ATR Activation Necessary But Not Sufficient for p53 Induction and Apoptosis in Hydroxyurea-Hypersensitive Myeloid Leukemia Cells

Abstract

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.

Cell cycle dependent transcription, a determinant factor of heterogeneity in cationic lipid‐mediated transgene expression

“…Furthermore, it has been reported that hydroxyurea treatment induces transcription factors such as p53 [36], which alters the transcription activity of certain types of proteins [37]. However, hydroxyurea had little effect on the expression level of GL3, stably expressed in HeLa cells under the regulation of the CMV promoter ( Figure 7).…”

Reverse engineering of drug induced DNA damage response signalling pathway reveals dual outcomes of ATM kinase inhibition

“…Among the two possibilities, the latter seems likely because cell cytotoxicity analysis revealed correlation between pATR induction and cell death during KU + Dox treatments pointing out to a pathway involving KU ┤ ATM = pATR → Apoptosis. As such, the role of ATR in apoptosis induction has already been reported following treatments with chemotherapeutic agents [78,79].…”

“…ATR activity has already been known to signal apoptosis in response to treatments with different chemotherapeutic agents [78,79]. Hence the inhibitory state of ATR levels at lower DNA damage suggests cellular signalling preference towards DNA repair.…”

Cyclooxygenase-2 Up-Regulates Ataxia Telangiectasia and Rad3 Related through Extracellular Signal-Regulated Kinase Activation

“…Similar to ATR, loss of Chk1 function results not only in mouse embryonic lethality at the developmental early stage but also causes cells to bypass the DNA damage and DNA replication checkpoints (33,35). p53 is also very well known to be important to maintain cell cycle fidelity, and ATR phosphorylates p53 at Ser 15 and Ser 37 (32,36). These data indicate that ATR, Chk1, and p53 are crucial for maintaining genomic integrity via regulation of the cell cycle in normal cells and in cells with damaged DNA.…”

“…IR is a well-known inducer of DNA double strand break. Hydroxyurea, a competitive inhibitor of ribonucleotide reductase, blocks DNA replication, resulting in ATR activation (13,32). HCT116-Mock and HCT116-COX-2 cells were exposed to graded doses of IR or hydroxyurea, and then cell viability was monitored by a clonogenic assay.…”