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We studied the DNA sequence of the entire coding region of ERCC1 gene, in five cell lines established from human Ž . ovarian cancer A2780, A2780rCP70, MCAS, OVCAR-3, SK-OV-3 , 29 human ovarian cancer tumor tissue specimens, Ž . Ž . one human T-lymphocyte cell line H9 , and non-malignant human ovary tissue NHO . Samples were assayed by Ž . PCR-SSCP and DNA sequence analyses. A silent mutation at codon 118 site for restriction endonuclease MaeII in exon 4 of the gene was detected in MCAS, OVCAR-3 and SK-OV-3 cells, and NHO. This mutation was a C ™ T transition, that Ž . codes for the same amino acid: asparagine. This transition converts a common codon usage AAC to an infrequent codon Ž . usage AAT , whereas frequency of use is reduced two-fold. This base change was associated with a detectable band shift on SSCP analysis. For the 29 ovarian cancer specimens, the same base change was observed in 15 tumor samples and was associated with the same band shift in exon 4. Cells and tumor tissue specimens that did not contain the C ™ T transition, did not show the band shift in exon 4. Our data suggest that this alteration at codon 118 within the ERCC1 gene, may exist in platinum-sensitive and platinum-resistant ovarian cancer tissues. q 1997 Elsevier Science B.V.

We assessed the possible role of the human repair genes, ERCC1 and ERCC3, in resistance to cisplatin-induced cytotoxicity. The UV repair-deficient Chinese hamster ovary (CHO) 43:3B [designated ERCC1(-)] cell line and its paired subline 83-J5, which is stably transfected with the human DNA excision repair gene ERCC1 [designated ERCC1(+)], were used in this study. UV repair-deficient CHO 27-1 cells [designated ERCC3(-)] and its paired subline designated 'ERCC3(+)', which is stably transfected with the human DNA excision repair gene ERCC3, were also used. In each pair of cell lines, we assessed cisplatin cytotoxicity, cellular drug accumulation and platinum-DNA adduct repair after 1 h drug exposures. Drug accumulation and DNA repair were assessed by atomic absorption spectrometry with Zeeman background correction. ERCC1(+) cells (IC50 = 4.0 microM) were 5-fold more resistant to cisplatin than ERCC1(-) cells (IC50 = 0.75 microM). ERCC1(+) cells repaired 25% of DNA lesions in cellular DNA within a 6 h time period following an IC50 drug exposure and repaired 48% over 24 h. No DNA repair was observed in ERCC1(-) cells during the same time periods. Both cell lines showed similar patterns of drug accumulation. For ERCC3(-) cells (IC50 = 54 microM) and ERCC3(+) cells (IC50 = 49 microM), the profiles of cisplatin sensitivity and cellular drug accumulation were similar. When treated with 50 microM cisplatin, these cells showed similar patterns of drug accumulation, and were equally efficient at forming and repairing lesions in cellular DNA. These data show that in UV repair-deficient CHO cells, ERCC1 confers resistance to cisplatin and confers the ability to remove platinum from cellular DNA. In contrast, ERCC3 does not influence cisplatin drug sensitivity or adduct repair capability. This suggests that ERCC1 may be a determinant of cisplatin resistance, whereas ERCC3 is probably not.

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