REPORT

Transactivation of the Metallothionein Promoter in Cisplatin-Resistant Cancer Cells: a Specific Gene Therapy Strategy

Didier Vandier, Vincent Calvez, Liliane Massade, Alain Gouyette, Lyn Mickley, Tito Fojo, Olivier Rixe

Affiliations of authors: D. Vandier, L. Mickley, T. Fojo, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD; V. Calvez, Service de Virologie, Hopital Pitié-Salpêtrière, Paris, France; L. Massade, Laboratoire de Toxicologie Moléculaire (U-490 INSERM), Paris; A. Gouyette, Laboratoire de Pharmacotoxicologie et Pharmacogénétique (UMR 8532 CNRS), Institut Gustave-Roussy, Villejuif, France; O. Rixe, Département d'Oncologie, Clinique Claude-Bernard, Metz, France.

Correspondence to: Tito Fojo, M.D., Ph.D., National Institutes of Health, Bldg. 10, Rm. 12C428, Bethesda, MD 20892 (e-mail: tfojo{at}helix.nih.gov).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Cisplatin (cis-diamminedichloroplatinum) is one of the most active agents against a broad range of malignancies, including ovarian cancer. Cisplatin resistance appears to be associated with several molecular alterations, including overexpression of metallothionein, a metal-binding protein. In the present study, we attempted to take advantage of metallothionein overexpression to overcome cisplatin resistance. Methods: Using a virus-free system (liposomes), we sought to express the suicide gene, thymidine kinase (TK), driven by the promoter of the human metallothionein IIa (hMTIIa) gene using the pMT-TK plasmid. We used cisplatin-resistant human ovarian carcinoma cells as a model. Results: We first analyzed metallothionein expression using a ribonuclease protection assay. In comparison to parental cells, the cisplatin-resistant cells were found to have increased expression of metallothionein messenger RNA (mRNA). Metallothionein overexpression in these cells was not associated with an increased copy number of the hMTIIa gene or with different transfection efficiencies. Furthermore, we showed by reverse transcription–polymerase chain reaction analysis that transfection of the pMT-TK plasmid results in a 56-fold higher expression of thymidine kinase mRNA in cisplatin-resistant cells compared with parental cells, consistent with increased metallothionein promoter-mediated transactivation in the cisplatin-resistant cells. Transfection of resistant cells with pMT-TK or a control plasmid (pCD3-TK) resulted in a marked sensitization to ganciclovir, with a 50% cell growth-inhibitory concentration (IC50) of 20 µg/mL and 9 µg/mL, respectively. Transfections of the cisplatin-sensitive cells resulted in no sensitization to ganciclovir with pMT-TK (IC50 200 µg/mL) and a high sensitization with pCD3-TK (IC50 = 6 µg/mL). Conclusion: These studies suggest that pMT-TK gene therapy may provide an alternative treatment for cisplatin-refractory ovarian tumors.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ovarian carcinoma has the highest fatality rate of the gynecologic malignancies (1). Resistance to available chemotherapeutic drugs is the major obstacle to effective, potentially curative chemotherapy in this cancer. Cisplatin (cis-diamminedichloroplatinum) is one of the most active drugs used for the treatment of solid tumors and is an integral component of many ovarian cancer treatment protocols. Although aggressive treatment of ovarian cancer patients with cisplatin-containing regimens is associated with clinical response rates of 40%–80%, chemotherapy is curative in only a fraction of these patients (2). Thus, the majority of ovarian cancer patients die of their disease.

Tumor cells are either resistant to cisplatin de novo or acquire resistance to this agent after an initial response (3). Several potential mechanisms of resistance have been described in vitro and in vivo, and these may partially explain the failure of cisplatin chemotherapy (48). The importance and the occurrence of these phenomena depend on the cell type and on the level of resistance.

