Role of polyamines in p53-dependent apoptosis of intestinal epithelial cells.
Although p53 is known to play a critical role in the proliferation of gastrointestinal epithelia, the role of the Mdm2/p53 pathway in response to inducers of apoptosis in intestinal epithelial cells is unknown. Our data show that camptothecin (CPT)-induced apoptosis correlated with increased p53, p21Cip1, and Mdm2 protein levels, with a simultaneous increase in ATR Ser428, p53 Ser15 and Mdm2 Ser166 phosphorylation in IEC-6 cells. Increased p53 levels and its phosphorylation increased Bax protein, caspase-9, -3 activation and apoptosis. However, TNF-alpha/CHX-mediated apoptosis was independent of p53 protein levels and phosphorylation. The translation inhibitor, cycloheximide (CHX), prevented CPT-induced apoptosis. CHX completely prevented CPT-induced p53 phosphorylation and synthesis of p21Cip1, Bax and Bcl-xL proteins without altering p53 levels. The p53 activator, RITA, augmented CPT-induced apoptosis. The Mdm2 antagonist, Nutlin-3, significantly increased apoptosis, which was accompanied by increased p53, Mdm2 and p21Cip1 protein levels. The ATM/ATR kinase inhibitor, CGK733, blocked CPT-induced p53 Ser15 phosphorylation and protected cells from CPT-induced apoptosis. Inhibition of ornithine decarboxylase (ODC) with alpha-difluromethylornithine (DFMO) and subsequent depletion of intracellular polyamines increased p53 protein, Mdm2 Ser166 phosphorylation and conferred resistance to CPT-induced apoptosis. However, polyamine depletion had no effect on p53 phosphorylation. Nutlin-3 reversed the protective effect of DFMO and sensitized cells to CPT-induced apoptosis. These results suggest that p53 stabilization and accumulation in response to polyamine depletion predominantly modulate cell cycle checkpoints via p21Cip1 expression and inhibit transcription of target genes responsible for apoptosis. In contrast, phosphorylation and stabilization of p53 in response to DNA-damage lead to apoptosis, which indicates different roles of p53 during DNA damage and polyamine depletion.
Bhattacharya
Sujoy
S
University of Tennessee Health Science Center, 894 Union Ave., Nash Building, Memphis, TN 38163, USA.
Ray
Ramesh M
RM
Johnson
Leonard R
LR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000502:physiology; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D004249:DNA Damage; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology; D009955:Ornithine Decarboxylase / Q000502:physiology; D065108:Ornithine Decarboxylase Inhibitors; D010766:Phosphorylation; D011073:Polyamines / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000502:physiology; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
D051017:Apoptosis Regulatory Proteins; C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D003513:Cycloheximide; C497208:Mdm2 protein, rat; D051736:Proto-Oncogene Proteins c-mdm2; C494920:Akt1 protein, rat; D051057:Proto-Oncogene Proteins c-akt; D048031:JNK Mitogen-Activated Protein Kinases; D009955:Ornithine Decarboxylase; D002166:Camptothecin