p73alpha is a candidate effector in the p53 independent apoptosis pathway of cisplatin damaged primary murine colonocytes

Abstract

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 p73alpha. 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 p73alpha in the absence of p53 and collaboration with p53 in wt colonocytes.

Figure 1

5′-Bromo-2′-deoxyuridine (BrdU) incorporation into untreated and cisplatin treated wild-type (wt) and p53 null colonocytes. Cells were incubated with BrdU for four hours and immunodetection was performed using rat anti-BrdU antibody. Positive cells were detected by immunocytochemistry and a total of 500 cells counted in triplicate in at least three different experiments. Results are mean (SEM), and show a higher BrdU incorporation in p53 null colonocytes, regardless of treatment and timepoint (p  =  0.015, ANOVA).

Figure 2

Apoptosis in wild-type (wt) and p53 null colonocytes exposed to cisplatin. The Feulgen stain and a 0.3% light green counterstain were used to assess apoptosis in cisplatin treated cells and their corresponding untreated controls. Apoptotic nuclei in a total of 500 cells were counted in triplicate, in at least three experiments. The mean percentages of apoptotic cells (SEM) are shown.

Figure 3

Expression of nuclear p53 and p21 in untreated and cisplatin treated colonocytes detected by immunocytochemistry using an avidin–biotin peroxidase technique. Positive cells were detected with diaminobenzidine and haematoxylin was used as nuclear counterstain. p53 expression is upregulated after exposure to cisplatin (A and B), whereas p21 levels do not change significantly in cisplatin treated wt cells (C and D) and p53 null cells (E and F).

Figure 4

p53 expression in untreated and cisplatin damaged colonocytes. p53 is expressed under baseline conditions but its expression is significantly upregulated after exposure to cisplatin at all timepoints (p<0.05, Mann-Whitney U test). The figure shows the mean (SEM) percentage of p53 positive cells in a total of 500.

Figure 5

Immunofluorescent detection of nuclear p73 in primary colonocytes using Alexa-488 conjugated secondary antibody. Nuclear accumulation of p73 is seen after treatment with cisplatin in both wild-type (wt) and p53 null cells using the rabbit polyclonal H-79 antibody. DAPI was used as a nuclear counterstain. The cells shown were exposed to cisplatin for 24 hours. Images were captured using a Hamamatsu chilled CCD camera and Zeiss fluorescent microscope.

Figure 6

Immunofluorescent detection of nuclear p73 in primary colonocytes using Alexa-488 conjugated secondary antibody. Nuclear accumulation of p73 is seen after treatment with cisplatin in both wild-type (wt) and p53 null cells using the sheep polyclonal Ab77 antibody. DAPI was used as a nuclear counterstain. The cells shown were exposed to cisplatin for 24 hours. Images were captured using a Hamamatsu chilled CCD camera and Zeiss fluorescent microscope.

Figure 7

Reverse transcription polymerase chain reaction (RT-PCR) showing p73 expression in wild-type (wt) and p53 null untreated (UNT) and cisplatin (cis) treated colonocytes. (A) Amplification with primers spanning exons 11–14 in murine primary colonocytes. The primers could also detect the presence of the C-terminus of p73β, but no band of the predicted size was obtained. (B) RT-PCR analysis for N-terminus detection of full length p73 using primers for exon 3–4. (C) RT-PCR showing the absence of ΔN variants in primary colonocytes. NIH-3T3 cDNA proved that the primers recognise a product of the correct size. (D) The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal qualitative and semiquantitative control.