 kinase[23]) all

play roles in RAS-induced transformation of cells.[16] In a

survey among genes being upregulated and downregulated

downstream of HRAS, 61 were BRAF/MAPKK dependent

and 116 were BRAF/MAPKK independent.[24] Some examples

of these upregulated genes were FOS-related antigen

1 (FRA1),[25] cyclin D1 (CCND1),[26] matrix metalloproteinase

9 (MMP9),[27] cyclo-oxygenase 2 (COX2; gene name:

PTGS2),[28] and urokinase plasminogen activator receptor

(uPAR; gene name: PLAUR),[29] which are mostly responsible

for invasion, metastasis, and epithelial-mesenchymal transfor-

mation of cells.[24]

Transcription of the above-mentioned genes is induced by a

range of transcription factors such as activator protein 1 (AP1)

complex (FOS/JUN), SP1, nuclear factor kappa B (NF-kB),ETS domain transcription factor (ELK1), serum-responsive

factor (SRF), and activating transcription factor 2 (ATF2).

Several of these gene regulators, including ELK1, SRF, the

leucine zipper JUNprotein, ATF2, andNFkB, are also inducedby the activated RAS signaling pathway.[30]

Considering the importance of the RAS signaling pathway

in the transformation of colorectal cells,[31,32] there have been

some efforts in exploiting this pathway for colon cancer gene

therapy.[16,33,34] The first promoter element recognized to be

responsive to RAS oncogenes was the polyomavirus (Py)

enhancer.[35] Therefore, in an effort for colon cancer-specific

gene therapy, the Py element containing ETS and AP1 binding

motifs was cloned upstream of the pro-apoptotic genes BAX,

caspase 8 (CASP8), and protein kinase G (PKG; PRKG1) as

suicide genes. These neighboring and overlapping binding sites

were more effective than either of them alone.[34] This was the

only study exploiting RAS-responsive elements for selective

killing of colon cancer cells, although this promoter did not

contain other binding regions responsive to activated RAS,

such as NFkB or SP1.

We hypothesized that an unaltered promoter of a gene up-

regulated by the activated RAS signaling pathway could con-

tain most of the important responsive elements to this pathway.

Therefore, inserting a natural promoter upstream of a ther-

apeutic gene may result in a more effective and controlled

expression in transformed and normal cells, respectively.

uPAR (PLAUR) is one of the genes downstream of the

activated RAS signaling pathway. uPARpromotes the hydrolysis

132 Teimoori-Toolabi et al.

ª 2010 Adis Data Information BV. All rights reserved. Biodrugs 2010; 24 (2)

of urokinase plasminogen activator (uPA) on the surface of

cells. Therefore, it has a role in the degradation and regenera-

tion of the basementmembrane andmigration of the cells.[36] In

addition, after the attachment of uPA to uPAR, a signaling

pathway is triggered in the cells, leading to wound healing,

inflammation, vascularization,[37] and metastasis of cancerous

cells.[38] PLAUR/uPAR is abnormally overexpressed in dif-

ferent tumors such as melanoma,[39] glioblastoma, ovarian

cancer,[40] breast cancer,[41] hepatocellular carcinoma,[42] pan-

creas,[43] head and neck cancers,[44] and gastric and colon

carcinoma.[45] Its expression is correlated directly with the in-

vasiveness and metastasis potential of these types of cancers. In

colorectal tumors that have a larger volume[46] and higher duke

stage,[47] uPAR expression is higher.

Different studies have verified the influence of the RAS

signaling pathway on uPAR expression.[29] The effect ofKRAS

mutation on uPAR expression is so profound that targeted

disruption ofKRAS in the human colorectal carcinoma cell line

HCT116 leads to a 50–85% decrease in uPAR expression.[48] In

addition, transfection of the human ovarian carcinoma cell line

OVCAR-3 with activated RAS increased the uPAR promoter

activity over 20-fold.[29] RAS effectors such asMAPKK[49] and

Jun N terminal kinase-1 (JNK1)[29] have been shown to be

necessary for uPAR expression.[50] Upregulation of the

PLAUR gene is mainly dependent on transcriptional induction,

although other factors such as mRNA stabilization and post-

translational modifications cannot be ignored.[51]

In this study, we placed the unaltered PLAUR promoter

upstream of bacterial b-galactosidase (LacZ) as a reporter andTK as a suicide gene. The effects of these constructs were

studied in two colon cancer cell lines (SW480 and HCT116),

normal human umbilical vein endothelial cell (HUVEC), and a

mix of cells isolated from normal colon tissue.

The application of natural promoters might have the ad-

vantage of mimicking the natural state of the promoters in the

cells by simultaneously utilizing different binding motifs and

the repressor elements. The results obtained after using this

promoter could also be employed to compare the performance

of a natural promoter with artificial promoters so far studied.

Materials and Methods

DNA Extraction

Genomic DNA was extracted from white blood cells in ve-

nous blood of a healthy person by the proteinase K method.[52]

The person signed a consent form before donating her blood.

No history of familial cancers or cancer onset in relatives before

the age of 60 years was reported. Plasmid DNA and viral DNA

from HSV were extracted using Mini Prep Plasmid Extraction

Kits and a QiaAmp Viral DNA Extraction Kits, respectively

(Qiagen GmbH, Hilden, Germany).

Fragment Amplifications

Cytomegalovirus (CMV) and PLAUR promoters were am-

plified from pcDNA3.1+ (Invitrogen Corporation, Carlsbad,

CA, USA) and human genomic DNA, respectively. The TK

gene was amplified from the HSV-1 genome. Primer sequences

are given in table I. All amplified fragments were studied by

bidirectional sequencing after cloning into pTZ57r/T (Fer-

mentas, Vilnius, Lithuania).

