DNA Damage Recognition Proteins and Their Involvement in Cisplatin Resistance

cis-Diamminedichloroplatinum(II) (CDDP) is a chemotherapeutic agent widely used in the treatment of various types of cancer. Its mechanism of cytotoxicity is unclear although it is believed that DNA is the critical target. CDDP binds to DNA forming a variety of adducts including intrastrand adducts, interstrand adducts, monofunctional adducts and DNA-protein crosslinks. This thesis presents evidence that there are protein(s) present in mammalian cells which recognise CDDP-damaged DNA, To assay for these DNA damage recognition proteins (DDRPs) conditions for two very separate assays were developed. The gel mobility shift assay, which detects protein complexes under non-denaturing conditions, identified two retardation complexes which bound to CDDP damaged DNA in human, murine and feline tumour cell extracts. Binding of these complexes is shown to CDDP treated oligonucleotide of 54 base pairs but not to a CDDP treated oligonucleotide of 27 base pairs, therefore suggesting binding is dependent on having normal DNA duplex. The other system used in the detection of the DDRPs is the South-Western assay. This allowed the detection of proteins of sizes 25, 50, 100KD binding to CDDP treated DNA. The proteins in the South-Western system are run under denaturing conditions. It is not entirely clear as to whether the proteins detected in both systems are the same or whether they represent entirely different species. CDDP has been reported to bind to DNA and cause areas of singlestrandedness around the adducts. The results presented in this thesis demonstrate that the 50KD and 100KD DDRP which bind to CDDP treated double-stranded DNA may also have an affinity for single-stranded DNA. The 25KD DDRP, however, only recognises double-stranded DNA treated with CDDP suggesting that it is recognising the CDDP adducts and not the areas of single-strandedness generated around the adducts. Resistance to CDDP proves a major problem area in treatment regimes. Many cell lines resistant to CDDP have been derived in vitro by multiple exposures to the drug. Many mechanisms of resistance to CDDP have been suggested from these lines. If a role of the DDRPs was to process damage in the DNA then cell lines resistant to CDDP may show an increase in expression of the DDRPs. This thesis presents evidence that an ovarian tumour cell line resistant to CDDP in comparison to its parental line shows an increase in the binding to the 50KD and 100KD DDRPs. Work in chapter 5 presents the isolation of CDDP resistant cell lines, by acute exposure to the drug, with an increase of up to seven fold resistance levels. Evidence is presented for the resistant clones being of a mutational origin. Resistant variants occur at a frequency of 3.2x10e-6 per viable cell. This frequency can be increased to 3.4x10e-5 by treatment of the cells with the chemical mutagen ethyl methane sulphonate, EMS. The CDDP resistant phenotype is maintained after six months growth in drug free medium. This single step selection may provide clones which are more clinically relevant than the lines isolated by multiple exposures to CDDP. They may therefore provide a superior model for the study of drug resistance mechanisms to CDDP. However examination of the DDRPs showed no detectable difference in the resistant clones derived from the A2780 human ovarian tumour cell line. The thesis therefore presents evidence of the existence of DDRPs in mammalian cells. The role of these damage recognition proteins will be discussed.