Adenovirus-Mediated Suicide SCLC Gene Therapy Using the Increased

Activity of the hTERT Promoter by the MMRE and SV40 Enhancer

Joon-Seok SONG

Institute of Biotechnology, Korea University, Seoul 136-701, Korea

Received August 4, 2004; Accepted October 26, 2004

Telomerase is a ribonucleoprotein complex of which

the function is to add telomeric repeats to chromosomal

ends. Telomerase consists of two essential components,

the telomerase RNA template (hTR) and the catalytic

subunit (hTERT). hTERT is expressed only in cells and

tissues positive for telomerase activity, i.e., tumor or

stem cells. The aim of this study was to use increased

telomerase promoter activity in small-cell lung cancer

(SCLC) gene therapy. The hTERT promoter and Myc–

Max response elements (MMRE) in pGL3-Control

vector containing SV40 enhancer resulted in strong

expression of the luciferase gene only in telomerase

positive and myc overexpressing SCLC cell line but not

in normal human cell line. To investigate the possibility

of the utilization of the MMRE, hTERT promoter, and

SV40 enhancer in targeted SCLC gene therapy, adeno-

virus vector expressing HSV-TK controlled by the

MMRE, hTERT promoter, and SV40 enhancer for the

induction of telomerase positive and myc-overexpress-

ing cancer specific cell death was constructed. SCLC

cells infected with Ad-MMRE-hT-TK-enh were signifi-

cantly suppressed and induced apoptosis more than

those of Ad-hT-TK or Ad-hT-TK-enh infected cells.

Telomerase and c-myc are activated in 60�80% of

SCLC, so the increased activity of telomerase promoter

can be used for targeted SCLC gene therapy. These

results show that the MMRE, hTERT promoter, and

SV40 enhancer can be used in SCLC targeted cancer

gene therapy.

Key words: hTERT promoter; Myc–Max response ele-

ments; SV40 enhancer; small cell lung

cancer; targeted cancer gene therapy

Compared with other types of lung cancer, small-celllung cancer (SCLC) has a greater tendency to be widelydisseminated by the time of diagnosis and is highlyaggressive, clinically characterized by rapid growth,frequent invasion, and metastasis.1) Initially, SCLC maysensitive to chemo- and radiotherapy, but many of themwill become resistant to any therapy. About 85% ofSCLC tumors shows overexpression of the myc family2)

and that makes the myc genes a good target for SCLC

treatment.Telomerase is highly active in small-cell and non-

small-cell lung cancers,3) colorectal and gastric can-cers,4) hepatocellular carcinomas,5) hematologic malig-nancies,6) giant cell tumors of bone,7) prostate cancers,8)

breast cancers,9) and ovarian carcinomas.10) Telomeraseis a ribonucleoprotein which consists of several compo-nents. Among these, two components are known as totheir functions: the RNA component (hTR) and thetelomerase catalytic subunit (hTERT). hTR acts as atemplate for telomere synthesis.11) The expression ofhTR is ubiquitous in all types of human cells, regardlessof the status of telomerase activity, but hTERT isexpressed only in cells and tissues positive for telomer-ase activity, i.e., tumor or fetal cells.12–14)

Recently, the promoter region of cancer specifichuman telomerase reverse transcriptase was cloned andcharacterized.15,16) The promoter of hTERT was GC richand lacked both TATA and CAAT boxes. Interestingly,transient expression assays indicated that transcriptionof hTERT was significantly activated in cancer cell linesbut repressed in normal primary cells. Overexpression ofc-Myc resulted in a significant increase in the transcrip-tional activity of the core promoter.15,16) Myc familyproteins, groups of the helix-loop-heilx/leucine zipperfamily including c, N, and L-Myc, form heterodimerswith a partner protein, Max. This Myc–Max proteincomplex binds to the CACGTG sequence and activatestranscription.17,18)