Plasmid Design and Construction

In this study, six vectors were constructed by using

pUCLTRLacZ, which encodes a complete bacterial LacZ gene

between the human T lymphotropic virus type 1 (HTLV-1)

promoter long terminal repeat (LTR) and the bovine growth

hormone polyadenylation signal (BGH-PolyA).[53] The LTR

was digested out and replacedwith CMVorPLAUR promoters

Table I. Primers for real-time PCR/cloning and their sequences

Primers Sequences of primers

b-Actin forward primer

for real-time PCR

50-CCAAGGCCAACCGCGAGAAG-30

b-Actin reverse primer

for real-time PCR

50-CACCGGAGTCCATCACGATGC-30

TK forward primer

for real-time PCR

50-AAACGCCTCCGTCCCATG-30

TK reverse primer

for real-time PCR

50-GGTCGCAGATCGTCGGTATG-30

TK forward primer

for cloning

50-ATCCATGGCTTCGATCCCCTGCCA-30

TK reverse primer

for cloning

50-TCCTCGAGTCATAGCGCGGGTTCCTTC-30

CMV promoter

forward primer

50-AATAAGCTTCGATGTACGGGCCAGA-30

CMV promoter

reverse primer

50-GGTAAGCTTAAGTTTAAACGCTAG-30

uPAR promoter

forward primer

50-AAGCTTTGCGAAAGAGCGAGTCAGCC-30

uPAR promoter

reverse primer

50-AAGCTTGCATGAGCCACCTCATCTGACC-30

CMV = cytomegalovirus; TK= thymidine kinase; uPAR= urokinase plasmino-

gen activator receptor (PLAUR ).

The uPAR (PLAUR) Promoter in Specific Colon Cancer Gene Therapy 133


to obtain pUCCMVLacZ and pUCUPARLacZ constructs,

respectively. The HindIII digested plasmid was also re-ligated

to construct the pUCLacZ plasmid. In addition, LacZ was

digested out frompUClacZ byXhoI andNcoI without breaking

the PolyA tail, and the TK gene was inserted in its place to con-

struct the pUCTK plasmid. CMV or PLAUR promoters were

also placed upstream of the TK gene to construct pUCCMVTK

or pUCUPARTK. Schematic figures of constructed plasmids are

given in figure 1. The promoterless plasmids, pUCLacZ or

pUCTK, were used as negative controls (mock plasmid).

Hind III (402)

Hind III (402)

Hind III (402)Hind III (402)

Hind III (402)

Hind III (402)

Hind III (1413)

Hind III (1110)

Hind III (1110)

Hind III (1415)

TK

TK

ClaI (2365)

ClaI (2062)

ClaI (1345)

LacZ

LacZ

LacZ

TK

EcoRI (4898)

EcoRI (4595)

EcoRI (3878)

EcoRI (2858)

pUCUPARLacZ7131 bp

pUCCMVLacZ6828 bp

pUCCMVTK4786 bp

pUCLacZ6111 bp

pUCTK4078 bp

pUCUPARTK5091 bp

BGH PolyA

BGH PolyA

BGH PolyA

BGH PolyA

BGH PolyA

BGH PolyA

XhoI (4642)

XhoI (4339)

XhoI (3622)XhoI (1587)

XhoI (2600)

XhoI (2295)

EcoRI (4609)

EcoRI (4306)

EcoRI (3589)EcoRI (1845)

EcoRI (2553)

uPAR promoter

CMV promoterCMV promoter

Afl II (1107)

Afl II (1107)

uPAR promoter

Fig. 1. Map of plasmids, indicating the names of each plasmid and some of their restriction sites. The right plasmids are thymidine kinase (TK)-expressing plasmids

(with pUCTK as the backbone); the left plasmids are b-galactosidase (b-gal)-expressing plasmids (with pUCLacZ as the backbone). BGH PolyA=bovine growth

hormone polyadenylation site; CMV= cytomegalovirus promoter; LacZ= bacterial b-gal gene; uPAR= urokinase plasminogen activator receptor (PLAUR).



Cell Lines and Culturing

HUVEC and SW480 cell lines were obtained from theNational

Cell Bank of Iran (NCBI, Pasteur Institute of Iran, Tehran, Iran).

The HCT116 cell line was obtained from American Type Culture

Collection (ATCC, Manassas, VA, USA). SW480 and HCT116

cell lines were cultured in high glucose Dulbecco’s modified Eagle’s

medium (DMEM) with 10% fetal bovine serum (FBS) plus peni-

cillin 100U/mL, streptomycin 100mg/mL, and L-glutamine

2mmol/L. The HUVEC line was cultured in a medium containing

Ham’s F12 : high glucose DMEM (1 :1) with the same supple-

ments as the SW480 andHCT116 cell lines. Themedia ofHCT116,

SW480, and HUVEC lines were changed every 3, 5, and 6 days,

respectively. All reagents were GIBCO� products purchased from

Invitrogen (Life Technologies Corporation, Carlasbad, CA,USA).

Isolating Primary Cells from Normal Colon Tissue

The normal colon tissue specimen was freshly obtained from

a 27-year-old man undergoing surgery at Imam Khomeini

Hospital (Tehran University of Medical Sciences, Tehran,

Iran). He had undergone surgery because of voluvulus in his

colon. He had no history of familial cancer and none of his first-

degree relatives had died from colorectal carcinoma. Before

donating his tissue, he had signed a consent form. The patho-

logic specimen taken from his colon tissue was not indicative of

any malignancy, and the colon cancer biomarker CEA was not

positive in this patient. This procedure was approved by the

ethical committee of Pasteur Institute of Iran.

The obtained tissue was immediately transferred to the la-

boratory and washed with phosphate buffer saline (PBS). After

washing, it was chopped into 1mm pieces. The chopped pieces

were washed with PBS and treated with collagenase 200U/mL

(Life Technologies Corporation) in a CO2 incubator (New

Brunswick Scientific, Edison, NJ, USA) for about 2 hours.

