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).
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ABSTRACT |
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INTRODUCTION |
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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 (67 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).
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MATERIALS AND METHODS |
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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 BglIIPvuII 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) HindIIIBamHI 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 23 hours, followed by autoradiography for 1 day (26).
Preparation and administration of liposomenucleic 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 TrisHCl (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 TrisHCl (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 doseresponse curves related to histograms (see Fig. 4) (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.
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RESULTS |
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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. 4). 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).
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DISCUSSION |
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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.
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NOTES |
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We thank Dr. G. Lambert for critical reading of the manuscript and Zhirong Zhan for help with the polymerase chain reaction.
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REFERENCES |
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1
Parker SL, Tong T, Bolden S, Wingo PA. Cancer statistics, 1996. CA Cancer J Clin 1996;46:527.
2 Ozols RF, Young, RC. Ovarian cancer: where to next? Semin Oncol 1991;18:30710.[Medline]
3 Fram RJ. Cisplatinum and platinum analogues: recent advances. Curr Opin Oncol 1992;4:10739.[Medline]
4 Andrews PA, Howell SB. Cellular pharmacology of cisplatin: perspectives on mechanisms of acquired resistance. Cancer Cells 1990;2:3543.[Medline]
5 Koropatnick J, Kloth DM, Kadhim S, Chin JL, Cherian MG. Metallothionein expression and resistance to cisplatin in a human germ cell tumor cell line. J Pharmacol Exp Ther 1995;275:16817.[Abstract]
6 Timmer-Bosscha H, Mulder NH, de Vries EG. Modulation of cis-diamminedichloroplatinum (II) resistance: a review. Br J Cancer 1992;66:22738.[Medline]
7 Vikhanskaya F, Clerico L, Valenti M, Stanzione MS, Broggini M, Parodi S, et al. Mechanism of resistance to cisplatin in a human ovarian-carcinoma cell line selected for resistance to doxorubicin: possible role of p53. Int J Cancer 1997;72:1559.[Medline]
8 Dong Y, Berners-Price SJ, Thorburn DR, Antalis T, Dickinson J, Hurst T, et al. Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy. Biochem Pharmacol 1997;53:167382.[Medline]
9 Kelley SL, Basu A, Teicher BA, Hacker MP, Hamer DH, Lazo JS. Overexpression of metallothionein confers resistance to anticancer drugs. Science 1988;241:18135.[Medline]
10 Lazo JS. Metallothioneins and cisplatin resistance. In: Howell SB, editor. Platinum and other metal coordination compounds in cancer chemotherapy. New York (NY): Plenum Press; 1991. p. 31522.
11 Mathe G, Kadani Y, Segiguchi M, Eriguchi M, Fredj G, Peytavin G, et al. Oxalato-platinum or 1-OHP, a third-generation platinum complex: an experimental and clinical appraisal and preliminary comparison with cis-platinum and carboplatinum. Biomed Pharmacother 1989;43:23750.[Medline]
12 Rixe O, Ortuzar W, Alvarez M, Parker R, Reed E, Paull K, et al. Oxaliplatin, tetraplatin, cisplatin and carboplatin: spectrum of activity in drug resistant cell lines and in the cell lines of the National Cancer Institute's Anticancer Drug Screen Panel. Biochem Pharmacol 1996;52:185565.[Medline]
13 Kelland LR, Misitry P, Abel G, Loh SY, O'Neill CF, Murrer BA, et al. Mechanism-related circumvention of acquired cis-diamminedichloroplatinum (II) resistance using two pairs of human ovarian carcinoma cell lines by amine/amine platinum (IV) dicarboxylates. Cancer Res 1992;52:385764.[Abstract]
14 O'Dwyer PJ, Hamilton TC, LaCreta FP, Gallo JM, Kilpatrick D, Halbherr T, et al. Phase I trial of buthionine sulfoximine in combination with melphalan in patients with cancer. J Clin Oncol 1996;14:24956.[Abstract]
15 Kojima M, Kikkawa F, Oguchi H, Mizuno K, Maeda O, Tamakoshi K, et al. Sensitization of human ovarian carcinoma cells to cis-diamminedichloroplatinum (II) by amphotericin B in vitro and in vivo. Eur J Cancer 1994;30:7738.