In vitro and in vivo studies show that overexpression of metallothionein is involved in cisplatin resistance: The level of metallothionein expression and the level of cisplatin resistance are related, and transfection of the gene encoding for metallothionein increases cisplatin resistance (9). Metallothioneins are metal-binding proteins of low molecular weight (6–7 kd); they are cysteine rich and have an important role in the homeostasis of trace metals, such as Zn2+ and Cu2+, and participate in the detoxification of metals, such as Cd2+ and Hg2+. Metallothioneins are transcriptionally induced by these metals through metal-responsive elements located in the 5`-regulatory regions of the human metallothionein genes. How metallothionein confers resistance is unclear, although binding of cisplatin to metallothionein seems to divert the drug from its nuclear target and to inhibit its cytotoxicity (10).

During the past decade, several approaches have been investigated to circumvent cisplatin resistance and include the following: 1) the development of non-cross-resistant platinum relatives (1113); 2) the use of pharmacologic modulators, such as buthionine sulfoximine (14); and 3) the restoration of cisplatin accumulation with amphotericin B (15). To date, however, these strategies must still be considered experimental or clinically unproved.

As an alternative, Blaese et al. (16) described a procedure in which tumor cells were engineered to express a foreign enzyme or suicide gene that converts a prodrug into a toxic metabolite, thereby killing the cells. Technical requirements for this system are as follows: 1) a highly efficient delivery of the transgene into tumor cells and 2) a relatively high specific expression of the suicide gene in tumor cells. A commonly used system employs the gene encoding herpes simplex virus thymidine kinase (HSV-TK) as the suicide gene and the anti-herpesvirus drug ganciclovir as the prodrug. Ganciclovir is not metabolized substantially by normal mammalian cells, but it is phosphorylated by HSV-TK to a deoxynucleotide analogue that kills cells undergoing DNA synthesis. In animal models, use of the HSV-TK system has been reported to produce complete tumor ablation (17), and some initial clinical reports are encouraging (18). One of the most important challenges of the HSV-TK and ganciclovir system may be how to enhance its specificity for tumor cells. The specificity of tumor cell killing could be substantially improved by using promoters that restrict expression of the HSV-TK to the tumor cells. This approach has been explored in several laboratories (1921).

In this study, we explored one approach to treat cisplatin-resistant cells. We describe the feasibility of using the human metallothionein IIa (hMTIIa) promoter to direct expression of a suicide gene (HSV-TK).


    MATERIALS AND METHODS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cells. A2780(1A9) is a single-cell clone of the human ovarian carcinoma cell line A2780 that was derived from a patient before treatment. A2780-E(80) is a cisplatin-resistant subline that was developed in vitro by intermittent, incremental exposure of the A2780(1A9) cells to cisplatin, starting at a dose of 0.5 µM and gradually advancing to a final concentration of 80 µM. All cell lines were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 U/L penicillin, and 50 µg/L streptomycin (Life Technologies, Inc. [GIBCO BRL], Cergy Pontoise, France) in a 5% CO2 incubator at 37°C. The A2780-E(80) subline was maintained in RPMI-1640 medium containing 80 µM cisplatin.

Plasmids. pTK-ß-gal and pCMV-ß-gal plasmids containing the lac-Z reporter gene were purchased from Clontech Laboratories, Inc. (Palo Alto, CA). We also used as a positive control the pCD3-TK plasmid containing the HSV-TK gene under the control of the constitutive cytomegalovirus (CMV) promoter. Briefly, the thymidine kinase-coding region was excised from the plasmid pMK, which contains the suicide gene under the control of the mouse MTI promoter region (22) as a 1.8-kilobase BglII–PvuII fragment, and ligated with a 5.4-kilobase fragment of pcDNA3 (Invitrogen Corp., Carlsbad, CA), obtained by digestion with BamHI and EcoRV (23). The pMT-TK plasmid containing the HSV-TK gene driven by the hMTIIa promoter was provided by M. Karin (University of Southern California School of Medicine, Los Angeles). Briefly, an 800-base-pair (bp) HindIII–BamHI DNA fragment containing the promoter region of the hMTIIa gene was inserted between the HindIII site of pBR322 and the BglII site of the HSV-TK gene (24) to generate plasmid pMT-TK (25). Plasmid DNAs were purified by ion exchange chromatography (Qiagen, Courtaboeuf, France).