The mixture of tissue and collagenase was then centrifuged

and seeded into 2–4 wells of a 12-well plate (Nunc, Roskilde,

Denmark) and cultured in a mixture of DMEM :Ham’s F12

(1 : 1) media (Life Technologies Corporation) supplemented

with L-glutamine 4mmol/L, penicillin 200U/mL, streptomycin

200 mg/mL, fungizone 2.5 mG/mL (Life Technologies Corpora-

tion), and gentamycin 40 mg/mL for about 5 days. In the

next four passages, fungizone and gentamycin were elimi-

nated from the supplement and the concentration of penicillin

and streptomycin were decreased to 100U/mL and 100 mg/mL,

respectively.

The cells that were proven to be free from any bacterial or

fungal contamination (data not shown) were seeded in a treated

plate with collagen from rat tail (Roche Applied Science,

Mannheim, Germany). After two passages, they were trypsi-

nized from the plate and seeded in non-collagenated plates or

flasks for further experiments. The mixture of these adherent

cells obtained from normal colon tissue will be henceforth re-

ferred to as normal colon cells (NCCs).

Delivery of DNA to the Cells

Delivery of DNA to cells was optimized (unpublished data)

by using different lipid-based transfection methods, namely

Lipo-fectamine� 2000 (Life Technologies Corporation), Effec-

tene, Polyfect and Superfect (Qiagen, Hilden, Germany). Opti-

mized transfection in SW480 cells was achieved using Effectene

and a DNA/reagent ratio of 400ng/4mL, and in HCT116,

HUVECs, and NCCs using Polyfect with the DNA/reagentratios of 1600ng/8mL, 800ng/12mL, and 1600ng/8mL, respec-tively. These ratios are optimized for each well of a 24-well plate.

One day before transfection, 2 · 105 of SW480, HCT116,

and HUVEC lines, and 1.5 · 105 of NCCs were seeded in each

well of a 24-well plate. When the confluency reached 70–80%,

they were transfected using the relevant transfection methods.

b-Galactosidase (b-gal) Staining

Forty-eight hours after transfection, the cells were fixed with

glutaraldhyde 0.5% (Sigma, Ronkonkoma, NY, USA). After

washing with PBS, the cells in a 24-well plate were stained with

3 mL of ferrocyanide potassium 400mmol/L (Fluka, Buchs,

Switzerland), 3 mL of MgCl2 200mmol/L (Merck KGaA,

Darmstadt, Germany), 15 mL of Xgal 20mg/mL (Fermentas),

and 276 mL of PBS. The percentage of positively stained cells in

each well was estimated by counting the blue cells among the

total number of cells in at least five different fields under an

inverted microscope (400· zoom).

b-gal ELISA

The day before transfection, 4 · 105 SW480, HCT116, and

HUVEC cells, and 3 · 105 NCCs were seeded in each well of a

12-well plate. When the confluency of each well reached

70–80%, the cells were transfected with the respective optimum

method of transfection. For normalization of transfection in

the b-gal ELISA, cells were transfected with a mixture of the

main reporter (pUCMVLacZ, pUCUPARLacZ, or mock

plasmid) and a chloramphenicol acetyltransferase (CAT)-

expressing construct (pRc/CMV2CAT [Invitrogen]) with the

ratio of 9 : 1. Forty-eight hours after transfection, cells were



lysed with 250 mL lysis buffer. Bacterial b-gal expression was

studiedwith the b-gal ELISA kit (RocheApplied Science) using

200 mL of cell extracts, measuring the optical density (OD) at

405 nm (490 nm as background) every 2 minutes with a micro-

plate reader (BioTek, Winooski, VT, USA) and calculating the

maximum slopes of OD change. The raw levels of b-gal ex-pression were calculated according to the standard curves.

These curves were drawn by plotting the maximum slopes of

OD change in different serial dilutions of standard b-galenzyme (provided by the manufacturer).

Chloramphenicol Acetyltransferase ELISA

A 50 mL aliquot of the cell extract prepared for b-gal ELISAwas mixed with 150 mL of sample buffer and then added to each

well of CAT ELISA plate (Roche Applied Science). After car-

rying out the ELISA according to the manufacturer’s instruc-

tion, the OD was measured at 405 nm (490 nm as background)

every 2 minutes, and the maximum slopes of OD change were

calculated. The raw levels of CAT expression were calculated

according to the standard curves. These curves were drawn

by plotting the maximum slopes of OD change in different

serial dilutions of standard CAT enzyme (provided by the

manufacturer).

RNA Extraction

Twenty-four hours after transfecting the cells with suicide

plasmids, cells in each well of the 12-well plate were washed

with PBS and then RNA was extracted with 1mL of TriPure

(Roche Applied Science).[52] The optical density ratio of ex-

tracted RNA was measured at 260 and 280 nm and the quality

of RNAwas considered suitable when the 260 : 280 ratio ranged

between 1.5 and 1.8.

cDNA Synthesis

The extracted RNA was treated with DNAase to remove the

contaminating plasmid DNA and converted to cDNA using

M-MuLV as reverse transcriptase enzyme and oligo dT as the

primer.[52]All reagentswereobtained fromRocheAppliedScience.

Real-Time PCR

The expression of TK in transfected cells with TK-expres-

sing plasmids was assessed at cDNA level, using SYBR�Green

PCRMasterMix in anABI 7300machine (Applied Biosystems/Life Technologies, Carlsbad, CA, USA). This PCR amplified a

141 bp fragment from cDNA of the TK gene. b-Actin expres-

sion as a reference gene was also evaluated at cDNA level

by amplifying 134 bp fragment from its cDNA. In order to

detect any nonspecific amplification such as primer dimers,

which creates additional separate peaks from the desired am-

plicon, dissociation curve analysis was performed after each

amplification process. Cycle threshold (CT) values of TK

and b-actin were calculated by the software automatically.