It is known that HSV-TK phosphorylates the nontoxicprodrug ganciclovir (GCV), which then becomes phos-phorylated by endogenous kinases to GCV-triphosphate,causing chain termination and single-strand breaks uponincorporation into DNA.19) The mechanisms linking TK-and GCV-induced DNA damage to apoptosis, however,are largely unknown. Recently, it was found that TK-GCV induces accumulation of p53 and increases cellsurface expression of death receptors, likely involvingp53-mediated translocation of CD95 associated withCD95-L-independent formation of a death-inducingsignaling complex (DISC), eventually leading to apop-tosis involving the Fas-associated death domain protein(FADD) and caspases.20)

To whom correspondence should be addressed. Tel/Fax: +82-2-926-3316; E-mail: antibody@korea.ac.kr

Biosci. Biotechnol. Biochem., 69 (1), 56–62, 2005

Because specific therapeutic gene expression isimportant in cancer gene therapy, targeted cancer genetherapy has the aim of concentrating the target ther-apeutic gene expression into the specific target tissue.Then it can minimize secondary effects and maximizethe therapeutic index. Therefore, the feasibility of usingthe hTERT promoter to induce tumor-specific transgeneexpression in cancer gene therapy has been expected.The promoter of hTERT, telomerase catalytic subunitsactivated in 80�90% of lung cancers is fascinating as acancer specific promoter. Although there are severalexamples of use of the hTERT promoter for targetedcancer gene therapy using adenovirus,21,22) the weakactivity of this promoter hinders this approach to clinicalresearch. So it is necessary to increase the activity of thispromoter.23) In this study, it was attempted to maintainthe cancer specific character of the hTERT promoter andto increase the activity by use of the Myc–Max responseelements and transcriptional enhancer of SV40 virus.

Materials and Methods

Cell culture. Wi-38 (human normal fibroblast) andQBI-293A (Quantum-Appligene, U.S.A., a human cellline transformed by adenovirus 5 DNA) cells weregrown in DMEM (Gibco BRL, Germany) supplementedwith 10% FCS (HyClone, Logan, U.S.A.), penicillin (50units/ml), and streptomycin (50 mg/ml) in the presenceof 5% CO2. NCI-H417 (small-cell lung cancer cells)

were grown in RPMI1640 (Gibco BRL, Germany)supplemented with 10% FCS, penicillin (50 units/ml),and streptomycin (50 mg/ml) in the presence of 5% CO2.All cell lines except QBI-293A were obtained from theAmerican Type Culture Collection (Manassas, VA).

Construction of report plasmids. Sense and antisenseoligonucleotides for Myc–Max response elements weresynthesized (Takara, Japan) as described by Eiseman’sGroup.17) Two oligomers were annealed as described byKishimoto’s group.18) This annealed fragment includesrestriction endonuclease sites of Kpn I and Bgl II at theend of 50 and 30 respectively (Fig. 1A). This fragmenthas four repeats containing a core sequence CACGTG,which is the binding site of Myc–Max heterodimers. ThehTERT promoter region was amplified by PCR usingLA-Taq polymerase (Takara, Japan) on the HeLagenomic DNA. The hTERT forward (50-agatctagt-ggattcgcgggcacaga-30) and reverse (50-aagcttagggcttccc-acgtgcgcag-30) primers, both introducing Bgl II andHind III site (underlined in the sequences respectively)were used. This region is the hTERT promoter regionfrom �204 to +56, which contains the core promoterand two E-boxes.16) PCR was carried out for 35 cycles at94 �C for 1min, at 66 �C for 1min, and at 72 �C for1min. pGL3-MMRE-hT-enh, which has MMRE,hTERT promoter and SV40 enhancer was constructedby replacing SV40 promoter of pGL3-Control plasmidwith hTERT promoter and inserting MMRE fragments

Fig. 1. Structure of Myc–Max Response Elements (MMRE, A) pGL3-MMRE-hT-TK-enh (B) and Luciferase Reporter Plasmids (C).

