Abstract
Cisplatin-resistant A2780DDP human ovarian carcinoma cells, 1 and 3 h following cisplatin treatment, display 3.2-6.1-fold enhancement of mRNA expression for oncogenes c-fos and c-H-ras and for enzymes necessary for DNA synthesis and repair. In contrast, c-myc mRNA was partially decreased. Increased transcription for the aforementioned genes accounts for the higher levels of mRNA since the enhancement is susceptible to actinomycin D pretreatment. Cyclosporin A (CSA) has been shown to reverse resistance to a variety of antineoplastic agents. An 18-h treatment with CSA yielded a 6-fold improved sensitivity to cisplatin in A2780DDP cells. When A2780DDP cells are pretreated with CSA followed by cisplatin, there is no increase in mRNA for c-fos and c-H-ras and the genes for thymidine synthesis and DNA repair. Their expression approaches the levels demonstrated in A2780S cells. In contrast, c-myc mRNA was elevated 7.4-fold in the presence of CSA in A2780DDP cells. Long term weekly exposures of A2780DDP cells to CSA resulted in the evolution of a revertant cell line, A2780DDP/CSA, with decreased c-fos and dTMP synthase mRNA. Thus, cisplatin treatment in A2780DDP cells differentially induces expression of certain nuclear oncogenes (c-fos/c-myc), as well as genes possibly involved in the repair of cisplatin-mediated DNA damage, an induction suppressed by CSA treatment.
Highlights
A2780DDP human ovarian carcinoma cells, 1 and 3 h following cisplatin treatment, display 3.2-6.1-fold enhancement of mRNA expression for oncogenes c-fos and c-H-r-as and for enzymes necessary for DNA synthesis and repair
We have demonstrated that Cyclosporin A (CSA) corrects resistance to cisplatin and in so doing alters the expression of oncogenes and the DNA synthesis genes
At the 3-h mark, the expression of enzymes involved in dTTP synthesis, and DNA synthesis and repair (DNA pol /3) in A2780DDP cells was elevated 3.2.fold over control levels (Fig. 1, lanes I and 2)
Summary
?;he phosphoglycerate kinase gene was obtained from Dr J. CDNAs were obtained from American Type Culture Collection (Rockville, MD). Ml) for 8 weeks in the absence of cisplatin treatment The cells were washed, and fresh medium was added to the dishes. Further quantitation of the RNA samples was done by ethidium bromide staining of the gels and reprobing the filters with the phosphoglycerate kinase gene [4]. The entire sequence of experiments (including growth of A2780DDP cells, drug treatment, and Northern blotting and hybridization) was performed and the results reproduced in more than three separate experiments. Quantitation of the Northern filters was performed by Radioanalytic Imaging Systems, Ambis, San Diego, CA
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