The difference between these CT values was calculated

(DCT). Thereafter, according to the previously published mate-

rials,[54] this DCT was compared with DCT of pUCCMVTK-

transfected cells as the calibrator, and the amount of target gene

expression in pUCUPARTK-transfected cells was calculated

based on the 2-DDCT formula. The sequences of primers are

provided in table I.

The efficiency of amplifications was evaluated by testing

serial dilutions of cDNA. Their efficiencies were the same at the

same concentration of cDNA on which the real-time PCR ex-

periments were performed.

Cytotoxicity Assay

Four and eight hours after transfection with Polyfect and

Effectene reagents, respectively, cells were trypsinized and 104

cells were plated into each well of a flat-bottomed, 96-well

cell culture plate. The next day, ganciclovir (Roche, Basel,

Switzerland) in different concentrations (0, 20, 40, 60, 80, and

100 mg/mL) was added to cell medium. Four days after trans-

fection, the media were replaced with 100 mL of fresh DMEM

supplemented with 10% FBS plus 50 mL of the Cell Prolifera-

tion Kit II (XTT) mixture (Roche Applied Science), following

the manufacturer’s instructions. Four hours after incubating

the plate in CO2 incubator, the OD of cell medium was mea-

sured at 490 nm (690 nm as background) using a microplate

reader (BioTek).

Analyzing the Dead Cells by Staining the Cells with M30

CytoDEATH� Antibody

Twenty-four hours after transfecting HCT116 and SW480

cell lines with suicide and mock plasmids, ganciclovir 40 mg/mL

was added to the cell medium. Twenty-four hours later, cells

were trypsinized, washed with PBS and stained with M30

CytoDEATH� antibody (Roche Applied Science) according

to the manufacturer’s instructions. Thereafter, cells were ana-

lyzed by flow cytometer with at least 10 000 events per reading

in a PAS machine (Partec GmbH, Munster, Germany), using

FloMax� software (Partec GmbH).



The ganciclovir concentrations and cell-harvesting time for

flow cytometry experiments were optimized by collecting the cells

at different time intervalswith different ganciclovir concentrations.

Analyzing the Dead Cells by Staining the Cells

with Annexin-Propidium Iodide (PI)

Twenty-four hours after transfecting SW480 and HCT116

with suicidal plasmids (pUCCMVTK, pUCUPARTK, and

mock plasmid), they were treated with ganciclovir 40 mg/mL.

24 hours later they were stained with the Annexin-Propidium

iodide (PI) staining kit (Roche Applied Science) according to

the manufacturer’s protocol. In order to reduce the negative

effect of trypsinization on staining the cells with Annexin-PI,

the cells were washed with PBS after trypsinization. Thereafter

they were analyzed with at least 10 000 events per reading in a

PAS machine, using FloMax� software (Partec GmbH).

Statistical Methods

All experiments were repeated at least three times. The results

were analyzedwith the Student’s t-test and Spearman correlation

coefficient using SPSS� 12.0 (SPSS Inc., Chicago, IL, USA).

Results

uPAR (PLAUR) Promoter is Active in SW480 and HCT116 Cells

Semi-quantitative b-gal staining was used preliminarily to

assess the transfection rate in different cell lines. This staining

method also gave an estimate of uPAR (PLAUR) promoter

activity in different cells.

The maximum transfection rates for HCT116, SW480,

HUVECs, and NCCs were 50%, 35%, 10%, and <10%, re-

spectively. This judgment was based on the percentage of

stained cells among the total number of cells after transfection

with positive control plasmid (pUCCMVLacZ).

Semi-quantitative analysis showed that the uPAR promoter

was active in both colon cancer cell lines (HCT116 and SW480)

but not in theHUVEC line andNCCs. Figures 2 and 3 show the

staining results of transfected cell lines andNCCs with different

plasmids.

After transfectionwith pUCUPARLacZ, 0.5%, 5%, 0%, and

0% of SW480, HCT116, HUVECs, and NCCs were positively

stained, respectively. With HUVECs and NCCs as the rep-

resentatives of nontransformed cells, the uPAR promoter did

not induce b-gal expression. Measuring b-gal expression with a

quantitative method such as ELISA was needed to prove that

pUCCMVLacZ transfected pUCUPARLacZ transfected Mock plasmid transfected

HU

VE

CS

W48

0H

CT

116

Fig. 2. b-Galactosidase (b-gal) staining in the human colorectal carcinoma (HCT116), human colon adenocarcinoma (SW480), and human umbilical vein

endothelial cell (HUVEC) lines transfected with pUCCMVLacZ (positive control), pUCUPARLacZ, and mock plasmids; the blue cells are the cells that have

expressed b-gal. All photographs were taken under an inverted microscope (400· zoom) with a motic image processor.



this observation is not related to the low transfection rate of

these cells.

uPAR Promoter Drives Expression of Reporter Gene

Specifically in Colon Cancer Cell Lines

b-gal ELISA as a quantitative method was used to detect the

activity of the uPAR promoter in comparison to CMV

promoter in different cells. The transfection rate of all cells

was normalized by cotransfecting pRc/CMV2CAT (CAT-

expressing plasmid) along with the main reporter plasmids

(pUCCMVLacZ, pUCUPARLacZ or negative control).

For this purpose, the b-gal maximum slope was divided by

the CAT maximum slope of the same cell lysate. The results of

the ELISA can be seen in figure 4. The estimates of raw

b-gal and CAT expression level are also provided in this

figure.

HCT116 and SW480 cell lines transfected with pUCCMV-

LacZ expressed b-gal at significantly higher levels than

mock-transfected cells (p = 0.003 and 0.007, respectively).

In both cell lines, the uPAR promoter induced significantly

higher b-gal expression than the negative control plasmid. The

ratio of activity of the uPAR promoter in comparison with that

of the CMV promoter in HCT116 and SW480 was 0.178 and

0.02, respectively. This ratio is lower in SW480 and this is

because of the lower activity level of KRAS signaling pathway

in this cell line.