A core sequence CACGTG, which is the binding site of Myc–Max heterodimers, is indicated bold and in underlined type in (A).

hTERT Promoter Activity Increased by MMRE and SV40 Enhancer 57

(Fig. 1C). Direct dideoxynucleotide sequencing ensuredcorrect sequence and direction.

Construction of recombinant adenovirus Ad-MMRE-hT-TK-enh. pGL3-MMRE-hT-TK-enh, which hasMMRE, hTERT promoter, and SV40 enhancer wasconstructed by replacing the luciferase gene of pGL3-MMRE-hT-enh plasmid with HSV-TK (Fig. 1B). Thenthe MMRE-hTERT promoter-HSV-TK-SV40 enhancercassette was digested with Kpn I and Sal I, and ligatedwith Kpn I and Sal I digested pShuttle (Ad transfervector; Quantum-Appligene, U.S.A.) and named pS-MMRE-hT-TK-enh. Then it was co-transformed withpAdEasy-1 (Quantum-Appligene, U.S.A.) into BJ5183E. coli cells using electroporation methods. After selec-tion on kanamycin plates, 40 colonies were selected andscreened for recombinants by plasmid size and restric-tion enzyme analysis. Viral DNAs of candidates weretranfected in QBI-293A cells using FuGENE�6 (Roche,Germany) according to the manufacturer’s protocol.This constructed adenovirus vector was named Ad-MMRE-hT-TK-enh. The constructed adenovirus werethen purified from the lysates of infected QBI-293Acells by two rounds of CsCl gradient centrifugation.24)

Then the titers of the virus stock were determined byplaque dilution assay.25) The recombinant virus werestored at �80 �C until use. The control recombinantadenovirus Ad-hT-TK and Ad-hT-TK-enh using pGL3-Promoter and pGL3-Control, which do not have SV40enhancer and MMRE respectively, were constructed inthe same way above. The negative control vector(Ad5.CMV-Null; Ad-CMV) was purchased from Quan-tum-Appligene. Ad-CMV is an empty vector, whichcontains no coding sequences between the CMVpromoter and PA (the poly adenylation site).

Luciferase assay. The selective expression of theluciferase gene in tumor cells was determined byluciferase reporter plasmid using FuGENE�6 (Roche,Germany) according to the manufacturer’s protocol.Briefly, 105 cells seeded in a 35-mm tissue culture dishwere exposed to a transfection mixture containing 2 mgof luciferase reporter plasmids and 0.5 mg of pSV-�-galactosidase control plasmid vector (Promega, U.S.A.).48 h after transfection, luciferase assays were performedaccording to the manufacturer’s protocols (Promega,U.S.A.) and measured with a TD-20/20 Luminometer(Turner Designs, Sunnyvale, CA). The Simian virus 40(SV40) promoter (pGL3-Promoter or pGL3-Control)was used as a positive control. hTERT promoterplasmids (pGL3-hT, pGL3-hT-enh) were constructedby replacing SV40 promoter with hTERT promoter. Theluciferase activity of the pGL3-Promoter or pGL3-Control plasmid in each cell line was considered to be100%. A �-Galactosidase assay was also performed withthe same cell extracts to standardize for transfectionefficiency. All of the data shown in this study wereobtained from at least three independent experiments.

Adenovirus infection with GCV treatment in vitro. 105

cells were plated in six-well plates prior to infection.The Ad-CMV, Ad-hT-TK, Ad-hT-TK-enh, and Ad-MMRE-hT-TK-enh adenovirus were applied in 1ml ofmedia at a 50 moi 24 h later. Cells were incubated at37 �C for 3 h followed by the addition of 2ml mediacontaining a 50 mM concentration of GCV (Ganciclovir;InvivoGen, San Diego, CA). Cell cultures were replen-ished every day with fresh medium containing GCV. Allof the data shown in this study were obtained from atleast three independent experiments.