Ribonuclease (RNase) protection analysis. RNA was extracted from the cell with the use of guanidinium thiocyanate solubilization and centrifugation at 24000 rpm for 18 hours at 15°C over a cesium chloride cushion. A 5- to 20-µg sample of total RNA was hybridized with 5 x 105 cpm antisense RNA probe for RNase protection analysis. Briefly, hybridization consisted of heat denaturation of the RNA at 85°C for 5 minutes, followed by overnight incubation of the probe and sample at 45°C. Subsequently, samples were digested with RNase-A and RNase-T1 and then incubated in proteinase K and sodium dodecyl sulfate (SDS). Samples were then extracted with phenol and chloroform, ethanol precipitated, lyophilized, and resuspended in a 90% formamide loading buffer. The samples were separated on a 6% polyacrylamide gel at 1500 V for 2–3 hours, followed by autoradiography for 1 day (26).

Preparation and administration of liposome–nucleic acid complexes. We mixed 2 µg of plasmid and 10 µL of lipofectamine (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD) in 1 mL of serum-free RPMI-1640 medium, according to the manufacturer's and published instructions (27). Six-well plates were seeded with 5 x 105 cells 1 day before use and rinsed with RPMI-1640 medium just before transfection.

Detection of ß-galactosidase expression. Cells expressing ß-galactosidase were detected 48 hours after transfection by blue staining, as previously described (28). We determined the transfection efficiency by counting the fraction of stained cells in 25 fields at high magnification (35 cells per field) in three independent transfection experiments.

Analysis of the hMTIIa gene by polymerase chain reaction (PCR) analysis. Genomic DNA was isolated and purified from both A2780(1A9) cells and the cisplatin-resistant A2780-E(80) subline by use of the Wizard Genomic DNA Purification Kit (Promega, Charbonnieres, France). DNAs were amplified from serial dilutions (0.1 ng, 1 ng, 5 ng, 10 ng, 50 ng, 100 ng, 500 ng, and 1 µg) of each sample in 50-µL tubes containing 10 mM Tris–HCl (pH 8.3), 5.5 mM MgCl2, 100 µM of each deoxynucleotide triphosphate (dNTP), and 0.1 U/µL of DNA polymerase. After denaturation for 2 minutes at 94°C, the DNAs were amplified by PCR (35 cycles: 1 minute at 92°C, 1 minute at 60°C, and 1 minute at 72°C). The following primers were used for PCR amplification to obtain a 352-bp fragment of the hMTIIa gene: EMT2-5: 5`-CGGTCCTCACGGGATGGGGAAAACCACCACCACG-3` (sense); EMT2-3: 5`-GCAGGGCGTCCCGGCAGCCGGCGGGCGATTGG-3` (antisense). Similar conditions were used to amplify the ß-globin gene with the following primers and to obtain a 230-bp fragment: B1-5: 5`-CAAATGGCTCCTCTTTCTTGCTC-3` (sense); B2-3: 5`-GCAGAGTGAACCAGAAGGTTTACAG-3` (antisense).

