p53 and DNA Damage-Induced Apoptosis in Ovarian Tumour Cell Lines

McIlwrath, Amanda Jane (1997) p53 and DNA Damage-Induced Apoptosis in Ovarian Tumour Cell Lines. MSc(R) thesis, University of Glasgow.

Abstract

The A2780 human ovarian adenocarcinoma cell line was used to establish methodologies for the detection and quantitation of ionising radiation- and cisplatin- induced apoptosis. Detection of apoptosis in A2780 cells by field inversion gel electrophoresis of DNA was found to be reliable and accurate. Apoptosis was quantitated using fluorescent end-labelling of DNA fragments by TUNEL/PI staining followed by FACs analysis. Cells displaying apoptotic morphology (dense chromatin, pyknotic nuclei around the cell margin, apoptotic bodies) when viewed by confocal microscopy were found to be those which also displayed increased green fluorescence due to TUNEL staining. A2780 cells engaged apoptosis in response to DNA damage generated by both ionising radiation and cisplatin. The DNA of apoptotic A2780 cells degraded in a non- random manner to produce DNA fragments of two sizes: one of 50kbp and one of 300- 1000kbp. The DNA did not degrade further to produce internucleosommal fragments. Maximal apoptosis occurred at either 72h or 96h after both cisplatin and radiation. Ionising radiation-induced apoptosis was reduced in the A2780/cp70 cell line compared to A2780 cells. Cisplatin-induced apoptosis was reduced in nine out of ten cisplatin resistant cell lines derived in vitro from A2780 cells. A higher level of Bcl-2 was observed in A2780/cp70 and this may contribute to the lack of apoptosis seen in these cells. The role of p53 in ionising radiation- and cisplatin-induced apoptosis in A2780 cells was investigated using a dominant negative mutant TP53 (codon 143, val to ala)- transfected cell line, A2780/mp53. A2780/mp53 cells underwent less apoptosis than the vector-transfected control cell line after equal doses of cisplatin or ionising radiation. These results showed that DNA damage caused by cisplatin or ionising radiation resulted in cell death by apoptosis in A2780 cells which was reduced in resistant cell lines. In addition they indicate a role for functional p53 in the ionising radiation- and cisplatin-induced apoptotic responses of A2780 cells.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Additional Information: Adviser: Bob Brown
• Keywords: Oncology
• Date of Award: 1997
• Depositing User: Enlighten Team
• Unique ID: glathesis:1997-74877
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 15:43
• Last Modified: 27 Sep 2019 15:43
• URI: https://theses.gla.ac.uk/id/eprint/74877