DNA Damage Recognition and p53 in Cisplatin Resistance

Edlin, Angela Rosalyn Margaret (1995) DNA Damage Recognition and p53 in Cisplatin Resistance. PhD thesis, University of Glasgow.

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Abstract

Cis-dichlorodiammineplatinum(II) (cisplatin) is widely used as an anti-cancer agent to treat various tumours particularly ovarian, testicular, bladder cancers and tumours of the head and neck. Although the precise mechanism of cisplatin-mediated cytotoxicity remains elusive, DNA damage is thought to be the therapeutically important interaction. Cisplatin resistance is an obstacle to effective treatment of cancer patients. Such resistance may arise due to a variety of intracellular changes including enhanced repair of DNA lesions caused by cisplatin and reduced susceptibility to DNA damage-induced apoptosis. The tumour suppressor protein, p53 is required for DNA damage-induced apoptosis in certain cell types. Cellular levels of p53 protein are increased subsequent to DNA damage due to post-translational alterations in p53. Induction of p53 protein levels by 1muM cisplatin or transplatin peaks at 24 hours for the cisplatin sensitive human, ovarian adenocarcinoma cell line, A2780. Levels of p53 protein increase in the cisplatin resistant derivative, A2780cp70 for at least 48 hours following treatment with transplatin and at least 72 hours following treatment with cisplatin. Also, certain cisplatin resistant cell lines contain higher basal levels of p53 protein compared to the cisplatin sensitive parental cells from which they were derived. Results of Western blotting experiments show an 8 fold higher p53 concentration in the A2780cp70 cell line than A2780, the cisplatin sensitive parental line. This difference in p53 protein levels does not reflect a mutation of the p53 gene, as direct PCR sequencing of p53 from A2780 and A2780cp70 showed both lines to have a wild-type p53 sequence. Furthermore, the p53 from A2780cp70 has less cross-reactivity with mutant-specific anti-p53 antibodies in immunoprecipitation experiments than cell lines with known mutations of the p53 gene. No evidence for increased MDM2 expression is observed in A2780cp70 suggesting that the p53 protein is not being stabilised by binding to the MDM2 protein in this cell line. Although increased levels of Hsp70 protein are observed in these cells, this may simply reflect a lack of transcriptional repression of the Hsp70 promoter in these cells due to the p53 being non-functional. Altered p53-mediated transcriptional transdiCivwdXion in A2780cp70 was examined by use of a luciferase reporter construct and by Northem blotting of endogenous genes, known to be transcriptionlly activated by p53. A2780cp70 cells have 10 fold lower WAF-1 mRNA levels than A2780 cells indicating reduced p53 transcriptional activity in the resistant cell line. Introduction of a mutant p53 gene into A2780 cells also provokes a reduction in basal p53 levels with a 3 fold decrease detected. Reduced function of p53 in the A2780cp70 cell line would be consistent with reduced entry to the apoptotic pathway in these cells. This may be one reason why A2780cp70 cells are more cisplatin resistant than A2780 cells. The DNA damaging agents, cisplatin and transplatin, induced WAF-1 mRNA levels 4 fold by 24 hours after drug removal in A2780 cells. Ionising radiation provoked a more rapid induction of 4.5 fold by 4 hours after drug removal in A2780 cells. This is consistent with induced p53 protein levels detected in A2780 after similar treatments. A2780cp70 cells, by comparison, showed only slight induction of WAF-1 following treatment with these agents. Clonogenic assay data shows that a pretreatment with cisplatin does not alter sensitivity to a second cisplatin dose. Thus intracellular alterations evoked by cisplatin treatment are not involved in a protective response. DNA damage recognition proteins (DRPs) have been proposed to have a role in drug resistance, possibly by an involvement in DNA repair although other functions are possible. Partial purification of a DRP which binds to cisplatin-damaged DNA was carried out. Using nuclear proteins, soluble in 2% tri-carboxylic acid for anion exchange chromatography and heparin column chromatography, this DRP was purified 183 fold. Using recombinant HMG proteins and using anti-HMG protein antibodies to retard protein-DNA complexes in the gel mobility shift assay, the DRP activity was shown to be identical to HMGl. Evidence is also presented showing that p53 does not directly bind to regions of cisplatin-induced DNA damage. However this does not eliminate a possible link between DRP-mediated DNA damage recognition and the p53 response to DNA damage.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Robert Brown
Keywords: Biochemistry, Pharmacology
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-74930
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:07
Last Modified: 27 Sep 2019 15:07
URI: https://theses.gla.ac.uk/id/eprint/74930

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