The human ovarian cell line A2780 was exposed to either cisplatin (10 ,uM) or 5-fluorouracil (5FUra) (5 ,uM) for 1 hr. Cytotoxicity was less than 14% with either agent alone. Cisplatin (10 j#M) and 5FUra (5 jzM) in combination for 1 hr caused a 76% reduction in cell growth. Thymidine (dThd, 10 jM), if given concomitantly with the combination of cisplatin and 5FUra. completely protected the tumor cells. A 30-min exposure to cisplatin increased the intracellular pools of 5,10-methylenetetrahydrofolate and tetrahydrofolate 2.5-fold.The capacity of intact cells to form 5-fluorodeoxyuridylate (FdUMP)-thymidylate (dTMP) synthase complex when incubated with fluorodeoxyuridine (FdUrd) was enhanced 2.5-fold when the cells were pretreated with cisplatin. These experiments demonstrate that cisplatin can increase the availability of the reduced folate necessary for tight binding of FdUMP to dTMP synthase, thus enhancing the cytotoxicity of the cisplatin and 5FUra combination.5-Fluorouracil (5FUra) and cis-diamminedichloroplatinum-(II) (cisplatin) in combination have been shown to have synergistic cytotoxicity against both murine and human neoplasms (1, 2). The mechanism ofthis synergy has not been elucidated. SFUra is metabolized within cells to 5-fluorodeoxyuridine monophosphate (FdUMP). FdUMP can covalently bind to dTMP synthase in the presence of 5,10-methylenetetrahydrofolate (CH2-H4folate) and inhibit DNA synthesis by depleting the cells of dTMP. 5FUra is also metabolized to 5-fluorouridine triphosphate (FUTP), which is incorporated into RNA. One or both of these 5FUra metabolites account for the antineoplastic activity of SFUra in experimental models (3). The antitumor activity of the second agent, cisplatin, may be explained in part by DNA crosslinking (4, 5), interactions with the cell-surface nucleic acids (6) or with the plasma membrane (7-9). Also, cisplatin can inhibit methionine uptake into tumor cells and cause perturbation of the methionine pools (7-9). Cells may respond by increasing methionine biosynthesis and increasing the pools of folate cofactors (10). The interaction of these two chemotherapeutic drugs was studied in patients with human ovarian cancer (11). Data presented in this report suggest that cisplatin treatment does increase the CH2-H4folate pool and the binding of FdUMP to dTMP synthase. The resultant inhibition of dTMP synthesis appears to be responsible for the cytotoxicity of the SFUra and cisplatin combination. MATERIALS AND METHODSThe following chemicals were obtained from these companies: thymidine, Sigma; SFUra, Hoffmann-La Roche; deoxyuridine, P-L Biochemicals; cisplatin and 5-fluorodeoxy- Cell Culture. The human ovarian cancer cell line A2780 was obtained from R. Ozols, National Cancer Institute (12) and maintained in RPMI 1640 with 10% (vol/vol) fetal calf serum, penicillin (62.9 mg/liter) and streptomycin (100 mg/ liter) (GIBCO). Cells were exposed to chemotherapeutic agents for only 60 min in RPMI 1640 medium and 10% (vol/vol) fetal calf serum and then washed three times wi...
The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A278ODDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos-and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase (3, topoisomerase I, and metallothionein HA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A278ODDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase (3, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.
In this study a ribozyme (catalytic RNA) was designed to site specifically cleave the mRNA of the activated H-ras gene expressed in human bladder carcinoma EJ cells. The optimal conditions for catalytic cleavage by the ribozyme were demonstrated in vitro. A synthetic DNA encoding the ribozyme was cloned into a mammalian expression vector (pH beta APr-1) and transfected into EJ cells. The expressed ribozyme significantly altered the morphology and suppressed the growth of EJ cells in vitro. These cell lines were examined for their malignant potential in athymic (nude) mice by an orthotopic (transurethral) implantation model, which recapitulates the invasive potential of various bladder carcinomas. EJ tumors expressing the H-ras ribozyme were characterized by a marked reduction in tumor take and invasion compared to those formed by control EJ cells. These differences resulted in almost a twofold increase in survival of mice implanted with ribozyme-containing EJ cells. These results further elucidate the role of ras genes in tumorigenicity and invasion, as well as introduce ribozymes as a new class of anticancer agents.
Scientists have been working on strategies to selectively turn off specific genes in diseased tissues for the past thirty years. In the 1980's, oligodeoxynucleotides (ODNs) with unique chemistries were tested with model systems both in vitro and in vivo with varying degrees of success. In the 1990's, ribozymes with both antisense and catalytic properties were successfully introduced to the field. Ribozymes were shown to selectively knock down targeted genes in human tumors grown in mice but delivery issues for these therapeutic anti-genes limited their clinical utility. Short interfering RNA (siRNA) is currently the fastest growing sector of this anti-gene field for target validation and therapeutic applications. The siRNA field may have an opportunity to impact the clinic faster than antisense and ribozymes if the scientists can overcome the previous anti-gene limitations. Fortuitously, there have been a several developments involving the expansion of our genomic knowledge coupled with the rapid dissemination of disease genes by the digital revolution. This convergence of the knowledge of the human genome with the speed of digital communication will help facilitate swift changes in the detection and treatment of human illnesses. The anti-gene field is positioned to exploit this timely union of two distinct technologies. Anti-gene molecules have an opportunity to become a successful technology in understanding the human genome, as well as, enabling the development of efficacious gene therapy for human diseases in the near future. This review will characterize the advances in this field and address the challenges to the success of for the anti-gene technology.
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