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p73 is a candidate effector in the p53 independent apoptosis pathway of cisplatin damaged primary murine colonocytes

Sir Alastair Currie Cancer Research UK Laboratories, Division of Pathology, Molecular Medicine Centre, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK

Correspondence to:
Professor D J Harrison
CRUK Laboratories, Division of Pathology, University of Edinburgh, Teviot Place, Edinburgh, EH8 9AG, UK; David.Harrison{at}ed.ac.uk Aims: Colonocytes were derived from wild-type (wt) and p53 deficient mice to investigate p53 dependent and independent death pathways after cisplatin treatment, and the role of p53 in growth regulation of primary, untransformed epithelial cells.

Methods: Wt and p53 null colonocytes were exposed to cisplatin and DNA synthesis, apoptosis, and p53, p21, and p73 expression were investigated after six, 12, and 24 hours. Major p73 isoforms were identified by reverse transcription polymerase chain reaction (RT-PCR).

Results: Cisplatin treated wt cells exhibited cell cycle arrest, whereas p53 null cells continued to synthesise DNA, although both cell types died. Apoptosis was significantly higher in cisplatin treated wt and p53 null colonocytes than in controls at all timepoints, although apoptosis was lower in cisplatin treated p53 null colonocytes than in wt cells. p53 expression was upregulated in cisplatin treated wt colonocytes. p21 expression was high and remained unchanged in cisplatin treated wt cells, although it was reduced in the absence of p53. p73 was investigated because it could account for p53 independent p21 expression and p53 independent death. RT-PCR detected full length p73. p73 transcript levels remained unchanged, whereas p73 protein accumulated in the nucleus of cisplatin treated cells, irrespective of genotype.

Conclusions: p53 is essential for cell cycle arrest, but not apoptosis in primary murine colonocytes. Apoptosis is reduced in cisplatin treated p53 null cells. Nuclear accumulation of endogenous p73 after cisplatin treatment suggests a proapoptotic role for p73 in the absence of p53 and collaboration with p53 in wt colonocytes.

Keywords: p73; p53; primary colonocytes; cisplatin

Abbreviations: BrdU, 5'-bromo-2'-deoxyuridine; N, N-terminally truncated variant; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; RT-PCR, reverse transcription polymerase chain reaction; TA, transactivation competent; wt, wild-type