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. 2012 Sep 6;31(36):4076-84.
doi: 10.1038/onc.2011.585. Epub 2011 Dec 19.

Hypoxic activation of ATR and the suppression of the initiation of DNA replication through cdc6 degradation

L Martin  1 , M Rainey, C Santocanale, L B Gardner
Affiliations

Hypoxic activation of ATR and the suppression of the initiation of DNA replication through cdc6 degradation

L Martin et al. Oncogene. .

Abstract

Many severely hypoxic cells fail to initiate DNA replication, but the mechanism underlying this observation is unknown. Specifically, although the ataxia-telangiectasia-rad3 related (ATR) kinase has been shown to be activated in hypoxic cells, several studies have not been able to document down-stream consequences of ATR activation in these cells. By clearly defining the DNA replication initiation checkpoint in hypoxic cells, we now demonstrate that ATR is responsible for activating this checkpoint. We show that the hypoxic activation of ATR leads to the phosphorylation-dependent degradation of the cdc25a phosphatase. Downregulation of cdc25a protein by ATR in hypoxic cells decreases CDK2 phosphorylation and activity, which results in the degradation of cdc6 by APC/C(Cdh1). These events do not occur in hypoxic cells when ATR is depleted, and the initiation of DNA replication is maintained. We therefore present a novel mechanism of cdc6 regulation in which ATR can have a central role in inhibiting the initiation of DNA replication by the regulation of cdc6 by APC/C(Cdh1). This model provides insight into the biology and therapy of hypoxic tumors.

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Conflict of interest statement

Conflicts of Interest

The authors declare no conflicts of interest

Figures

Fig 1
Fig 1
ATR inhibits the initiation of DNA replication in hypoxic cells. A. U2OS cells were rendered hypoxic for 8 hrs or 24 hrs, and pulsed with thymidine prior to the collection of DNA and fractionation as described in the text. Lighter fractions represent smaller, newly synthesized DNA molecules. B. Chk2 expression in normoxic (N), hypoxic (Hy) and 10 Gy irradiated (XRT) MEFs and, as control, AT−/− MEFs. Phosphorylation of Chk2 manifests as a mobility shift. C. Expression of Chk1-serine 345 phosphorylation and rad17-serine 645 phosphorylation in cells rendered hypoxic and also rendered hypoxic and treated with 10 mM caffeine. D. Incoroporation of thymidine into DNA in cells rendered hypoxic for eight hours, with and without 10 mM caffeine. Experiments were replicated twice; fold changes are displayed. E. ATR expression in U2OS cells expressing three different shRNA sequences (top) and extent of ATR depletion as indicated by increasing loading of proteins (bottom). F. ATR expression in wild-type HCT116 cells, HCT116 ATRflox/− cells expressing a tamoxifen inducible cre recombinase, and HCT116 ATRflox/− cells treated with tamoxifen for 24 hrs. G. Chk1-serine 345 phosphorylation in cells described in (F) rendered hypoxic. H. Thymidine incorporation in U2OS and HCT116 depleted cells rendered hypoxic for eight hours. Experiments were replicated three times. Average ± standard error is displayed.
Fig 2
Fig 2
cdc25a protein and CDK2 activity are regulated by ATR in hypoxic cells. A. U2OS and HCT117cells with and without ATR depletion as demonstrated in Fig 1, were rendered hypoxic (Hy) or maintained as normoxic (N) and cdc25a protein expression was assessed. Experiments were replicated and fold changes displayed. B. cdc25a mRNA expression in U2OS cells rendered hypoxic for four and eight hours. Experiments were done in triplicate and average ± standard error displayed. C. Expression of Cdc25a in normoxic and hypoxic cells stably expressing a wild-type cdc25a construct or a cdc25a S76A construct. Experiments were done in triplicate and fold changes displayed. D. CDK2 tyrosine 15 phosphorylation in normoxic and hypoxic U2OS cells expressing either wild-type cdc25a or cdc25a S76A, and in E. normoxic and hypoxic control and ATR depleted U2OS cells. Experiments were replicated and fold changes displayed F. CDK2 activity and expression in normoxic and hypoxic U2OS cells expressing wild-type cdc25a or cdc25a S76A G. CDK2 activity and expression in normoxic and hypoxic control and ATR depleted U2OS cells.
Fig 3
Fig 3
ATR down-regulates Cdc6 in hypoxic cells by APC/CCdh1. A. Soluble and chromatin bound cdc6 protein expression in normoxic (N) and hypoxic (Hy) control and ATR depleted U2OS cells. Experiments were done in triplicate and fold changes displayed. B. cdc6 mRNA expression in hypoxic U2OS cells. Experiments were done in triplicate average ± standard error is displayed. C. Cdc6 expression and phosphorylation of cdc6 serine 106 in normoxic and hypoxic wild-type and ATR deleted cells. Note lanes have been removed from this figure, but exposure is consistent. D. Transient transfected 293T cells, expressing either wild-type or a phosphomimetic (DDD) form of cdc6 were rendered hypoxic and assessed for cdc6 expression Experiments were replicated and fold changes displayed E. cdc6 protein expression in normoxic and hypoxic U2OS cells expressing wild-type cdc25a or a stable cdc25a S76A construct. Experiments were done in triplicate and fold changes displayed F. U2OS cells were rendered hypoxic or normoxic for eight hours, the last three with either MG132 (10 mM) or MLN, and cdc6 expression was assessed. G. Normoxic and hypoxic U2OS cells were depleted for Huwe1 or cdh1 and cdc6 expression was assessed. Experiments were replicated and fold changes displayed H. 293T cells, expressing either control plasmid, wild-type or a phosphomimetic (DDD) form of cdc6 were rendered hypoxic and thymidine incorporation was assessed. Experiments were done in triplicate and average ± is displayed.
Fig 4
Fig 4
Chromatin bound MCM2 expression and phosphorylation is affected by hypoxic activation of ATR. A. MCM2 expression in hypoxic (Hy) and normoxic (N) cells was assessed in cytoplasmic, chromatin bound, and nucleoplasm cell compartments. Chromatin bound MCM2 was also treated with nuclease to confirm proper fractionation. B. Soluble and chromatin bound MCM2 in normoxic and hypoxic U2OS control and ATR depleted cells (note, panel is a composite). Proteins were resolved so that two bands were apparent, the bottom one consistent with a phosphorylated form, as C. demonstrated by its disappearance with lambda phosphatase treatment. D. Soluble and Chromatin bound phosphorylated forms of MCMC2 in normoxic and six hours hypoxic U2OS control and ATR depleted cells.
Fig 5
Fig 5
A model for the hypoxic regulation of the initiation of DNA replication.
See this image and copyright information in PMC

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