Early Chk1 Phosphorylation Is Driven by Temozolomide-Induced, DNA Double Strand Break- and Mismatch Repair-Independent DNA Damage
Figure 3
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.