Mohammed Kashani-Sabet

Therapeutic applications of ribozymes.

The demonstration that RNA can be cleaved by cis or trans ribozymes (catalytic RNAs, RNA enzymes) has potentially important therapeutic implications. Since their discovery in the 1980s, the biochemistry and conserved sequences of ribozymes have been well characterized. Ribozymes are effective modulators of gene expression because of their simple structure, sitespecific cleavage activity, and catalytic potential. The targets of ribozyme-mediated gene modulation have ranged from cancer cells to foreign genes that cause infectious diseases. Additional target sites for ribozymes are in initial phases of development and design. Ribozymes have been targeted against a myriad of genes, including oncogenes (ras, BCR-ABL, c-fos) and drug resistance genes, as well as the human immunodeficiency virus-type I genome. These ribozymes have cleaved the target RNAs in vitro and altered the cellular pathology. Currently, the therapeutic application of ribozymes to human diseases is limited by gene transfer systems. It is anticipated that ribozymes ultimately will play an important role in human gene therapy.

OLIGONUCLEOTIDE-MEDIATED MODULATION OF MAMMALIAN GENE EXPRESSION

The notion that oligonucleotides can modulate gene‐specific expression was established more than a decade ago. Recent advances in molecu‐lar genetics have broadened the armamentarium used to manipulate gene expression in biological systems including triplex DNA, antisense RNA/DNA, and ribozymes (catalytic RNA). These oligonu‐cleotides demonstrated important early application to the elucidation of cellular signaling pathways. More recently, studies with these agents have probed their utility as potential therapeutic agents, especially in the realm of cancer. With the implementation of gene therapy in early clinical trials, oligonucleotide‐ mediated suppression of gene expression has emerged as an important strategy for gene therapy. This review will discuss the current knowledge in this field, focusing on the biology of triplex DNA, antisense oligonucleotides, and ribozymes.—Scanlon, K. J., Ohta, Y., Ishida, H., Kijima, H., Ohkawa, T., Kaminski, A., Tsai, J., Horng, G., Kashani‐Sabet, M. Oligonucleotide‐mediated modulation of mammalian gene expression. FASEB J. 9, 1288‐1296 (1995)

Cisplatin resistance in human cancers

Cancer chemotherapeutic agents primarily act by damaging cellular DNA directly or indirectly. Tumor cells, in contrast to normal cells, respond to cisplatin with transient gene expression to protect and/or repair their chromosomes. Repeated cisplatin treatments results in a stable resistant cell line with enhanced gene expression but lacking gene amplification for the proteins that will limit cisplatin cytotoxicity. Recently, several new human cell lines have been characterized for cisplatin resistance. These cell lines have led to a better understanding of the molecular and biochemical basis of cisplatin resistance. The c-fos proto-oncogene, a master switch for turning on other genes in response to a wide range of stimuli, has been shown to play an important role in cisplatin resistance both in vitro and in patients. Based on these studies, new strategies have been developed to circumvent and/or exploit clinical cisplatin resistance.

The utility of an antifos ribozyme in reversing cisplatin resistance in human carcinomas

The results presented here demonstrate that expression of a fos ribozyme limits Fos protein synthesis and enhances sensitivity of A2780DDP cells to antineoplastic agents, including cisplatin. Moreover, the reversal of this resistance is associated with down-regulation of dTMP synthase, DNA polymerase beta, topoisomerase I and hMTII-A, genes previously linked to DNA synthesis and repair. Thus these studies further implicate the role of the c-fos gene in DNA synthesis through modulation of expression of dTMP syntase, DNA polymerase beta and topoisomerase I. Finally, the use of ribozymes to circumvent drug resistance suggests their potential utility as agents to inhibit tumor cell growth.

Differential oncogene amplification in tumor cells from a patient treated with cisplatin and 5-fluorouracil

Peritoneal cells were derived from a patient (PK) with adenocarcinoma of the colon during the course of cisplatin/5-fluorouracil (5-FUra) treatment. Resistance to cisplatin and 5-FUra, characterized by a lack of response to chemotherapy and continued growth of the tumor, was concomitantly associated with a 2-4-fold increase in DNA copy number for dTMP synthase and dihydrofolate reductase. There was a corresponding amplification in DNA copy number of the c-myc (2X), H-ras (4X), and c-fos (15X) oncogenes. Cytogenetic studies revealed an iso (13q) chromosome, but failed to show any double minutes or homogeneously staining regions. In addition, drug-resistant tumor cells from PK and another patient (HG) displayed enhanced expression of dTMP synthase, c-fos and DNA polymerase beta when compared to normal colon tissue and the HCT8 human colon carcinoma cell line. These results suggest that elevated oncogene DNA and gene expression may be involved in the development of cisplatin resistance.

