Camptothecin induced expression and phosphorylation of p53 regulates apoptosis in epithelial cells

Abstract

p53 is an established tumor suppressor protein. In addition to preventing tumor growth, p53 plays a key role in cell cycle regulation and in the cellular response to cytotoxic stress and DNA damage. Upon induction of p53, a host of target genes are transcriptionally activated, including p21waf1, GADD45, bax, bcl‐2. At this time, the specific signals required for p53‐mediated cell cycle arrest and/or apoptosis remain complex. Polyamine depletion of intestinal epithelial cells (IEC‐6) increased p53 and p21 protein levels and these cells were resistant to TNF‐α‐, γ‐ irradiation‐, and camptothecin (CPT)‐induced apoptosis. CPT induced p53 protein levels in a time dependent manner, however, its phosphorylation decreased significantly. Increased p53 levels correlated with p21 levels indicating transcriptional activity of p53. The inhibitors of transcription (Actinomycin D) and translation (Cycloheximide) prevented CPT‐induced apoptosis. Although, cycloheximide (CHX) completely prevented p21 synthesis, p53 levels were unaltered. RITA (p53 activator III) which blocks p53‐MDM2 interaction and thereby p53 ubiquitination significantly increased apoptosis. Actinomycin D significantly increased p53 protein levels and its phosphorylation. However, p21 expression was completely abolished. These results suggest a predominant role of p53 protein in CPT‐induced apoptosis. In addition, we used retinal pigment epithelial cells (RPE), which are highly resistant to a variety of apoptosis inducers, and compared the p53 status and phosphorylation in response to CPT. Supported by NIDDK grant DK‐16505.