Cell death detection ELISA. Cytoplasmic histone-associated-DNA-fragments (mono-and oligonucleo-somes) after induced cell death by Ad-MMRE-hT-TK-enh and GCV were determined by Cell Death DetectionELISAPLUS (Roche, Germany). Briefly, the Ad-CMV,Ad-hT-TK, Ad-hT-TK-enh, and Ad-MMRE-hT-TK-enhinfected cell lysates were placed in a streptavidin-coatedmicro-plate. A mixture of anti-histone-biotin and anti-DNA-POD was added and incubated for 2 h at 15–25 �C.After removal of unbound antibodies by a washing step,POD was determined photometrically at 405 nm withABTS as substrate.

Results

Variation of transcriptional activity of hTERT pro-moter in SCLC and normal cells

To show the variation of transcriptional activity ofhTERT promoter in SCLC and normal cells, a transienttransfection of luciferase repoter plasmid was performed(Fig. 2). The transcriptional activity of pGL3-Promoterplasmid in each cell line was considered to be 100%.pGL3-hT showed significant transcriptional activity inSCLC at about 138% of pGL3-Promoter. In contrast,there was no significant transcriptional activity in the

Fig. 2. Specific Expression of Luciferase Genes Imparted by the

hTERT Promoter pGL3-hTERT Plasmid Consisted in hTERT

Promoter.

Luciferase genes were introduced into normal fibroblast (Wi-38)

and a small cell lung cancer cell line (NCI-H417). Relative

luciferase activity was standardized with control plasmid pGL3-

Promoter transfection. The means from at least three independent

experiments are shown; bars, SD.

58 J.-S. SONG

telomerase activity negative normal fibroblast cell line(Wi-38). These results showed that SCLC had strongtranscriptional activity for hTERT promoter. A highlysensitive TRAP assay26) also showed that the NCI-H417cell line had strong telomerase activity, but not thenormal cell line (Wi-38; data not shown).

Effect of MMRE and SV40 enhancer on hTERTpromoter activity

To determine if plasmid vectors containing MMREand SV40 enhancer could produce higher levels oftransgene expression than those without them, a MMREand a SV40 enhancer were inserted upstream anddownstrem from the hTERT promoter respectively(Fig. 1C). As shown in Fig 3B, pGL3-MMRE-hT-enhwith a MMRE and SV40 enhancer produced a more than6-fold higher expression of luciferase than pGL3-hTwith only the hTERT promoter in QBI-293A cells, andpGL3-hT-enh with a SV40 enhancer increased the levelof expression more than 3-fold higher than pGL3-hTplasmid. But in the case of normal fibroblast Wi-38,there was no significant increase in hTERT promoteractivity by MMRE and SV40 enhancer (Fig. 3A). Toshow increased activity of the hTERT promoter by theMMRE sequence, c-Myc was overexpressed in QBI-293A as well as normal fibroblast Wi-38 cells, andluciferase assays were performed. In QBI-293A cells,the introduction of c-Myc expression vectors (pCMV-c-myc) resulted in more than 2-fold activation. Recently,c-Myc has been reported to activate telomerase pro-moter in normal epithelial and fibroblast cells.27)

Similarly, in this study, c-Myc overexpression increasedmore than 3-fold the activation of hTERT transcriptionin Wi-38 (Fig. 3A). NCI-H417 SCLC cells are known tooverexpress c-Myc protein.28) As shown in Fig. 4,

MMRE and SV40 enhancer increased the activity ofhTERT promoter more than 17-fold without them inNCI-H417. These results showed that MMRE sequencesand SV40 enhancer activated the hTERT promoter andcan be used in SCLC treatment.

