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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 independent of the TMZ-induced generation of ROS.MGMT-deficient U87 cells were pre-incubated with vehicle or the ROS scavenger NAC (400 µM, 2 hours) after which the cells were incubated with vehicle (untreated, U) or TMZ (100 µmol/L, 3 hours). TMZ was removed, vehicle or NAC was added, and the cells were harvested 1–24 hours following TMZ exposure for analysis of ROS based on the oxidation of 2,7-dichloro-dihydrofluorescein diacetate to an intracellular fluorescent compound (A, DAPI-stained cells as indicated), or pChk1 and β-actin expression by Western blot (B). 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-g004: TMZ-induced Chk1 activation is independent of the TMZ-induced generation of ROS.MGMT-deficient U87 cells were pre-incubated with vehicle or the ROS scavenger NAC (400 µM, 2 hours) after which the cells were incubated with vehicle (untreated, U) or TMZ (100 µmol/L, 3 hours). TMZ was removed, vehicle or NAC was added, and the cells were harvested 1–24 hours following TMZ exposure for analysis of ROS based on the oxidation of 2,7-dichloro-dihydrofluorescein diacetate to an intracellular fluorescent compound (A, DAPI-stained cells as indicated), or pChk1 and β-actin expression by Western blot (B). 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 number of factors in addition to DNA damage have been suggested to play a role in TMZ-induced G2 arrest including most recently the generation of ROS by TMZ [35], [36]. To address the possibility that TMZ-induced ROS activate Chk1 independently of MGMT status and MMR capability, MGMT-deficient, MMR-proficient U87 cells were pre-exposed to vehicle or the reactive oxygen scavenger NAC, after which the cells were exposed to TMZ and monitored for ROS and Chk1 activation. TMZ-treated control cells began accumulating ROS within 1 hour after drug exposure (Fig. 4A, upper panel) and consistent with the data in Fig. 2B, activated Chk1 within 3 hours of TMZ exposure (Fig. 4B, upper panel). The pretreatment of the cells with NAC completely blocked the accumulation of ROS (Fig. 4A, lower panel). Elimination of the build-up of TMZ-induced ROS, however, had no effect on Chk1 phosphorylation, which occurred within 3 hours of TMZ exposure and was temporally and quantitatively indistinguishable from that noted in the TMZ-treated U87 cells in which ROS were generated (Fig. 4B, lower panel). These results show that although TMZ generates ROS in a time frame consistent with Chk1 phosphorylation, ROS do not cause Chk1 phosphorylation.


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 independent of the TMZ-induced generation of ROS.MGMT-deficient U87 cells were pre-incubated with vehicle or the ROS scavenger NAC (400 µM, 2 hours) after which the cells were incubated with vehicle (untreated, U) or TMZ (100 µmol/L, 3 hours). TMZ was removed, vehicle or NAC was added, and the cells were harvested 1–24 hours following TMZ exposure for analysis of ROS based on the oxidation of 2,7-dichloro-dihydrofluorescein diacetate to an intracellular fluorescent compound (A, DAPI-stained cells as indicated), or pChk1 and β-actin expression by Western blot (B). 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-g004: TMZ-induced Chk1 activation is independent of the TMZ-induced generation of ROS.MGMT-deficient U87 cells were pre-incubated with vehicle or the ROS scavenger NAC (400 µM, 2 hours) after which the cells were incubated with vehicle (untreated, U) or TMZ (100 µmol/L, 3 hours). TMZ was removed, vehicle or NAC was added, and the cells were harvested 1–24 hours following TMZ exposure for analysis of ROS based on the oxidation of 2,7-dichloro-dihydrofluorescein diacetate to an intracellular fluorescent compound (A, DAPI-stained cells as indicated), or pChk1 and β-actin expression by Western blot (B). 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 number of factors in addition to DNA damage have been suggested to play a role in TMZ-induced G2 arrest including most recently the generation of ROS by TMZ [35], [36]. To address the possibility that TMZ-induced ROS activate Chk1 independently of MGMT status and MMR capability, MGMT-deficient, MMR-proficient U87 cells were pre-exposed to vehicle or the reactive oxygen scavenger NAC, after which the cells were exposed to TMZ and monitored for ROS and Chk1 activation. TMZ-treated control cells began accumulating ROS within 1 hour after drug exposure (Fig. 4A, upper panel) and consistent with the data in Fig. 2B, activated Chk1 within 3 hours of TMZ exposure (Fig. 4B, upper panel). The pretreatment of the cells with NAC completely blocked the accumulation of ROS (Fig. 4A, lower panel). Elimination of the build-up of TMZ-induced ROS, however, had no effect on Chk1 phosphorylation, which occurred within 3 hours of TMZ exposure and was temporally and quantitatively indistinguishable from that noted in the TMZ-treated U87 cells in which ROS were generated (Fig. 4B, lower panel). These results show that although TMZ generates ROS in a time frame consistent with Chk1 phosphorylation, ROS do not cause Chk1 phosphorylation.

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