P53 control over cell cycle progression at G2

• Main Author: Stewart, Nancy G.
• Language: en_US
• Published: 2007
• Online Access: http://hdl.handle.net/1993/1557

Tp53 (the human form) is the most commonly mutated tumour suppressor gene found in a large variety of cancers, and appears to be involved in several functions, including DNA damage response, related suppression of inappropriate cell growth, and apoptosis. Wild type p53 is capable of inducing cell cycle arrest at specific points in the cell cycle, an ability lost by most p53 mutants. The first block occurs at the restriction point before commitment to DNA replication (S). In this thesis, I examined whether p53 can also induce cell cycle arrest at the G2/M boundary of the cell cycle, and by what mechanism this is accomplished. To study this I used the REF52 cell line and the temperature sensitive p53 mutant, p53val$\sp{135}.$ Cells were enriched in S phase before the temperature shift. REF52 cells expressing mutant p53val135 alone or with activated H-ras gene arrest primarily at the G1/S and G2/M parts of the cell cycle at the restrictive temperature, as determined by flow cytometric analysis. This suggested that the antiproliferative activity of p53 may be involved in regulation of the cell cycle at the G2/M restriction point as well as transit through G1/S and initiation of DNA synthesis. I further demonstrated that Cdc2 kinase was inactive in p53 induced G2 arrested cells. G2 arrest appears to operate through a mechanism controlling the phosphorylation state of Cdc2. Cells arrested by p53 maintain phosphorylation of the regulatory tyrosine 15 amino acid on Cdc2 kinase. The levels of Cdc2 protein as well as the relevant cyclin, B1 were unaffected. Addition of caffeine, a known checkpoint inhibitor, both prior to and after establishment of G2 arrest can overcome this event, but not the G1 arrest. xamination of one Cdc2 kinase regulatory phosphatase, Cdc25C, showed the protein hypophosphorylated in p53 induced G2 REF52 arrested cells. This protein became phosphorylated upon addition and release by caffeine in these cells. From this I conclude the p53 induces arrest at G2 by regulation of phosphorylation and activation of Cdc2 kinase, and completion of the cell cycle.