Analysis of DNA and RNA of HSV-TK by quantitative PCR. Genomic DNA and total cellular RNA were extracted and purified from both A2780(1A9) cells and the cisplatin-resistant A2780-E(80) subline by use of the Wizard Genomic DNA Purification Kit and the SV Total RNA Isolation System (Promega). DNA and RNA were prepared from untransfected cells and from cells transfected with either pMT-TK and/or pCD3-TK. Quantitative PCR for the HSV-TK gene was performed as previously described (29). Briefly, RNAs were reverse transcribed and DNAs were amplified from serial dilution of the samples in a 50-µL tube containing 10 mM Tris–HCl (pH 8.3), 5.5 mM MgCl2, 100 µM of each dNTP, and 0.1 U/µL of DNA polymerase. After denaturation for 2 minutes at 94°C, the DNAs were amplified by PCR (32 cycles: 1 minute at 94°C, 1 minute at 62°C, and 2 minutes at 68°C). The following primers were used for PCR amplification to obtain an 855-bp fragment of the HSV-TK gene: 5`-CGGTCCTCACGGGATGGGGAAAACCACCACCACG-3` (sense) and 5`-GCAGGGCGTCCCGGCAGCCGGCGGGCGATTGG-3` (antisense). pCD3-TK and total RNA from A2780(1A9) cells transfected with pMT-TK were used, respectively, as PCR and reverse transcription (RT)–PCR reference standards. Dilutions were performed to ensure that the PCR was in the exponential range. The levels of both reference standards were arbitrarily assigned a value of 10, and all other values were determined relative to this value. All quantitations were performed by densitometry.

Ganciclovir sensitivity assays. Forty-eight hours after transfection, medium was removed and replaced daily with ganciclovir (Cymevan, 500 mg; Roche Laboratories, Neuilly-sur-Seine, France) at a range of concentrations (i.e., 0, 10, 50, 100, or 200 µg/mL). Cells were incubated at 37°C for 7 days with ganciclovir, after which time cell viability was quantified by the tetrazolium salt assay (30). The percent survival was calculated from the ratio of the absorbance measured in HSV-TK-transfected cells compared with similarly treated pCMV-ß-gal-transfected cells. The cellular sensitivity to ganciclovir, expressed as the concentration of ganciclovir that inhibited cell survival by 50% (IC50), was determined from dose–response curves related to histograms (see Fig. 4Go) (curves not shown). The IC50 was calculated, assuming the survival rate of untransfected and non-ganciclovir-treated cells to be 100%. The results are expressed as means with 95% confidence intervals from three independent experiments.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Detection of hMTIIa messenger RNA (mRNA) in parental and cisplatin-resistant cell lines. With the use of an RNase protection assay, expression of hMTIIa mRNA was investigated in total RNA from parental cells and from the cisplatin-resistant cell line, maintained with or without drug. As shown in Fig. 1Go, fragments corresponding to hMTIIa mRNA were protected by RNA from the cisplatin-resistant cell lines but not by RNA from parental cells. Fragments are observed as a result of mismatches between monkey and human MTIIa mRNA. We have previously shown increased metallothionein expression in A2780-E(80) cells (31). We sought to clarify to what extent this increase was constitutive. We examined the effects on metallothionein expression of removing or adding a higher concentration of cisplatin to determine whether the increased expression was constitutive or inducible. As shown in Fig. 1Go, increased expression was seen in the A2780-E(80) cells maintained in 80 µM cisplatin, compared with parental A2780(1A9) cells. Addition of 160 µM cisplatin (+CP) resulted in a slight further increase, while removal of cisplatin for 2 weeks (-CP) led to, at most, a minimal decrease in several experiments. These results suggested that the increased expression observed in A2780-E(80) cells was largely constitutive in nature.



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Fig. 1. Ribonuclease (RNase) protection analysis. Single-stranded RNA, complementary to the monkey metallothionein IIa (MTIIa) gene, was synthesized and used in an RNase protection analysis to detect human MTIIa (hMTIIa) messenger RNA. The fragments generated result from mismatches at four residues, which are sites of difference between the human and monkey sequences. Lane 1: parental A2780(1A9) human ovarian cancer cell line. Lane 2: cisplatin-resistant ovarian cancer cell line A2780-E(80), maintained in 80 µM cisplatin. Lane 3: A2780-E(80) + CP; the cells, which were carried in 80 µM cisplatin, were treated with 160 µM cisplatin. Lane 4: A2780-E(80) - CP; the cells were maintained without drug for 2 weeks. Because previous results [(31); Rixe O, Fojo T: unpublished results] have demonstrated threefold to greater than 50-fold differences in expression of actin, vimentin, ß-tubulin, {alpha}-tubulin, desmin, and cytokeratin, precluding their use as internal controls, a gel was run concurrently to demonstrate comparability of RNA loading (bottom panel).