Compensating for the lower transfection rate by normal-

ization with CAT expression, the obtained results in HUVEC

showed that pUCUPARLacZwas similar tomock plasmid and

did not promote expression of b-gal, whereas pUCCMVLacZ

expressed b-gal at a significantly higher level than pUCU-

PARLacZ and mock plasmid.

In NCCs, which were a mixture of different cells isolated

from normal colon tissue, b-gal expression under the control of

the CMV promoter was significantly higher than that driven by

themock plasmid and pUCUPARLacZ. In contrast, the uPAR

promoter did not induce a significant level of b-gal expressioncompared with mock plasmid.

Therefore, it can be concluded that the uPAR promoter was

not active in normal cells (HUVECs andNCCs), whereas CMV

promoter activity was significantly measurable in both normal

cell types.

uPAR Promoter Induces Expression of Thymidine Kinase

in Colon Cancer Cell Lines

The expression of TK in SW480 and HCT116 cell lines was

assessed at the RNA level by real-time PCR. Considering that

the efficiency of real-time PCR for TK and b-actin genes was

similar, the mean – standard deviation (SD) ratio of TK ex-

pression by pUCUPARTK in comparison to pUCCMVTK

was 0.016219 – 0.004081 in SW480 and 0.022664 – 0.016727 in

HCT116. This test proved that in both colon cancer cell lines,

TK can be expressed under the control of the uPAR promoter.

pUCUPARTK Construct Induces Cell Death in Colon

Cancer Cell Lines

Cytotoxicity assays were carried out to measure the cyto-

toxic effects of pUCCMVTK and pUCUPARTK in different

concentrations of ganciclovir. Viability of pUCCMVTK-

transfected SW480 cells in the presence of ganciclovir 20 mg/mL

or higher was significantly lower than the untreated cells

(p = 0.036 at 20 mg/mL). This decline in viability in pUCU-

PARTK-transfected SW480 cells was significant in the

presence of ganciclovir 40 mg/mL or higher (p = 0.025 at

40 mg/mL). Treating mock-transfected SW480 cells with

100 mg/mL (or lower concentrations) of ganciclovir did not

significantly reduce their viability (p = 0.255198 at 100 mg/mL).

As can be seen in figure 5, treating mock-transfected SW480

cells with different concentrations of ganciclovir not only did

pUCCMVLacZ pUCUPARLacZ Mock plasmid

Fig. 3. b-Galactosidase (b-gal) staining in normal colon cells transfected with pUCCMVLacZ (positive control), pUCUPARLacZ, and mock plasmids; the blue

cells are the cells that have expressed b-gal. Photographs were taken with a fixed Sony camera (400· zoom).



not reduce their viability, but in fact increased it in some con-

centrations. Thismay be related to the growth-promoting effect

of nonphosphorylated ganciclovir on this cell line. The viability

of SW480 cells after transfection with pUCCMVTK and

pUCUPARTK decreased to <70% and about 70% of negative

control, respectively.

Viability of pUCCMVTK- and pUCUPARTK-transfected

HCT116 cells in the presence of ganciclovir 20 mg/mL and

higher was significantly lower than untreated cells (p = 0.009and p = 0.027, respectively, in 20 mg/mL of ganciclovir). Com-

pared with untreated cells, a significant decline in the viability

of mock-transfected HCT116 cells was observed when they

were treated with 80 mg/mL of ganciclovir or higher (p = 0.021

at 80 mg/mL). At lower concentrations of ganciclovir, no

significant reduction in cell viability was seen in the mock-

transfected HCT116 line. The lowest cell viability of

pUCCMVTK- and pUCUPARTK-transfected HCT116 cells

was about 70%, while the viability ofmock-transfected cells was

diminished to 80% of untreated cells.

The cytotoxic effect of suicide constructs seemed more im-

pressive in SW480 in comparison with HCT116. This may be

related to the toxic effect of nonphosphorylated ganciclovir on

HCT116 andgrowth-promoting effect of this prodrug for SW480.

Spearman correlation analysis also showed that in

pUCCMVTK- and pUCUPARTK-transfected SW480 cells,

increasing the ganciclovir concentration decreased the cell

3.5

2.35

0.9

1.2

4.64.8

0.001

0.003

0.0004

pUCLacZpUCUPARLacZpUCCMVLacZ

Max

imum

slo

pe o

f OD

cha

nge/

norm

aliz

ed e

xpre

ssio

n of

β-g

al

HCT116

3.0

2.5

2.0

1.5

1.0

0.5

0

12

2.6

0.22 0.25

2.8 2.9

0.009

0.007

0.008


Max

imum

slo

pe o

f OD

cha

nge/

norm

aliz

ed e

xpre

ssio

n of

β-g

al

SW480

10

8

6

4

2

0

4.5

<0.1 <0.05

0.52

0.1 <0.1 <0.1<0.05 <0.1

0.1

0.1

0.01178

0.01248

0.28


Max

imum

slo

pe o

f OD

cha

nge/

norm

aliz

ed e

xpre

ssio

n of

β-g

al

HUVEC

4.0

3.0

2.0

1.5

1.0

0.5

0

2.5

3.5

2.5

0.017157

0.01227

0.2295


Max

imum

slo

pe o

f OD

cha

nge/

norm

aliz

ed e

xpre

ssio

n of

β-g

al

NCC

2.0

1.5

1.0

0.5

0

Fig. 4. Results of b-galactosidase (b-gal) ELISA. The first bar in each group is indicative of raw b-gal expression, the second bar shows raw chloramphenicol

acetyltransferase (CAT) expression, and the third bar is indicative of normalized b-gal expression; theY axis demonstratesmeasured optical density (OD) of raw

b-gal and CAT expression and the ratio of b-gal :CAT expression of normalized b-gal expression. The figures over the bars of b-gal and CAT maximum slopes

are estimates of b-gal and CAT raw-expression levels (ng/mL), calculated according to the standard curve (curves drawn by plotting the maximum slopes of

b-gal or CAT serial dilutions). The numbers over the linking lines between the different bars are the p-values, calculated by one-tailed Student’s t-tests. The line

within each bar represents the standard error. HCT116= human colorectal carcinoma cell line; HUVEC=human umbilical vein endothelial cell; NCC= normal

colon cells; SW480= human colon adenocarcinoma cell line.



viability significantly (p = 0.001 and 0.005, respectively);

whereas, in mock-transfected SW480 cells, this correlation was

not significant (p = 0.46). The correlation coefficients between

viability and ganciclovir concentration were -0.629 and

-0.679 for pUCCMVTK- and pUCUPARTK-transfected

cells, respectively.

