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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 associated with TMZ-induced ALD.U87 or U87+MGMT cells (A, B) were incubated with TMZ (100 µmol/L, 3 hours), ABT888 (5 µM, continuous), or both (one hr ABT pre-incubation followed by the 3 hr TMZ exposure). Cells were then washed and incubated in drug-free (for TMZ groups) or ABT-containing (for the ABT or ABT+TMZ groups ) media and harvested 1–24 hrs later for analysis of tail moment by single cell alkaline gel electrophoresis (A), or pChk1, Chk1, MGMT, and β-actin expression by Western blot (B). Tail moment values are the mean+standard error for three independent experiments. Similar tail moment and Western blot analyses were performed in G55 cells and G55 cells depleted of MGMT by a one hr pre-exposure to BG (5 µM) 1–24 hrs post TMZ (100 µmol/L, 3 hours) exposure (C). 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-g005: TMZ-induced Chk1 activation is associated with TMZ-induced ALD.U87 or U87+MGMT cells (A, B) were incubated with TMZ (100 µmol/L, 3 hours), ABT888 (5 µM, continuous), or both (one hr ABT pre-incubation followed by the 3 hr TMZ exposure). Cells were then washed and incubated in drug-free (for TMZ groups) or ABT-containing (for the ABT or ABT+TMZ groups ) media and harvested 1–24 hrs later for analysis of tail moment by single cell alkaline gel electrophoresis (A), or pChk1, Chk1, MGMT, and β-actin expression by Western blot (B). Tail moment values are the mean+standard error for three independent experiments. Similar tail moment and Western blot analyses were performed in G55 cells and G55 cells depleted of MGMT by a one hr pre-exposure to BG (5 µM) 1–24 hrs post TMZ (100 µmol/L, 3 hours) exposure (C). 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: The observation that both MGMT-proficient and MGMT-deficient cells exhibit Chk1 activation following TMZ exposure implies that lesions unrelated to O6MG must play a role in the early activation of Chk1. In addition to O6MG, TMZ induces N7G and 3 meA DNA lesions, both of which are substrates for rapid removal by the base excision repair system but not by MGMT [37]. To address the possibility that some form of DNA damage induced by either N7G, 3 meA could underlie the TMZ-induced activation of Chk1, the paired MGMT proficient/deficient U87 and G55 cells, all of which are base excision repair- proficient [38], were exposed to TMZ, and DNA damage in the form of single strand breaks was revealed by single cell gel electrophoresis assays performed under denaturing alkaline pH conditions. The amount of ALD increased rapidly in MGMT-proficient U87+MGMT (open diamonds, Fig. 5A) and G55 cells (open diamonds, Fig. 5C) in the first hour post TMZ exposure, peaking at 3 hours and returning to control levels by 12 to 24 hours post drug. This ALD was not caused by O6MG lesions, their repair, or their downstream consequences because MGMT-mediated repair of O6MG does not involve base removal or stand breakage [38], [39], and because early induction of ALD following TMZ was also noted in cells (U87 and G55+BG) incapable of repairing O6MG (closed diamonds, Fig. 5A and C). The onset and disappearance of the ALD in MGMT-proficient cells was, however, temporally consistent with the pattern of ATR activation and Chk1 phosphorylation in these cells (Fig. 2B–D), and with the known base excision and strand cleavage processes involved in base excision repair of N7G and 3 meA lesions. Furthermore, although pre-incubation of MGMT-proficient U87+MGMT cells with the base excision repair/PARP inhibitor ABT888 alone had no effect on the extent of ALD (open squares, Fig. 5A) or Chk1 phosphorylation (Fig. 5B, middle panel), combined exposure to ABT888 plus TMZ significantly slowed the disappearance of ALD (open triangles, Fig. 5A) and prolonged TMZ-induced Chk1 activation (Fig. 5B, lower panel) relative to cells treated with TMZ or ABT888 alone. These results suggest that the early phosphorylation of Chk1 following TMZ exposure appears to be a consequence of the single-strand DNA damage (but not double-stand DNA damage) generated by the repair of TMZ-induced N7G and 3 meA lesions. In MGMT-proficient cells capable of processing TMZ-induced N7G and 3 meA lesions as well as O6MG lesions, elimination of ALD parallels the return of pChk1 to control levels. In contrast, in MGMT-deficient cells capable of processing only N7G and 3 meA lesions, unrepaired O6MG lesions and their consequences appear to lead to the sustained ALD and Chk1 phosphorylation noted up to 24 hours post-TMZ in these cells (Fig. 5A–C).


