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Early Chk1 phosphorylation is driven by temozolomide-induced, DNA double strand break- and mismatch repair-independent DNA damage.

Ito M, Ohba S, Gaensler K, Ronen SM, Mukherjee J, Pieper RO - PLoS ONE (2013)

Bottom Line: Furthermore, TMZ-induced early phosphorylation of Chk1 was noted in glioma cells regardless of whether they were MGMT-proficient or MGMT-deficient, and regardless of their MMR status.Early Chk1 phosphorylation was not associated with TMZ-induced reactive oxygen species, but was temporally associated with TMZ-induced alkalai-labile DNA damage produced by the non-O6-methylguanine DNA adducts and which, like Chk1 phosphorylation, was transient in MGMT-proficient cells but persistent in MGMT-deficient cells.These results re-define the TMZ-induced DNA damage response, and show that Chk1 phosphorylation is driven by TMZ-induced mismatch repair-independent DNA damage independently of DNA double strand breaks, Chk2 activation, and cell cycle arrest, and as such is a suboptimal biomarker of TMZ-induced drug action.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Surgery, University of California-San Francisco, San Francisco, California, United States of America.

ABSTRACT
Temozolomide (TMZ) is a DNA methylating agent used to treat brain cancer. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair during DNA replication and trigger cycles of futile mismatch repair (MMR). Futile MMR in turn leads to the formation of DNA single and double strand breaks, Chk1 and Chk2 phosphorylation/activation, cell cycle arrest, and ultimately cell death. Although both pChk1 and pChk2 are considered to be biomarkers of TMZ-induced DNA damage, cell-cycle arrest, and TMZ induced cytotoxicity, we found that levels of pChk1 (ser345), its downstream target pCdc25C (ser216), and the activity of its upstream activator ATR, were elevated within 3 hours of TMZ exposure, long before the onset of TMZ-induced DNA double strand breaks, Chk2 phosphorylation/activation, and cell cycle arrest. Furthermore, TMZ-induced early phosphorylation of Chk1 was noted in glioma cells regardless of whether they were MGMT-proficient or MGMT-deficient, and regardless of their MMR status. Early Chk1 phosphorylation was not associated with TMZ-induced reactive oxygen species, but was temporally associated with TMZ-induced alkalai-labile DNA damage produced by the non-O6-methylguanine DNA adducts and which, like Chk1 phosphorylation, was transient in MGMT-proficient cells but persistent in MGMT-deficient cells. These results re-define the TMZ-induced DNA damage response, and show that Chk1 phosphorylation is driven by TMZ-induced mismatch repair-independent DNA damage independently of DNA double strand breaks, Chk2 activation, and cell cycle arrest, and as such is a suboptimal biomarker of TMZ-induced drug action.

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TMZ-induces delayed DNA DSB and cell cycle arrest in an MGMT-dependent manner.MGMT-deficient U87 cells or U87 cells engineered to over-express MGMT (U87+MGMT) were incubated with vehicle (untreated) or TMZ (100 µmol/L, 3 hours) after which drug was removed and cells were harvested 24, 48, and 72 hours later. A) representative photomicrographs of cells analyzed for phospho-H2A.X foci (a measure of DNA DSB) following immunofluorescent staining with anti-Ser-139-phosphorylated H2A.X antibody. Right panel of pairs, cells counterstained with DAPI to visualize nuclei. B) Analysis of tail moment (a measure of DNA fragmentation) in cells embedded in agarose, lysed, electrophoresed under neutral conditions, and stained using SYBR Green. Each point represents the mean+standard error of at least one hundred cells per treatment. *, p<.05. C) histograms of cells incubated with propidium iodide and analyzed for DNA content by FACS.
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pone-0062351-g001: TMZ-induces delayed DNA DSB and cell cycle arrest in an MGMT-dependent manner.MGMT-deficient U87 cells or U87 cells engineered to over-express MGMT (U87+MGMT) were incubated with vehicle (untreated) or TMZ (100 µmol/L, 3 hours) after which drug was removed and cells were harvested 24, 48, and 72 hours later. A) representative photomicrographs of cells analyzed for phospho-H2A.X foci (a measure of DNA DSB) following immunofluorescent staining with anti-Ser-139-phosphorylated H2A.X antibody. Right panel of pairs, cells counterstained with DAPI to visualize nuclei. B) Analysis of tail moment (a measure of DNA fragmentation) in cells embedded in agarose, lysed, electrophoresed under neutral conditions, and stained using SYBR Green. Each point represents the mean+standard error of at least one hundred cells per treatment. *, p<.05. C) histograms of cells incubated with propidium iodide and analyzed for DNA content by FACS.

Mentions: A 3 hour exposure to an IC50 concentration of TMZ [15] resulted in the formation of H2AX foci (a surrogate measure of DSB) beginning 2 days after drug treatment (Fig. 1A, upper panels). The fragmented DNA resulting from the DSB could also be detected in the same time frame using a single cell Comet assay performed under neutral pH conditions (Fig. 1B), as could the resulting accumulation of cells with a 4 n DNA content consistent with G2 arrest (Fig. 1C, upper panels). None of these effects were noted in isogenic U87 cells which were engineered to over-express MGMT and which repaired O6MG lesions before the O6MG lesions could mispair and cause DNA damage. Consistent with previous reports, elevated levels of pChk1 and pChk2 were also noted 2 and 3 days after TMZ exposure in MGMT-deficient U87 cells but not in U87 cells over-expressing MGMT (Fig. 2A). These results are consistent with the prevailing view that phosphorylation of Chk1and Chk2 following TMZ exposure is a biomarker of TMZ-induced DNA damage and its downstream therapeutic consequences [34].