This analysis also showed significant reverse correlation

between viability and ganciclovir concentration in

pUCCMVTK- and pUCUPARTK-transfected HCT116 cell

line. The correlation coefficients were -0.424 and -0.363 for

pUCCMVTK- and pUCUPARTK-transfected cells, respec-

tively (p = 0.005 and 0.018, respectively). This correlation was

not significant in mock-transfected cells (p = 0.075). The via-

bility graphs are shown in figure 5.

The required concentration of ganciclovir for decreasing cell

viability was equivalent in pUCUPARTK- and pUCCMVTK-

transfected HCT116 cells. In SW480 cells, a higher concentra-

tion of ganciclovir was required to reduce viability when the

cells were transfected with pUCUPARTK compared with

those tranfected with pUCCMVTK.

M30CytoDEATH�AntibodyDetectsApoptosis inpUCCMVTK-

Transfected HCT116 Cells but Not in SW480 Cells

The M30 CytoDEATH� antibody was used to detect the

apoptosis after administering a definite amount of ganciclovir

in pUCCMVTK-, pUCUPARTK-, and mock-transfected

cells. InHCT116, a one-tailed Student’s t-test showed that there

was a significant difference in the apoptosis rate (reflected in

stained cells with M30 CytoDEATH� antibody) between

treated (drug added) and untreated pUCCMVTK-transfected

cells (figure 6). Although the difference in percentage of

apoptotic cells between treated and untreated pUCUPARTK-

transfected cells was marked (4.52%– 0.016 [mean –SD]

in treated vs 2.67%– 0.003 in untreated cells), this difference

was not statistically significant. In mock-transfected HCT116,

the apoptosis rates between ganciclovir-treated and -untreated

cells were not significantly different or marked.

The mean (–SD) rates of apoptosis in ganciclovir-treated

pUCCMVTK- and pUCUPARTK-transfected SW480 cell

line were 18.6225%– 3.750586 and 10.0125%– 5.453356, re-spectively; whereas in untreated cells transfected with the

same plasmid, these rates were 16.04%– 4.059187 and

7.755%– 2.544871, respectively. Differences between the

apoptosis rate in treated and untreated cells were marked,

although not significantly different. Therefore, the M30

CytoDEATH� antibody may detect apoptosis in HCT116

cells expressing a considerable level of TK (pUCCMVTK in

this study), although it was not able to detect apoptosis in

SW480 transfected with the same plasmid.

Annexin-PI Staining Detects Apoptosis in SW480 and

HCT116 Cell Lines Transfected with Suicide Plasmid

Since theM30 CytoDEATH� antibody could only evaluate

one aspect of apoptosis (because it did not seem to be sufficiently

activated in SW480), Annexin-PI staining was considered

1.4

1.2

1.0

0.8

Per

cent

age

of v

iabi

lity

0.6

0 20 40GCV concentration

60 80 100

1.2

b

a

1.0

0.8

Per

cent

age

of v

iabi

lity

0.6

0.4

0 20 40GCV concentration

60 80 100

pUCCMVTKMock plasmidpUCUPARTK

HCT116 transfected with

pUCCMVTKMock plasmidpUCUPARTK

SW480 transfected with

Fig. 5. Viability graphs of (a) human colon adenocarcinoma (SW480) and

(b) human colorectal carcinoma (HCT116) cell lines transfected with

pUCUPARTK, pUCCMVTK (positive control), and mock plasmids after

treatment with different concentrations of ganciclovir (GCV).



for detecting apoptosis. The SW480 and HCT116 cell lines

transfected with suicide plasmids were stained with

Annexin-PI. Unexpectedly, the apoptosis rate was not sig-

nificantly different in treated and untreated SW480 cells trans-

fected with pUCCMVTK. However, in pUCUPARTK-

transfected cells, the apoptosis rate was significantly different

between treated and untreated cells. As expected, these ratios in

mock-transfected cells were not significantly different. In

pUCCMVTK- and pUCUPARTK-transfected HCT116, the

percentage of apoptotic cells between treated and untreated cells

was significantly different; whereas, in mock-transfected cells,

these rates were not significantly different. The graphs depicting

the results of staining these cells with Annexin-PI are shown in

figure 7. These results were repeated in two different sets of

experiments (data from one experiment is shown).

Discussion

A major problem with systemic administration of suicide-

gene constructs for cancer therapy is the cytotoxic effect of

the suicide gene/prodrug on normal cells, which can be as

debilitating as inducing hepatitis.[55] Studying the expression

a

b

300.033397

0.092066

0.180446

Mock plasmidpUCUPARTKpUCCMVTK

HCT116 cell line transfected with different plasmids

25

20

15

10

5

0

−5

−10

Per

cent

ages

of s

tain

ed c

ells

GCV-treatedUntreated

250.1931

0.24620.3478

pUCTKpUCUPARTKpUCCMVTK

Per

cent

ages

of s

tain

ed c

ells

SW480 cell line transfected with different plasmids

20

15

10

5

0

Fig. 6. Percentages of (a) human colon adenocarcinoma (SW480) and

(b) human colorectal carcinoma (HCT116) cells stained with the M30

CytoDEATH� antibody, measured by flowcytometry. Ganciclovir (GCV)

40mg/mL was added to the ‘GCV-treated’ cells, whereas the ‘untreated’ cells

were only transfected with the respective plasmids and was not treated with

GCV. The numbers over the linking lines between the two different bars

are the p-values of one-tailed Student’s t-tests. The line within each bar

represents the standard error.