 
Comparison of hMTIIa DNA copy number in parental and cisplatin-resistant cells. To examine the copy number of the hMTIIa gene in the resistant cells compared with the parental cells, we performed PCR using DNA from parental cells and the cisplatin-resistant subline. As shown in Fig. 2Go, with the use of ß-globin primers as an internal control, PCR revealed similar levels of hMTIIa DNA in both cell lines, suggesting that amplification of this gene had not occurred.



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Fig. 2. Polymerase chain reaction analysis of DNA from human ovarian cancer cell line A2780(1A9) and its cisplatin-resistant descendant A2780-E(80) for the presence of the human metallothionein IIa (hMTIIa) and ß-globin genes. DNA was extracted from cells and analyzed for gene copy number with the use of the oligonucleotide primers and conditions described in the "Materials and Methods" section. With the use of a serial dilution of DNA (A = 0.1 ng; B = 1 ng; C = 5 ng; D = 10 ng; E = 50 ng; F = 100 ng; G = 500 ng; H = 1 µg), the copy number of hMTIIa is shown to be similar in both cell lines when normalized to a control gene encoding for ß-globin.

 
Transfection efficiencies in both cell lines. To ensure that the extent of transfection in both parental cells and the cisplatin-resistant subline were comparable, we transiently transfected cells with the pCD3-TK plasmid and determined the relative copy number of the HSV-TK gene by quantitative PCR using HSV-TK primers. As shown in Fig. 3Go, A, varying amounts of DNA were analyzed in serial twofold dilutions, ensuring that amplification was in the exponential range. A twofold increase indicated that the amplification proceeded in the exponential range and that an accurate value could be obtained. The amounts used in this experiment were determined by use of carefully measured DNA concentrations to standardize for input DNA. As expected, a PCR product was not detected in DNA from control cells that had not been transfected, since HSV-TK is not a human gene. The HSV-TK PCR product in the pCD3-TK-transfected parental cell line was threefold higher than the PCR product with the use of DNA from transfected cisplatin-resistant cells. This result indicates comparable liposomal transfection of both plasmids, with a possibility of slightly more efficient transfection of the parental cell line.



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Fig. 3. A) Quantitative polymerase chain reaction (PCR) analysis of DNA from A2780(1A9) human ovarian carcinoma cells and their descendant, cisplatin-resistant A2780-E(80) cells untransfected and transfected with pCD3-TK plasmids that contain the herpes simplex virus-thymidine kinase (HSV-TK) gene driven by the constitutive cytomegalovirus (CMV) promoter. Genomic DNA was extracted 48 hours after the transfection. This composite demonstrates the PCR results of serial DNA dilution with the use of HSV-TK primers as described in the "Materials and Methods" section. The HSV-TK PCR product is threefold higher in transfected parental cells than in transfected cisplatin-resistant cells. B) Quantitative reverse-transcription (RT)–PCR of messenger RNA from A2780(1A9) and A2780-E(80) cell lines untransfected and transfected with pMT-TK that contains the HSV-TK gene driven by the human metallothionein IIa promoter (hMTIIa). Total cellular RNA was extracted 48 hours after the transfection of both cell lines with pMT-TK. Serial dilutions were prepared and assayed by RT–PCR by use of HSV-TK primers. The composite is organized as described in panel A. Values were standardized to a 28S ribosomal RNA control. Comparison of levels of HSV-TK expression showed the relative expression to be about 56-fold higher in the resistant cells, which overexpress metallothionein.