Ad-MMRE-hT-TK-enh confers more GCV sensitivitythan Ad-hT-TK or Ad-hT-TK-enh to SCLC cells but doesnot affect normal fibroblastsTo determine increased cancer specific cell death by

Ad-MMRE-hT-TK-enh, SCLC and normal fibroblastcell lines were infected with a moi of 50 of Ad-MMRE-hT-TK-enh, Ad-hT-TK-enh, Ad-hT-TK, and Ad-CMVwith 50 mM of GCV (Fig. 5). After 5 days of infection,

Fig. 3. Specific Increased Expression of Luciferase Gene Imparted by the MMRE, hTERT Promoter, and SV40 Enhancer.

pGL3-Con, pGL3-hT, pGL3-hT-enh, and pGL3-MMRE-hT-enh plasmids were introduced into normal fibroblast (A, Wi-38) and telomerase

positive control cell line (B, QBI-293A). After transfection with c-Myc expression plasmid (pCMV-c-myc), the next day both cell lines were

transfected with pGL3-MMRE-hT-enh plasmid (c-myc+mmhTenh). Relative luciferase activity was standardized with control plasmid pGL3-

Control transfection. The means from at least three independent experiments are shown (Con, pGL3-Control; hT, pGL3-hT; hTenh, pGL3-hT-

enh; mmhTenh, pGL3-MMRE-hT-enh; c-myc, pCMV-c-myc); bars, SD.

Fig. 4. SCLC Specific Increased Expression of Luciferase Gene

Imparted by the MMRE, hTERT Promoter, and SV40 Enhancer.

pGL3-Con, pGL3-hT, pGL3-hT-enh, and pGL3-MMRE-hT-enh

plasmids were introduced into SCLC cells (NCI-H417). The means

from at least three independent experiments are shown (Con, pGL3-

Control; hT, pGL3-hT; hTenh, pGL3-hT-enh; mmhTenh, pGL3-

MMRE-hT-enh); bars, SD.

hTERT Promoter Activity Increased by MMRE and SV40 Enhancer 59

almost all the tumor cells displayed typical cell death,while they did not in Wi-38 cells. Ad-MMRE-hT-TK-enh resulted in about 44% and 24% more cell death thanAd-hT-TK or Ad-hT-TK-enh infected cells respectively,but normal fibroblast Wi-38 was not affected by Ad-hT-TK, Ad-hT-TK-enh, or Ad-MMRE-hT-TK-enh.

Ad-MMRE-hT-TK-enh induced apoptosis of SCLCcellsThe apoptotic cells in the Ad-MMRE-hT-TK-enh

infected cells were analyzed by Cell Death DetectionELISAPLUS (Roche, Germany). The analysis showedthat the Ad-MMRE-hT-TK-enh treated SCLC cells hadundergone about 2.5- and 1.4-fold more apoptosis thanthat of Ad-hT-TK and Ad-hT-TK-enh respectively(Fig. 6). Consequently, the experiment clearly indicatedthat Ad-MMRE-hT-TK-enh increased cell death byapoptosis of SCLC cells.

Discussion

In cancer gene therapy, restricted expression of thetherapeutic gene in tumor is important. If the therapeuticgene is expressed in all cells, it will affect both tumorand normal cells. Using the tumor specific promotersystem will solve this problem, but true tumor specificpromoters are rare, and often these promoters are usefulonly for the particular types of cancers from which theyare derived. Although telomerase activity is undetect-able in most human somatic tissues, it is generallypresent in highly replicative tissues and in most humancancers. As such, its exclusive expression in most tumorcells makes it a very strong candidate for targeted cancergene therapy. There are two kinds of telomerase-associated cancer gene therapy for telomerase-positivetumors.29) First, tumor cell growth is inhibited by direct

inhibition of telomerase activity, resulting in apoptoticcell death or growth arrest.30,31) Telomerase consists oftwo major components, the RNA template (hTR) and thecatalytic subunit (reverse transcriptase, hTERT). hTR isconsidered to be good therapeutic targets.30,31) The otherapproach is telomerase-specific cancer gene therapyusing telomerase promoter as a means directly to killtelomerase positive tumors.21–23) Although there are noclearly known mechanisms that control hTERT tran-scription, it is known that c-Myc has an important role inpositive regulation of hTERT gene activation andtelomerase activation. In previous studies, the c-Myclevel directly influenced telomerase activity.32) Telo-merase activity in human leukemic cell lines is inhibitedby antisense c-Myc oligonucleotide.33) In the hTERTpromoter region, there are two typical E-boxes(CACGTG, �165 to �160 and 42 to 48) which areknown as potential binding sites of the c-Myc oncopro-tein.16)