35a

b

0.323477

0.003119

0.327883

pUCTKpUCUPARTKpUCCMVTK

Per

cent

ages

of a

popt

otic

cel

ls in

Ann

exin

-Pl s

tain

ing

SW480 cell line transfected with different plasmids

30

25

20

15

10

5

0

−5

16 0.008

0.022

0.2

Mock plasmidpUCUPARTKpUCCMVTK

Per

cent

ages

of s

tain

ed c

ells

HCT116 cell line transfected with different plasmids

14

10

12

8

6

4

2

0

−2

GCV-treatedUntreated

Fig. 7. Percentages of (a) apoptotic humancolon adenocarcinoma (SW480)

and (b) human colorectal carcinoma (HCT116) cells, as assessed by Annexin-

propidium iodide (PI) staining. Ganciclovir (GCV) 40mg/mL was added to the

‘GCV-treated’ cells, whereas the ‘untreated’ cells were only transfected with

the respective plasmids and was not treated with GCV. The numbers over the

linking lines are the p-values calculatedbyone-tailedStudent’s t-tests. The line

within each bar represents the standard error.



profiles of cancer cells and selecting the most cancer-specific

promoters for inducing the expression of suicide genes seems a

rational approach for overcoming these deleterious adverse

effects.[56]

Among various specific promoters activated in colon cancer

cell lines, CEA is the most extensively studied. In vitro studies

have shown that despite its activity in non-CEA producing

cells,[57] the activity of this promoter is too low to reach ther-

apeutic targets.[58] Results on the use of other colon cancer-

specific promoters (e.g. MUC1[59]) have been disappointing,

with the exception of the COX2 (PTGS2) promoter.[60] How-

ever, this promoter is unsuitable for use in systemic suicide

constructs, since PTGS2 is a stress gene and highly expressed in

white blood cells.

Exploring signaling pathways deregulated in transformed

cells and selecting the promoters downstreamof these pathways

for cancer-specific suicide gene therapy would be a more rea-

listic strategy. RAS is one of the most important pathways

activated in cancerous cells, especially in colon cancer. This is

mostly related to mutations in the KRAS gene, which play a

pivotal role in colorectal tumorgenesis and epithelial-

mesenchymal transformation.[61] Mutations in this gene predict

a more invasive and metastatic phenotype for this type of

cancer.[62]Mutations inKRAS alone lead to the transformation

of DLD-1 andHCT116 cell lines, whereas oncogenic mutations

in the tumor protein 53 (TP53) gene (in DLD-1) or DCC gene

(in HCT116) without mutation in KRAS do not lead to the

same phenotypic changes.[63] This has lead to the hypothesis

that KRAS downstream promoters are good targets to be used

in colon cancer-specific gene therapy.

There have been a few reports about using promoters

downstream of this pathway for suicide gene therapy. In one of

these approaches, the Py enhancer containing ETS and AP1

binding sites was inserted upstream of different apoptotic

genes. These two overlapping binding regions had a synergistic

effect in transformed cells, which was better than either of these

binding sites alone.[34]

uPAR (PLAUR) is one of the genes downstream of the

activated KRAS signaling pathway. All RAS effectors induce

uPAR expression through themain regulator,MEK/ERK.[50] In

addition, ligands bound to uPAR trigger activation of

signaling pathways in cells, such as the JAK/STAT2orMAPKK

pathways.[64-66] There is other evidence that uPAR expression is

also induced by the WNT signaling pathway, which is another

important signaling pathway activated in colon cancer.[67]

The uPAR promoter can be considered as a combination of

different binding motifs responsive to the KRAS signaling

pathway, although it does not contain TATA and CAT

boxes.[68] Binding elements in the uPAR promoter consist

of proximal and distal AP1 binding sites at -70[69] and -184(-190/-171)[70,71] bases from the transcription start site (TSS),

two SP1-responsive elements at -94[72] and -103,[73] bindingsites for SP1, SP3, and the AP2a-related factor from -152 to

-135[70,74] and a NFkB binding motif at -45.[75] Also, the Py

enhancer activator 3/ETS binding site at -248[76] and AP2aB at

-152 to -135[70] act as suppression motifs in this promoter.

Transactivation of some of these binding sites, such as the

AP2/SP1 site at -152, plays an essential role in inducing uPAR

expression in gastrointestinal tumors but not in normal gastro-

intestinal cells.[77] In addition, -190/-171 sequence is required

for the induction of gene expression by RAS/MAPKK path-

way.[48] The factors bound to -190/-171 and -152/-135 motifs

have functional synergism.[70] Considering that the unaltered

uPAR promoter introduces a collection of these different

binding sites, we opted to amplify the sequence from -878 to

+171 of the uPAR promoter and place it upstream of reporter

and suicide genes.

Two representative colon cancer cell lines were analyzed in

this study. The rationale behind choosing these two specific cell

lines was the activation of the WNT signaling pathway in

SW480 cells[78] and the dependency of transformation in the

HCT116 cell line on KRAS mutation.[63] These two pathways

are the most important pathways activated in colon can-

cer.[3,21,79] Studying the suicide construct in different types of

colon cancer cell lines will help predict the activity of this

specific construct in different types of colorectal cancers. The

lower activity of this promoter in SW480 cells was inevitable

because the uPAR protein is not highly expressed in this cell

line.[74] This can be related to lower activation level of the

KRAS signaling pathway in SW480 compared with HCT116.