Early Chk1 phosphorylation is driven by temozolomide-induced, DNA double strand break- and mismatch repair-independent DNA damage.

Ito M, Ohba S, Gaensler K, Ronen SM, Mukherjee J, Pieper RO - PLoS ONE (2013)

TMZ-induces delayed DNA DSB and cell cycle arrest in an MGMT-dependent manner.MGMT-deficient U87 cells or U87 cells engineered to over-express MGMT (U87+MGMT) were incubated with vehicle (untreated) or TMZ (100 µmol/L, 3 hours) after which drug was removed and cells were harvested 24, 48, and 72 hours later. A) representative photomicrographs of cells analyzed for phospho-H2A.X foci (a measure of DNA DSB) following immunofluorescent staining with anti-Ser-139-phosphorylated H2A.X antibody. Right panel of pairs, cells counterstained with DAPI to visualize nuclei. B) Analysis of tail moment (a measure of DNA fragmentation) in cells embedded in agarose, lysed, electrophoresed under neutral conditions, and stained using SYBR Green. Each point represents the mean+standard error of at least one hundred cells per treatment. *, p<.05. C) histograms of cells incubated with propidium iodide and analyzed for DNA content by FACS.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646831&req=5

pone-0062351-g001: TMZ-induces delayed DNA DSB and cell cycle arrest in an MGMT-dependent manner.MGMT-deficient U87 cells or U87 cells engineered to over-express MGMT (U87+MGMT) were incubated with vehicle (untreated) or TMZ (100 µmol/L, 3 hours) after which drug was removed and cells were harvested 24, 48, and 72 hours later. A) representative photomicrographs of cells analyzed for phospho-H2A.X foci (a measure of DNA DSB) following immunofluorescent staining with anti-Ser-139-phosphorylated H2A.X antibody. Right panel of pairs, cells counterstained with DAPI to visualize nuclei. B) Analysis of tail moment (a measure of DNA fragmentation) in cells embedded in agarose, lysed, electrophoresed under neutral conditions, and stained using SYBR Green. Each point represents the mean+standard error of at least one hundred cells per treatment. *, p<.05. C) histograms of cells incubated with propidium iodide and analyzed for DNA content by FACS.
Mentions: A 3 hour exposure to an IC50 concentration of TMZ [15] resulted in the formation of H2AX foci (a surrogate measure of DSB) beginning 2 days after drug treatment (Fig. 1A, upper panels). The fragmented DNA resulting from the DSB could also be detected in the same time frame using a single cell Comet assay performed under neutral pH conditions (Fig. 1B), as could the resulting accumulation of cells with a 4 n DNA content consistent with G2 arrest (Fig. 1C, upper panels). None of these effects were noted in isogenic U87 cells which were engineered to over-express MGMT and which repaired O6MG lesions before the O6MG lesions could mispair and cause DNA damage. Consistent with previous reports, elevated levels of pChk1 and pChk2 were also noted 2 and 3 days after TMZ exposure in MGMT-deficient U87 cells but not in U87 cells over-expressing MGMT (Fig. 2A). These results are consistent with the prevailing view that phosphorylation of Chk1and Chk2 following TMZ exposure is a biomarker of TMZ-induced DNA damage and its downstream therapeutic consequences [34].

Bottom Line: Furthermore, TMZ-induced early phosphorylation of Chk1 was noted in glioma cells regardless of whether they were MGMT-proficient or MGMT-deficient, and regardless of their MMR status.Early Chk1 phosphorylation was not associated with TMZ-induced reactive oxygen species, but was temporally associated with TMZ-induced alkalai-labile DNA damage produced by the non-O6-methylguanine DNA adducts and which, like Chk1 phosphorylation, was transient in MGMT-proficient cells but persistent in MGMT-deficient cells.These results re-define the TMZ-induced DNA damage response, and show that Chk1 phosphorylation is driven by TMZ-induced mismatch repair-independent DNA damage independently of DNA double strand breaks, Chk2 activation, and cell cycle arrest, and as such is a suboptimal biomarker of TMZ-induced drug action.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Surgery, University of California-San Francisco, San Francisco, California, United States of America.

ABSTRACT
Temozolomide (TMZ) is a DNA methylating agent used to treat brain cancer. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair during DNA replication and trigger cycles of futile mismatch repair (MMR). Futile MMR in turn leads to the formation of DNA single and double strand breaks, Chk1 and Chk2 phosphorylation/activation, cell cycle arrest, and ultimately cell death. Although both pChk1 and pChk2 are considered to be biomarkers of TMZ-induced DNA damage, cell-cycle arrest, and TMZ induced cytotoxicity, we found that levels of pChk1 (ser345), its downstream target pCdc25C (ser216), and the activity of its upstream activator ATR, were elevated within 3 hours of TMZ exposure, long before the onset of TMZ-induced DNA double strand breaks, Chk2 phosphorylation/activation, and cell cycle arrest. Furthermore, TMZ-induced early phosphorylation of Chk1 was noted in glioma cells regardless of whether they were MGMT-proficient or MGMT-deficient, and regardless of their MMR status. Early Chk1 phosphorylation was not associated with TMZ-induced reactive oxygen species, but was temporally associated with TMZ-induced alkalai-labile DNA damage produced by the non-O6-methylguanine DNA adducts and which, like Chk1 phosphorylation, was transient in MGMT-proficient cells but persistent in MGMT-deficient cells. These results re-define the TMZ-induced DNA damage response, and show that Chk1 phosphorylation is driven by TMZ-induced mismatch repair-independent DNA damage independently of DNA double strand breaks, Chk2 activation, and cell cycle arrest, and as such is a suboptimal biomarker of TMZ-induced drug action.

Show MeSH
Related in: MedlinePlus