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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-induced Chk1 activation is MMR-independent.Isogenically paired HCT cells differing only in MMR capabilities (MMR-deficient HCT116 and MMR-proficient HCT3-6) depleted of MGMT by pre-incubation with BG (20 µM, 2 hrs, G55+BG) were incubated with vehicle (U) or TMZ (100 µmol/L, 3 hours) after which TMZ was removed, vehicle or BG was replaced, and cells were harvested at 24–72 hours (A, C) or at earlier time points (1–24 hours, B, D) following TMZ exposure for analysis of H2AX foci (A, DAPI staining in right panel of pairs), DNA content by FACS (B), or pChk1, Chk1, hMLH1, MGMT, and β-actin expression by Western blot (C, D). Mean fold induction of protein expression was based on densitometric measurements and is shown (relative to untreated controls) below the relevant immunoreactive bands. *, p<.05.
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pone-0062351-g003: TMZ-induced Chk1 activation is MMR-independent.Isogenically paired HCT cells differing only in MMR capabilities (MMR-deficient HCT116 and MMR-proficient HCT3-6) depleted of MGMT by pre-incubation with BG (20 µM, 2 hrs, G55+BG) were incubated with vehicle (U) or TMZ (100 µmol/L, 3 hours) after which TMZ was removed, vehicle or BG was replaced, and cells were harvested at 24–72 hours (A, C) or at earlier time points (1–24 hours, B, D) following TMZ exposure for analysis of H2AX foci (A, DAPI staining in right panel of pairs), DNA content by FACS (B), or pChk1, Chk1, hMLH1, MGMT, and β-actin expression by Western blot (C, D). Mean fold induction of protein expression was based on densitometric measurements and is shown (relative to untreated controls) below the relevant immunoreactive bands. *, p<.05.

Mentions: Although TMZ-induced Chk1 activation appeared to precede Chk2 activation and the formation of the DNA DSB, it remained possible that TMZ-induced, MMR-dependent single-strand breaks, or a limited number of MMR-dependent DNA DSBs induced at very early time points, were responsible for the Chk1 phosphorylation noted, at least in the MGMT-deficient cells. To address this possibility, human cells deficient in MMR by virtue of chromosome 3 loss, and the same cells complemented for MMR activity by reintroduction of chromosome 3 and gain of hMLH1 expression, were depleted of MGMT by BG exposure, then incubated with TMZ and assayed for DNA damage, cell cycle arrest, and Chk1 activation. The MGMT-deficient, MMR-deficient HCT116 cells did not exhibit H2AX foci (Fig. 3A) and did not undergo G2 arrest (Fig. 3B) at 3 days following TMZ exposure, consistent with the results of previous studies with these cells [17] and with the inability of the cells to use the MMR system to process O6MG lesions into DNA damage. In contrast, MGMT-deficient, MMR-complemented HCT3-6 cells exhibited H2AX foci and cell cycle arrest 3 days following TMZ exposure (Fig. 3A and B), consistent with the role of MMR in converting TMZ-induced O6MG lesions into DNA DSB. As with the U87 and G55 cells examined, the MGMT-deficient, MMR-proficient HCT 3–6 cells exhibited significant induction of pChk1 (but not Chk1) levels both 2 and 3 days after TMZ exposure while the MGMT-deficient, MMR-deficient paired HCT116 cells did not (Fig. 3C). An examination at early time points, however, showed that both the MMR-proficient and MMR-deficient HCT cells exhibited Chk1 activation within 3–6 hours after TMZ exposure (Fig. 3D). Because MMR-deficient cells are incapable of processing TMZ-induced O6MG lesions into DNA damage, these results show that the TMZ-induced phosphorylation of Chk1 is independent of MMR and of the DNA damage generated by this system in response to TMZ.


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-induced Chk1 activation is MMR-independent.Isogenically paired HCT cells differing only in MMR capabilities (MMR-deficient HCT116 and MMR-proficient HCT3-6) depleted of MGMT by pre-incubation with BG (20 µM, 2 hrs, G55+BG) were incubated with vehicle (U) or TMZ (100 µmol/L, 3 hours) after which TMZ was removed, vehicle or BG was replaced, and cells were harvested at 24–72 hours (A, C) or at earlier time points (1–24 hours, B, D) following TMZ exposure for analysis of H2AX foci (A, DAPI staining in right panel of pairs), DNA content by FACS (B), or pChk1, Chk1, hMLH1, MGMT, and β-actin expression by Western blot (C, D). Mean fold induction of protein expression was based on densitometric measurements and is shown (relative to untreated controls) below the relevant immunoreactive bands. *, p<.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646831&req=5

pone-0062351-g003: TMZ-induced Chk1 activation is MMR-independent.Isogenically paired HCT cells differing only in MMR capabilities (MMR-deficient HCT116 and MMR-proficient HCT3-6) depleted of MGMT by pre-incubation with BG (20 µM, 2 hrs, G55+BG) were incubated with vehicle (U) or TMZ (100 µmol/L, 3 hours) after which TMZ was removed, vehicle or BG was replaced, and cells were harvested at 24–72 hours (A, C) or at earlier time points (1–24 hours, B, D) following TMZ exposure for analysis of H2AX foci (A, DAPI staining in right panel of pairs), DNA content by FACS (B), or pChk1, Chk1, hMLH1, MGMT, and β-actin expression by Western blot (C, D). Mean fold induction of protein expression was based on densitometric measurements and is shown (relative to untreated controls) below the relevant immunoreactive bands. *, p<.05.
Mentions: Although TMZ-induced Chk1 activation appeared to precede Chk2 activation and the formation of the DNA DSB, it remained possible that TMZ-induced, MMR-dependent single-strand breaks, or a limited number of MMR-dependent DNA DSBs induced at very early time points, were responsible for the Chk1 phosphorylation noted, at least in the MGMT-deficient cells. To address this possibility, human cells deficient in MMR by virtue of chromosome 3 loss, and the same cells complemented for MMR activity by reintroduction of chromosome 3 and gain of hMLH1 expression, were depleted of MGMT by BG exposure, then incubated with TMZ and assayed for DNA damage, cell cycle arrest, and Chk1 activation. The MGMT-deficient, MMR-deficient HCT116 cells did not exhibit H2AX foci (Fig. 3A) and did not undergo G2 arrest (Fig. 3B) at 3 days following TMZ exposure, consistent with the results of previous studies with these cells [17] and with the inability of the cells to use the MMR system to process O6MG lesions into DNA damage. In contrast, MGMT-deficient, MMR-complemented HCT3-6 cells exhibited H2AX foci and cell cycle arrest 3 days following TMZ exposure (Fig. 3A and B), consistent with the role of MMR in converting TMZ-induced O6MG lesions into DNA DSB. As with the U87 and G55 cells examined, the MGMT-deficient, MMR-proficient HCT 3–6 cells exhibited significant induction of pChk1 (but not Chk1) levels both 2 and 3 days after TMZ exposure while the MGMT-deficient, MMR-deficient paired HCT116 cells did not (Fig. 3C). An examination at early time points, however, showed that both the MMR-proficient and MMR-deficient HCT cells exhibited Chk1 activation within 3–6 hours after TMZ exposure (Fig. 3D). Because MMR-deficient cells are incapable of processing TMZ-induced O6MG lesions into DNA damage, these results show that the TMZ-induced phosphorylation of Chk1 is independent of MMR and of the DNA damage generated by this system in response to TMZ.

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