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 associated with TMZ-induced ALD.U87 or U87+MGMT cells (A, B) were incubated with TMZ (100 µmol/L, 3 hours), ABT888 (5 µM, continuous), or both (one hr ABT pre-incubation followed by the 3 hr TMZ exposure). Cells were then washed and incubated in drug-free (for TMZ groups) or ABT-containing (for the ABT or ABT+TMZ groups ) media and harvested 1–24 hrs later for analysis of tail moment by single cell alkaline gel electrophoresis (A), or pChk1, Chk1, MGMT, and β-actin expression by Western blot (B). Tail moment values are the mean+standard error for three independent experiments. Similar tail moment and Western blot analyses were performed in G55 cells and G55 cells depleted of MGMT by a one hr pre-exposure to BG (5 µM) 1–24 hrs post TMZ (100 µmol/L, 3 hours) exposure (C). 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-g005: TMZ-induced Chk1 activation is associated with TMZ-induced ALD.U87 or U87+MGMT cells (A, B) were incubated with TMZ (100 µmol/L, 3 hours), ABT888 (5 µM, continuous), or both (one hr ABT pre-incubation followed by the 3 hr TMZ exposure). Cells were then washed and incubated in drug-free (for TMZ groups) or ABT-containing (for the ABT or ABT+TMZ groups ) media and harvested 1–24 hrs later for analysis of tail moment by single cell alkaline gel electrophoresis (A), or pChk1, Chk1, MGMT, and β-actin expression by Western blot (B). Tail moment values are the mean+standard error for three independent experiments. Similar tail moment and Western blot analyses were performed in G55 cells and G55 cells depleted of MGMT by a one hr pre-exposure to BG (5 µM) 1–24 hrs post TMZ (100 µmol/L, 3 hours) exposure (C). 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: The observation that both MGMT-proficient and MGMT-deficient cells exhibit Chk1 activation following TMZ exposure implies that lesions unrelated to O6MG must play a role in the early activation of Chk1. In addition to O6MG, TMZ induces N7G and 3 meA DNA lesions, both of which are substrates for rapid removal by the base excision repair system but not by MGMT [37]. To address the possibility that some form of DNA damage induced by either N7G, 3 meA could underlie the TMZ-induced activation of Chk1, the paired MGMT proficient/deficient U87 and G55 cells, all of which are base excision repair- proficient [38], were exposed to TMZ, and DNA damage in the form of single strand breaks was revealed by single cell gel electrophoresis assays performed under denaturing alkaline pH conditions. The amount of ALD increased rapidly in MGMT-proficient U87+MGMT (open diamonds, Fig. 5A) and G55 cells (open diamonds, Fig. 5C) in the first hour post TMZ exposure, peaking at 3 hours and returning to control levels by 12 to 24 hours post drug. This ALD was not caused by O6MG lesions, their repair, or their downstream consequences because MGMT-mediated repair of O6MG does not involve base removal or stand breakage [38], [39], and because early induction of ALD following TMZ was also noted in cells (U87 and G55+BG) incapable of repairing O6MG (closed diamonds, Fig. 5A and C). The onset and disappearance of the ALD in MGMT-proficient cells was, however, temporally consistent with the pattern of ATR activation and Chk1 phosphorylation in these cells (Fig. 2B–D), and with the known base excision and strand cleavage processes involved in base excision repair of N7G and 3 meA lesions. Furthermore, although pre-incubation of MGMT-proficient U87+MGMT cells with the base excision repair/PARP inhibitor ABT888 alone had no effect on the extent of ALD (open squares, Fig. 5A) or Chk1 phosphorylation (Fig. 5B, middle panel), combined exposure to ABT888 plus TMZ significantly slowed the disappearance of ALD (open triangles, Fig. 5A) and prolonged TMZ-induced Chk1 activation (Fig. 5B, lower panel) relative to cells treated with TMZ or ABT888 alone. These results suggest that the early phosphorylation of Chk1 following TMZ exposure appears to be a consequence of the single-strand DNA damage (but not double-stand DNA damage) generated by the repair of TMZ-induced N7G and 3 meA lesions. In MGMT-proficient cells capable of processing TMZ-induced N7G and 3 meA lesions as well as O6MG lesions, elimination of ALD parallels the return of pChk1 to control levels. In contrast, in MGMT-deficient cells capable of processing only N7G and 3 meA lesions, unrepaired O6MG lesions and their consequences appear to lead to the sustained ALD and Chk1 phosphorylation noted up to 24 hours post-TMZ in these cells (Fig. 5A–C).

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