16 Blaese RM, Culver KW, Ishii H, Oldfield EH, Ram Z, Wallbridge S. Brain tumor treatment: significant contributions. Science 1992;258:1960.[Medline]
17 Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 1992;256:15502.[Medline]
18 Berger MS, Prados M, Van Gilder J, Warnick RE, Link CJ, Marcus SG, et al. Gene therapy for treatment of recurrent glioblastoma multiforme with in vivo transduction using the herpes simplex-thymidine kinase gene/ganciclovir system [abstract]. J Neurosurg 1997;86:378A.
19 Huber BE, Richards CA, Krenitsky TA. Retroviral mediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy. Proc Natl Acad Sci U S A 1991;88:803943.[Abstract]
20 Vile RG, Hart IR. Use of tissue-specific expression of the herpes simplex virus thymidine kinase gene to inhibit growth of established murine melanomas following direct intratumoral injection of DNA. Cancer Res 1993;53:38604.[Abstract]
21 Ring CJ, Harris JD, Hurst HC, Lemoine NR. Suicide gene expression induced in tumour cells transduced with recombinant adenoviral, retroviral and plasmid vectors containing the ERBB2 promoter. Gene Therapy 1996;3:1094103.[Medline]
22 Stuart GW, Searle PF, Chen HY, Brinster RL, Palmiter RD. A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci U S A 1984;81:731822.[Abstract]
23 Vandier D, Rixe O, Brenner M, Gouyette A, Besnard F. Selective killing of glioma cell lines using an astrocyte-specific expression of the herpes simplex virus-thymidine kinase gene. Cancer Res 1998;58:457780.[Abstract]
24 McKnight SL. The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase. Nucleic Acids Res 1980;8:594964.[Abstract]
25 Karin M, Haslinger A, Holtgreve H, Cathala G, Slater E, Baxter, JD. Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5`-flanking DNA. Cell 1984;36:3719.[Medline]
26 Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 1984;12:703556.[Abstract]
27 Zerrouqi A, Rixe O, Ghoumari AM, Yarovoi SV, Mouawad R, Khayat D, et al. Liposomal delivery of the herpes simplex virus thymidine kinase gene in glioma: improvement of cell sensitization to ganciclovir. Cancer Gene Ther 1996;3,6:38592.[Medline]
28 MacGregor GR, Nolan GP. Use of E. coli lacZ (ß-galactosidase) as a reporter gene, chap. 17. Methods in molecular biology. Vol 7. In: Murray EJ, Walker JM, editors. Gene transfer and expression protocols. Clifton (NJ): Humana Press; 1991. p. 21735.
29 Murphy LD, Herzog C, Rudick J, Fojo T, Bates SE. Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 1990;29:103516.[Medline]
30 Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, et al. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 1988;48:589601.[Abstract]
31 Schilder RJ, Hall L, Monks A, Handel LM, Fornace AJ Jr, Ozols RF, et al. Metallothionein gene expression and resistance to cisplatin in human ovarian cancer. Int J Cancer 1990;45:41622.[Medline]
32
Roth JA, Christiano RJ. Gene therapy for cancer: what have we done and where are we going? J Natl Cancer Inst 1997;89:2139.
33 Rosenfeld ME, Feng M, Michael SI, Siegal GP, Alvarez RD, Curiel DT. Adenoviral-mediated delivery of the herpes simplex virus thymidine kinase gene selectively sensitizes human ovarian carcinoma cells to ganciclovir. Clin Cancer Res 1995;1:157180.[Abstract]
34
Mesnil M, Piccoli C, Tiraby G, Willecke K, Yamasaki H. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins. Proc Natl Acad Sci U S A 1996;93:18315.
Manuscript received December 9, 1998; revised December 3, 1999; accepted January 21, 2000.
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