We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug. The Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation. This quantitation was done for total genomic lesions and at the level of individual genes. In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines. Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells. These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level. Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells. There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners. However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links. DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and b-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines. Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.The metal coordination complex cisplatin has been widely used in the treatment of ovarian, testicular, head and neck, bladder, and small-cell lung carcinomas (19). DNA is thought to be the most critical target for the antitumor action of cisplatin, although the compound can also react with other biologically important molecules such as RNA and proteins (25, 29). The major lesions produced by cisplatin are DNA intrastrand adducts (IA) between adjacent purines GpG or ApG. Minor lesions include IA between two guanine residues separated by one nucleotide residue (GpXpG), interstrand cross-links (ICL) between two adjacent guanine residues on opposite DNA strands, and DNA-protein crosslinks (13,27). The lesions of most therapeutic significance are thought to be IA and ICL.Platinum-based chemotherapy is the standard treatment in the initial therapy of advanced-stage ovarian cancer. Although up to 80% of patients will have an early favorable response to therapy, less than 20% of individuals will experience long-term disease-free survival (33). As a result of primary or acquired resistance, most ovarian cancer patients will eventually relapse with cisplatin-resistant disease (5). Attempts to overcome drug resistance are central to both clinical and basic molecular research ...

We have found that di-, tri-, tetra-, and hexa-9-acridinylamines are so efficiently associated with DNA during electrophoresis in polyacrylamide or agarose gels that they retard its migration. The retardation is roughly proportional to the reagent to base pair ratio, and the magnitude of the retardation indicates that a combined charge neutralization/helix extension mechanism is mainly responsible for the effect. Furthermore, DNA sequence dependent differences are observed. Thus, the pUC 19 restriction fragments (HaeIII or AluI), which in the native state comigrate upon gel electrophoretic analysis, could be separated in the presence of a diacridine, and specific DNA fragments responded differently to different diacridines. These results suggest that the effect also is due to a contribution from the DNA conformation and that the DNA conformation dynamics are influenced differently upon binding of different diacridines. We foresee three applications of this observation: (1) in analytical gel electrophoretic separation of otherwise comigrating DNA molecules, (2) in studies of polyintercalator-DNA interaction, and (3) in measurements of polyintercalator-induced DNA unwinding.

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Cisplatin is a chemotherapeutic agent known to cause DNA damage. The cytotoxicity of this drug is believed to result from the formation of DNA intrastrand adducts (IA) and DNA interstrand crosslinks (ICL). While there are many studies on DNA repair of cisplatin damage at the overall level of the genome in various human cell lines, there is little information on the gene-specific repair. In this report, we have measured the formation and repair of cisplatin induced DNA adducts in the dihydrofolate reductase (DHFR) and ribosomal RNA (rRNA) genes in three cell lines: normal human fibroblasts, Fanconi's anemia complementation group A (FAA) and Xeroderma pigmentosum complementation group A (XPA). It is generally thought that XPA cells lack nucleotide excision repair and that FAA cells are deficient in the repair of DNA ICL. We find that normal human fibroblast cells repair 84% of the ICL in the DHFR gene after 24 h, whereas XPA and FAA cell lines only repaired 32 and 50% of the ICL respectively. Furthermore, 69% of the cisplatin IA in the DHFR gene were repaired in 24 h in normal human fibroblasts compared to 22% for XPA and 24% for FAA cells. The repair of the rRNA gene was less efficient than in the DHFR gene, but the relative pattern between the different cell lines was similar to that of the DHFR gene. We thus find that FAA cells are deficient not only in the gene specific repair of cisplatin ICL, but also in the gene specific repair of the more common cisplatin IA. XPA cells are normally thought to be without any nucleotide excision repair capacity, but our data could support a slight ICL unhooking activity.

We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug. The resistant cell lines A2780/cp70 and 2008/c13*5.25 were compared with their respective parental cell lines, A2780 and 2008. Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation. This quantitation was done for total genomic lesions and at the level of individual genes. In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines. Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells. These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level. Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells. There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners. However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links. DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and delta-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines. Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.

We evaluated the formation and removal of (+)-3a,48-dihydroxy-la,2a-epoxy-1,2,3,4-tetrahydroben- 2). A similar strand bias for BPDE-induced mutations in the hypoxanthine phosphoribosyl transferase gene (HPRT) of human fibroblasts was described where 77% of the premutagenic guanine bases were also on this strand (3). Assuming that carcinogen modification of DNA in vivo occurs with similar frequency on both strands, several possible explanations for the apparent strand selectivity of induced mutations can be considered and tested. One explanation for the strand bias of mutations is that the selection for dihydrofolate reductase-deficient mutants is very stringent, causing mutations to occur on one strand preferentially (4). We recently devised a reversion assay to address this possibility (5). BcPHDE-induced forward mutations were transversions favored at triple purine sequences where the 5' base was altered (2). The assay used DHFRnonsense mutants that carry this target sequence on both strands at each of two stop codons, and all purine thymine mutations yield amino acid substitutions compatible with dihydrofolate reductase activity. Randomly induced mutations at these sites would produce a 2:1 ratio of substitutions on the nontranscribed versus the transcribed strand. Sequence analysis of 66 independent BcPHDE-induced revertants showed that the combined ratio of mutations affecting purines on the nontranscribed strand was 15:1. Hence, the strand bias here was independent of phenotypic selection.Analysis of induced mutations in rodent HPRT led to the suggestion that strand-biased mutations relate to the polarity of the marker gene with respect to DNA replication (6). Since strand-biased UV-induced mutations were found in the absence of DNA repair on the transcribed strand of HPRT (7), the misinsertion rate differences for replicative polymerases a and 6 may also contribute to the strand-bias phenomenon.

Abstract— Escherichia coli was transformed with a pUC 19 plasmid which contained a 4,5′,8‐trimethylpsoralen cross‐link specifically at position T410‐A411, and transformants in which the cross‐link had been repaired were scored on the basis of ampicillin resistance. Repair was only seen after induction of the SOS‐system by shortwave ultraviolet irradiation. The repair efficiency was 6%, and of 100 colonies analyzed only two was found to be mutated at the cross‐linked site: a T‐A transversion was observed at position 410. A repair mechanism which does not involve recombination is proposed.

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