The uPAR promoter was not active in two representatives

of normal cells (HUVECs and NCCs). Although the uPAR

promoter activity was weaker relative to the CMV promoter

even in HCT116 (reflected in the b-gal ELISA results), this

lower activity was probably compensated by the bystander

effect in cytotoxic experiments. In SW480, the activity of the

uPAR promoter was not strong enough to produce sufficient

lethal doses of phosphorylated ganciclovir from 20 mg/mL of

ganciclovir. The main reason for this observation is the lower

activity of the RAS signaling pathway in the SW480 cell line.

Another possible explanation is that a region for an unknown

suppressor element exists in the selected part of the promoter.

This element may be responsible for low uPAR promoter

activity in the SW480 cell line and, to a lesser extent, lower than

expected activity in HCT116 cells (compared with the CMV

promoter). This assumption is based on another observation in



which the expression induced by a longer promoter (from

-2300 bp of TSS) is slightly higher than the expression induced

by a shorter one (spanning 400 bp from TSS).[72] This hypoth-

esis should be confirmed in future studies. The different levels

of activity of the uPAR promoter observed in these two colon

cancer cell lines may have clinical implications. It may limit the

optimal application of these constructs in colon cancer cases

with a higher activation level of the KRAS signaling pathway.

TheM30 CytoDEATH� antibody was used to elucidate the

mechanism of cell death. This antibody showed that apoptosis

had occurred in HCT116 cells after transfection with a suicide

construct and treatment with ganciclovir, but failed to detect

this phenomenon in SW480 with the same treatment. This

discrepancy might have been due to the following: firstly, be-

cause the TK/ganciclovir system acts on proliferating cells,[80]

nonproliferating and/or low proliferating cells do not respond

well to its suicide effects. Therefore, in SW480 cells, which have

a longer replication time than HCT116, the apoptotic action

of TK/ganciclovir could not be detected by the M30

CytoDEATH� antibody. Secondly, TK/ganciclovir induces

both apoptosis and necrosis, which may explain the observed

discordance between the death rate assessed by M30

CytoDEATH� antibody and the cytotoxicity assays.[81] Fur-

thermore, the M30 CytoDEATH� antibody detects neoepi-

topes of cytokeratin 18 that has been cleaved by CASP3[82] in

the early phases of apoptosis. In TP53-deficient cell lines such

as SW480, the main mechanism of cytotoxicity induced by

TK/ganciclovir is not the activation of CASP3;[83] whereas, in

TP53-proficient cell lines such as HCT116, the mechanism

of cell death induced by TK/ganciclovir is CASP3-

dependent.[84,85] If this speculation is proven in further analysis,

M30 CytoDEATH� cannot be considered as a good detector

of apoptosis when the main mechanism of apoptosis is not

activation of CASP3.

In HCT116, Annexin-PI detected apoptosis in pUCU-

PARTK- and pUCCMVTK-transfected cells, although the

apoptosis rate in pUCUPARTK-transfected cells was very low.

This method failed to detect apoptosis in pUCCMVTK-

transfected SW480 cells, although it verified this phenomenon

in pUCUPARTK-transfected cells. It can be assumed that

the same concentration of ganciclovir in the presence of

different expression levels of TK protein induced different

mechanisms of cell death, at least in SW480 cells. For example,

in pUCCMVTK-transfected SW480 cells, 20 mg/mL of ganci-

clovir was sufficient for inducing cell death, whereas in

pUCUPARTK-transfected cells, 40 mg/mL was needed to in-

duce a significant level of cell death (observed in cytotoxic as-

says). Therefore, it can be hypothesized that 40 mg/mL of

ganciclovir might trigger necrosis in the presence of a higher

amount of TK, and apoptosis in the presence of a lower level of

this enzyme. These preliminary assumptions about the mecha-

nism of cell death induced by the TK/ganciclovir system should

be studied in detail. The results obtained after administering the

suicidal construct showed that assessing the cytotoxic effects of

suicide constructs with a metabolic assessment such as XTT

could give more direct measurement than surrogate markers

such as staining the cells withM30 CytoDEATH� antibody or

Annexin-PI.

Conclusions

We showed that the uPAR (PLAUR) promoter can be

considered as a potential candidate promoter for colon cancer-

specific gene therapy. The effect of these constructs in other cell

lines including colon, prostate, and pancreas cancer cell lines

should be studied in the future. The specificity of the uPAR

promoter must also be confirmed by its application in other

normal cells. In addition, this construct should be compared

with artificial promoters containing different binding motifs,

including AP1, SP1, NFkB, SP2, AP2a-related, and SP3 alone

or in combination with each other.

In vivo and ex vivomodels must also be included in the design

of further research to study the effectiveness of this system.

Since transfection efficiencies of different cell lines are diverse,

there should be a standardized way to introduce the suicide

constructs to different cell lines. In addition, applying these

suicide gene constructs in animal models requires a more effi-

cient and standardized deliverymethod. The application of viral

vectors would help to resolve the above-mentioned problem.

Acknowledgments

This research was funded by the Pasteur Institute of Iran. We hereby

thank Mrs Maryam Noorayee Kia (Head Nurse of 1st Surgery Ward,

Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran,

Iran), Mrs Leyli Ghaffarpour (Head Nurse of 1st Operating Room, Imam

Khomeini Hospital), and other nurses in these wards for their kind help

in obtaining the normal colon tissue. We also thank Dr Ahmad Kaviani

(1st Surgery Ward, Imam Khomeini Hospital) for his kind help in

obtaining the colon tissue during an operation.

The authors have no conflicts of interest that are directly relevant to the

content of this study.

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Correspondence: Dr Sirous Zeinali, Molecular Medicine Department,

Biotechnology Research Center, Pasteur Institute of Iran, 69th Pasteur

Street, Kargar Avenue, Tehran, 13169-43551, Iran.

E-mail: [email protected]



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