 


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Fig. 4. Effect of ganciclovir (GCV) concentration on cell survival. A2780(1A9) human ovarian carcinoma cells (A) and descendant A2780-E(80) cisplatin-resistant cells (B) were transfected with either a negative control plasmid pTK-ß-gal designed to express ß-galactosidase from the herpes simplex virus-thymidine kinase (HSV-TK) promoter, the positive control plasmid pCD3-TK, or the tested plasmid pMT-TK. Two days after transfection, cells were exposed for 7 days to a range of ganciclovir concentrations. Cell viability was assessed with the use of a tetrazolium salt assay in which MTT (i.e., 3-[4,5-dimethylthiazol-2-yl]-2–5-diphenyl tetrazolium bromide) is metabolized in live cells to form a formazan. Viability was reported relative to untreated cells as the mean with 95% confidence intervals of three independent experiments.

 
Transactivation of the hMTIIa promoter in cisplatin-resistant cells. The above results confirmed high levels of expression of the hMTIIa gene in the cisplatin-resistant cells, without gene amplification, and comparable transfection efficiencies. To investigate the possibility that the increased expression of the hMTIIa gene was a result of transactivation of the hMTIIa gene, we analyzed HSV-TK expression following transient transfection. After RNA isolation, quantitative RT–PCR was used to measure relative HSV-TK expression in RNA from pMT-TK-transfected resistant and parental cells. As previously described, varying amounts of input RNA were analyzed. The values were standardized to a 28S ribosomal RNA control. Expression of thymidine kinase was detected in both pMT-TK-transfected cell lines, with higher levels in the cisplatin-resistant subline A2780-E(80), even though the transfection efficiency was threefold higher in the parental cell line, as discussed above (Fig. 3Go, B). As expected, expression was not detected either in untransfected parental A2780(1A9) cells or in the untransfected A2780-E(80) cells. Relative levels of expression determined from the exponential portion of the quantitative PCR, following densitometry, showed the expression to be about 56-fold higher in the resistant cells, which overexpress metallothionein. Similarly, expression was increased 12-fold in an intermediate step in the selection, designated as the A2780-E(40) cell line, which is maintained in 40 µM cisplatin (data not shown). These results are consistent with enhanced transactivation of the hMTIIa promoter in the cisplatin-resistant subline compared with the parental line and support enhanced transactivation as a mechanism of hMTIIa overexpression in the resistant cells.

HSV-TK gene transfer and ganciclovir sensitivity. We next tested whether this preferential activation of the hMTIIa promoter could be exploited to preferentially target cisplatin-resistant cells for thymidine kinase-mediated toxicity. To that end, cells were transfected by use of lipofectamine with pMT-TK plasmid, which contains the HSV-TK gene under the control of the hMTIIa promoter, or with either one of two control vectors (pCD3-TK, a positive control in which the HSV-TK gene is under the control of the CMV promoter, or pTK-ß-gal, a negative control in which the ß-galactosidase gene is under the control of the HSV-TK promoter) (Fig. 4Go). No cytotoxicity was noted when cells transfected with pTK-ß-gal were treated with ganciclovir at concentrations up to 50 µg/mL. Transfection of resistant cells with pMT-TK or pCD3-TK resulted in marked sensitization to ganciclovir, with an IC50 of 20 µg/mL and 9 µg/mL, respectively. In contrast, transfections of the parental cells resulted in no sensitization to ganciclovir with pMT-TK (IC50 >200 µg/mL) but a high sensitivity with pCD3-TK (IC50 = 6 µg/mL).


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although conclusive proof is not yet available, substantial evidence exists suggesting that metallothioneins have an important role in cisplatin resistance. A previous study (9) has shown increased metallothionein expression in cisplatin-resistant cells compared with drug-sensitive parental cells. While the mechanisms responsible for the increased expression have not been fully elucidated, the observation that transcriptional induction of metallothionein is mediated by several metal-responsive elements in the 5` regulatory region suggests increased transcription as a possibility. Because of the latter possibility, in the present study, we used the hMTIIa promoter sequence to drive the expression of the HSV-TK suicide gene in transfected cells. Our results indicate that, after transfection, the cisplatin-resistant cells are more sensitive to ganciclovir.

