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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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pChk1 levels are elevated prior to the onset of TMZ-induced DNA DSB, Chk2 activation, and cell cycle arrest.Isogenically paired U87 cells differing only in MGMT expression (U87 or U87+MGMT)(A- C) or G55 cells expressing MGMT or depleted of MGMT by pre-incubation with the MGMT-depleting agent BG (20 µM, 2 hours, G55+BG)(C- E) 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, D) or at earlier time points (1–24 hours, B, C, E) following TMZ exposure for analysis of pChk1 (ser345), Chk1, pChk2 (thr68), Chk2, MGMT, pcdc25C (ser216), cdc25C, and β-actin expression by Western blot. For panel C, an ATR (or control IgG) immunoprecipitate was first prepared and incubated with the ATR substrate PHAS, after which a Western blot analysis of the extent of ATR-mediated PHAS ser/thr phosphorylation was performed. 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-g002: pChk1 levels are elevated prior to the onset of TMZ-induced DNA DSB, Chk2 activation, and cell cycle arrest.Isogenically paired U87 cells differing only in MGMT expression (U87 or U87+MGMT)(A- C) or G55 cells expressing MGMT or depleted of MGMT by pre-incubation with the MGMT-depleting agent BG (20 µM, 2 hours, G55+BG)(C- E) 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, D) or at earlier time points (1–24 hours, B, C, E) following TMZ exposure for analysis of pChk1 (ser345), Chk1, pChk2 (thr68), Chk2, MGMT, pcdc25C (ser216), cdc25C, and β-actin expression by Western blot. For panel C, an ATR (or control IgG) immunoprecipitate was first prepared and incubated with the ATR substrate PHAS, after which a Western blot analysis of the extent of ATR-mediated PHAS ser/thr phosphorylation was performed. 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: 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)

pChk1 levels are elevated prior to the onset of TMZ-induced DNA DSB, Chk2 activation, and cell cycle arrest.Isogenically paired U87 cells differing only in MGMT expression (U87 or U87+MGMT)(A- C) or G55 cells expressing MGMT or depleted of MGMT by pre-incubation with the MGMT-depleting agent BG (20 µM, 2 hours, G55+BG)(C- E) 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, D) or at earlier time points (1–24 hours, B, C, E) following TMZ exposure for analysis of pChk1 (ser345), Chk1, pChk2 (thr68), Chk2, MGMT, pcdc25C (ser216), cdc25C, and β-actin expression by Western blot. For panel C, an ATR (or control IgG) immunoprecipitate was first prepared and incubated with the ATR substrate PHAS, after which a Western blot analysis of the extent of ATR-mediated PHAS ser/thr phosphorylation was performed. 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-g002: pChk1 levels are elevated prior to the onset of TMZ-induced DNA DSB, Chk2 activation, and cell cycle arrest.Isogenically paired U87 cells differing only in MGMT expression (U87 or U87+MGMT)(A- C) or G55 cells expressing MGMT or depleted of MGMT by pre-incubation with the MGMT-depleting agent BG (20 µM, 2 hours, G55+BG)(C- E) 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, D) or at earlier time points (1–24 hours, B, C, E) following TMZ exposure for analysis of pChk1 (ser345), Chk1, pChk2 (thr68), Chk2, MGMT, pcdc25C (ser216), cdc25C, and β-actin expression by Western blot. For panel C, an ATR (or control IgG) immunoprecipitate was first prepared and incubated with the ATR substrate PHAS, after which a Western blot analysis of the extent of ATR-mediated PHAS ser/thr phosphorylation was performed. 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: 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