Suppression of H-ras-mediated transformation in NIH3T3 cells by a ras ribozyme

Murine NIH3T3 cells were used to study the effect of ribozymes on H-ras-mediated transformation. Parental 3T3 cells were transfected with the activated H-ras gene. H-ras-transformed cells had altered morphology and increased colony formation in soft agar in contrast to untransfected 3T3 cells. A hammerhead ribozyme (site-specific ribonuclease) designed to cleave codon 12 (GUC) of the activated H-ras RNA was expressed in transformed cells. 3T3 clones expressing the ras ribozyme displayed decreased expression of activated H-ras RNA. The ras ribozyme reversed the transformed phenotype to resemble that of untransfected 3T3 cells. Furthermore, 3T3 cells containing the ras ribozyme were shown to suppress transformation when they were subsequently transfected with activated H-ras. Insertion of a mutant ribozyme largely devoid of cleaving capacity into H-ras-transformed cells resulted in smaller reductions in H-ras gene expression and colony formation in soft agar when compared with the ras ribozyme. Finally, the ras ribozyme alone did not perturb normal 3T3 cell growth. This study suggests the possible utility of anti-oncogene ribozymes as suppressors of tumor cell growth as well as inhibitors of cellular transformation.

Mechanisms of cross-resistance to methotrexate and 5-fluorouracil in an A2780 human ovarian carcinoma cell subline resistant to cisplatin☆

Some properties of the human ovarian carcinoma line A2780 and a subline three times more resistant than the parent line to cisplatin are compared in this report. The rates of uptake and release of cisplatin were similar in the two cell lines. Resistance to cisplatin was associated with: (a) cross-resistance to 5-fluorouracil and methotrexate; (b) a 2.5-fold increase in thymidylate synthase, as measured by both enzyme activity and the capacity to complex 5-fluorodeoxyuridylate; and (c) an increase in the intracellular pools of 5,10-methylenetetrahydrofolate and tetrahydrofolate. These data suggest that cross-resistance to 5-fluorouracil and methotrexate in A2780 cells may be a consequence of increases in their respective target enzymes.

Inhibition of methionine uptake by methotrexate in mouse leukemia L1210 cells

SummaryMethionine-auxotrophic L1210 cells were used to study the effect of methotrexate (MTX) on methionine utake and metabolism. MTX was shown to inhibit amino acid transport systems and cause a decrease of methionine uptake into L1210 cells. Conversely, a nonmetabolizable amino acid analogue reduced MTX uptake into L1210 cells. MTX also blocked the transfer of the beta carbon from serine into methionine. Therefore, methionine deprivation may be an additional mechanism of action for MTX in methionine-auxotrophic tumor cells.

The Role of the c-fos Oncogene in Cisplatin Resistance

The c-fos oncogene has been proposed to play an important role not only in DNA synthesis (1) but also in resistance to the antineoplastic agent cis-diamminedichloroplatinum (cisplatin) (2). Administration of cisplatin to A2780 ovarian carcinoma cells was followed by a sequential induction of c-fos, dTMP synthase, and DNA polymerase. A2780 cells transfected with a plasmid containing the fos gene displayed morphological and pharmacological changes reflecting cisplatin resistance. A ribozyme (catalytic RNA) was designed to cleave c-fos mRNA. Cleavage conditions were optimized in vitro, and DNA encoding the fos ribozyme was cloned into the pMAMneo expression vector. The resultant plasmid was transfected into the A2780DDP cell line resistant to cisplatin, and the transformants assayed for enhanced sensitivity to cisplatin. These results suggest that c-fos plays a causal role in cisplatin resistance.

The role of methionine in methotrexate-sensitive and methotrexate-resistant mouse leukemia L1210 cells

SummaryA mouse L1210 leukemia cell line was made 25-fold resistant to methotrexate (MTX) and had altered methionine transport and metabolism. L1210 cells resistant to methotrexate also had a 50-fold decrease in the exogenous methionine requirement for optimal cell growth compared to the parent cells. This change in methionine requirement was associated with differences in methionine metabolism between MTX-sensitive and MTX-resistant cell lines. Analysis of amino acid transport systems revealed different K1 and Vmax properties of methionine and nonmetabolizable amino acid analogues. There was a greater than twofold decrease in the initial sodium-dependent uptake of methionine in the resistant cells. Amino acid competition experiments revealed altered substrate specificities in the resistant cells. The cellular alterations occurring upon resistance may result from methotrexatemembrane interactions, and have been previously observed in cisplatinum-resistant cells. Thus modulation of methionine metabolism may provide the biochemical basis for MTX and cisplatinum collateral resistance.