As a first step, we investigated the expression of the hMTIIa gene in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. We found that cisplatin-resistant cells expressed very high levels of the hMTIIa gene. The cisplatin-resistant cells expressed similar levels in the presence or absence of drug, suggesting that the high levels of expression largely represented a constitutive change independent of drug in the medium. Because increased expression can result from either gene amplification or increased transactivation, we examined the relative copy number of the hMTIIa gene by PCR, and we found no difference in the copy number of the hMTIIa gene. In contrast, there was a 56-fold increase in the transcription of the HSV-TK gene driven by the hMTIIa promoter in the cisplatin-resistant cells compared with parental cells. This increase could not be due to a better transfection efficiency into the cisplatin-resistant cells, since we found by PCR that liposomal transfection of HSV-TK was comparable in both cell lines and even slightly more efficient in the parental cell line. Considered together, these observations suggested that the acquired, constitutive increase in expression in the resistant cells was a result of increased transcription.

Although we observed a low level of hMTIIa-driven HSV-TK transcription in parental cells, suggesting a low level of hMTIIa transactivation, this finding was consistent with previous reports describing constitutive expression of the metallothionein gene in most cell lines. Although we did not detect metallothionein expression with our RNase protection analysis, it could have been detected by more sensitive methods, such as PCR. When the pMT-TK gene was transfected into both parental and cisplatin-resistant cell lines, treatment with ganciclovir demonstrated that the cisplatin-resistant cells were more sensitive to ganciclovir.

Several groups have proposed the use of plasmid-, retrovirus-, or adenovirus-mediated delivery of prodrug-activating genes for the treatment of localized tumor deposits (32). Guided by previous experience in which liposome-encapsulated plasmid DNA was successfully delivered to a wide variety of cells, we used a similar strategy and complexed the pMT-TK plasmid with cationic lipids. Although the liposomal strategy resulted in a relatively low transfection efficiency, a deterrent to its use in ovarian carcinoma in vivo, one would anticipate that this obstacle could be overcome, at least in part, with the use of one of several recombinant adenoviral vectors that have been utilized for direct in vivo delivery of genes into a variety of organs (33).

The observation that we could obtain 80% cell killing even though only 20%–30% of cells were transfected can be attributed to the bystander effect (17), in which thymidine kinase-expressing cells can be toxic to nearby nonexpressing cells by transfer of the phosphorylated product through gap junctions (34). This amplification of the toxicity due to thymidine kinase expression may prove critical to its success in treating tumors. Although, theoretically, it could also put nearby normal cells at risk, present knowledge indicates that this risk is minimal because gap junctions between cancerous and normal cells have not been observed.

Although these experiments need to be validated in vivo, they offer a promising approach for the specific killing of cisplatin-resistant tumor cells. Because the hMTIIa promoter limits expression to cisplatin-resistant cells, delivery systems other than direct injection of the plasmid into the tumor could be utilized. For example, intraperitoneal administration could be used to treat cisplatin-resistant ovarian peritoneal carcinomatosis. Intraperitoneal administration might enable expression of the HSV-TK gene in cisplatin-resistant tumor cells infiltrating normal tissues, without affecting the surrounding cells. Although normal cells would also express the transgene, the modest level of metallothionein expression in normal tissues and their low rate of cell division should render them relatively immune to ganciclovir treatment.


    NOTES
 
Supported by the "Association pour la Recherche contre le Cancer (Villejuif, France)," ARC 1808. D. Vandier was supported by the "Ligue Départementale du Val-de-Marne Contre le Cancer (Créteil, France)."

We thank Dr. G. Lambert for critical reading of the manuscript and Zhirong Zhan for help with the polymerase chain reaction.


    REFERENCES
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received December 9, 1998; revised December 3, 1999; accepted January 21, 2000.


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