To investigate the possibility of the utilization of thehTERT promoter, MMRE, and SV40 enhancer intargeted SCLC gene therapy, adenovirus vector con-taining HSV-TK gene under the control of MMRE,hTERT promoter, and SV40 enhancer was constructed.This virus was introduced into normal fibroblast andSCLC cancer cell lines. After treatment, the morphologyof the majority of the Ad-MMRE-hT-TK-enh infectedSCLC cells changed into apoptotic cells and underwentcell death, but not normal fibroblasts. The growth of Ad-MMRE-hT-TK-enh infected cancer cells was signifi-cantly more suppressed than that of Ad-hT-TK or Ad-hT-TK-enh (Fig. 5). Using a Cell Death DetectionELISAPLUS, the apoptotic cells of the Ad-MMRE-hT-TK-enh infected NCI-H417 cells were analyzed and itwas found that the Ad-MMRE-hT-TK-enh treatedcancer cells had undergone about 2.5- and 1.4-foldmore apoptosis than that of Ad-hT-TK or Ad-hT-TK-

Fig. 5. Exposure of Wi-38 and NCI-H417 Cells in Vitro to Ad-hT-

TK, Ad-hT-TK-enh, or Ad-MMRE-hT-TK-enh (50 moi) + GCV

(50 mM) and Determination of the Number of Viable Cells by Trypan

Blue Exclusion 5 Days Later.

The cells were seeded at 105 cells in 6-well dish 24 h prior to

infection. 50 mM of GCV alone was not effective in cell growth. The

means from at least three independent experiments are shown; bars,

SD.

Fig. 6. Detection of Nucleosomes in the Cytoplasm of NCI-H417

Cells Treated with Ad-MMRE-hT-TK-enh.

NCI-417 cells (105 cells) were treated with Ad-hT-TK, Ad-hT-

TK-enh, or Ad-MMRE-hT-TK-enh (50 moi) + GCV (50 mM) for 5days. Afterward lysis cells were centrifuged and the supernatent was

analyzed by ELISA. Ad-CMV infected cells were used as a negative

control. The positive control provided by the manufacturer was

used; bars, SD.

60 J.-S. SONG

enh (Fig. 6). Therefore, it is concluded that theadenovirus Ad-MMRE-hT-TK-enh suppressed SCLCcell growth and induced apoptosis and, as such, might bea useful method for suppressing tumor growth intargeted cancer gene therapy. The existing cancertreatment method has limitations for elevation of thesurvival rate of cancer patients and has serious sideeffects on normal tissues and organs because ofindiscriminate killing effects between normal and cancercells. Therefore the technology of gene therapy, whichcan kill only tumor cells, is another alternative that canincrease the rate of complete cure and the quality of life(QOL) of cancer patients. This is the first trial to useadenovirus vector, which has Myc–Max binding motifs,telomerase catalytic promoter hTERT, HSV-TK, andSV40 enhancer to suppress Myc and telomerase activitypositive SCLC cells and induce apoptotic cell death.Adenovirus vector consisted of MMRE, hTERT pro-moter, SV40 enhancer, and suicide gene HSV-TK hasthe feature of maintaining the cancer specific characterof the hTERT promoter and increased activated ex-pression of HSV-TK by MMRE and SV40 enhancer,and it might be useful for targeted SCLC gene therapy.

Acknowledgments

This work was supported by grant No. R08-2003-000-10007-0 from the Basic Research Program of theKorea Science and Engineering Foundation.

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