Characterisation of Novel Genetic Suppressor Elements Conferring Resistance to Cisplatin

Spraggon, Carolyn Louise (1996) Characterisation of Novel Genetic Suppressor Elements Conferring Resistance to Cisplatin. MSc(R) thesis, University of Glasgow.

Abstract

Cisplatin is a chemotherapeutic agent which is thought to exert its cytotoxic effects by causing DNA damage in the form of crosslinks. The precise molecular mechanisms involved in the transduction of this damage to a cytotoxic outcome are as yet not fully characterised. Resistance to cisplatin is a major clinical problem making elucidation of its mechanisms of action and mechanisms of resistance to it an important area of investigation. Genetic suppressor elements (GSE's) are small, randomly generated fragments of DNA which are isolated by conferring a selectable phenotype when expressed in target cells. They are powerful tools in the elucidation of recessive mechanisms of resistance to drug treatment. The work presented here describes the characterisation of GSE's generated by DNasel digestion of equalised HeLa total cDNA library and isolated by Kirschling and Roninson (Chicago, USA) by cisplatin selection. Characterisation of GSE's generated from the random digestion of a p53 cDNA library are also included. The GSE's isolated by Kirschling and Roninson have been transfected into the human ovarian adenocarcinoma cell line, A2780, and selected using cisplatin. Further examination of these cell lines revealed that the line generated by transfection of GSE 7.10 showed a high level of resistance to cisplatin (2-3.8 fold) and a high level of cross-resistance to an additional DNA damaging agent, ionising radiation. I have shown, by PCR amplification, that the GSE 7.10 construct is present in these transfectants and confirmed by DNA sequencing that it is 100% identical to a 188bp fragment of the human phosphoglycerate mutase-B gene. Analysis of the cell cycle checkpoints of these transfectants in response to DNA damaging agents has revealed that GSE 7.10 abrogates the G1/S checkpoint in response to ionising radiation, and suggests a reduction in the same checkpoint arrest in response to cisplatin. I have also purified and sequenced four of the p53 cisplatin GSE's and confirmed that they are fragments of the p53 gene.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Additional Information: Adviser: Robert Brown
• Keywords: Pharmacology
• Date of Award: 1996
• Depositing User: Enlighten Team
• Unique ID: glathesis:1996-74865
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 15:46
• Last Modified: 27 Sep 2019 15:46
• URI: https://theses.gla.ac.uk/id/eprint/74865