ATR activation necessary but not sufficient for p53 induction and apoptosis in hydroxyurea-hypersensitive myeloid leukemia cells.
Hydroxyurea (HU) is a competitive inhibitor of ribonucleotide reductase that is used for the treatment of myeloproliferative disorders. HU inhibits DNA replication and induces apoptosis in a cell type-dependent manner, yet the relevant pathways that mediate apoptosis in response to this agent are not well characterized. In this study, we employed the human myeloid leukemia 1 (ML-1) cell line as a model to investigate the mechanisms of HU-induced apoptosis. Exposure of ML-1 cells to HU caused rapid cell death that was accompanied by hallmark features of apoptosis, including membrane blebbing, phosphatidylserine translocation, and caspase activation. HU-induced apoptosis required new protein synthesis, was induced by HU exposures as short as 15 min, and correlated with the accumulation of p53 and induction of the p53 target gene PUMA. p53 induction in ML-1 cells was ATR dependent and downregulation of p53 through RNAi delayed HU-induced apoptosis. HU did not induce p53 or induce apoptosis in Molt-3 leukemia cells, even though exposure to HU induced a comparable level of DNA damage and robustly activated the ATR pathway. The microtubule inhibitor nocodazole suppressed HU-induced p53 accumulation in ML-1 cells suggesting that a microtubule-dependent event contributes to p53 induction and apoptosis in this cell line. Our findings outline an HU-induced cell death pathway and suggest that activation of ATR is necessary, but not sufficient, for stabilization of p53 in response to DNA replication stress.
Kumar
Sujatha
S
Department of Pharmacology, Molecular and Cellular Pharmacology Program, The University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706, USA.
Dodson
Gerald E
GE
Trinh
Anthony
A
Puchalski
Joel R
JR
Tibbetts
Randal S
RS
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000502:physiology; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology*; D007951:Leukemia, Myeloid / Q000378:metabolism* / Q000473:pathology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
D018797:Cell Cycle Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D016159:Tumor Suppressor Protein p53; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D006918:Hydroxyurea