Mismatched nucleotides as the lesions responsible for radiosensitization with gemcitabine: a new paradigm for antimetabolite radiosensitizers

Abstract

Radiation sensitization by 2 ¶,2 ¶-difluoro-2 ¶-deoxycytidine (dFdCyd) has correlated with dATP depletion [dFdCDPmediated inhibition of ribonucleotide reductase (RR)] and S-phase accumulation. We hypothesized that radiosensitization by dFdCyd is due to nucleotide misincorporations in the presence of deoxynucleotide triphosphate pool imbalances, which, if not repaired, augments cell death following irradiation. The ability of dFdCyd to produce misincorporations was measured as pSP189 plasmid mutations in hMLH1-deficient [mismatch repair (MMR) deficient] and hMLH1-expressing (MMR proficient) HCT116 cells. Only MMR-deficient cells showed a significant increase in nucleotide misincorporations (2-to 3-fold increase; P V 0.01) after radiosensitizing concentrations of dFdCyd F 5 Gy radiation, which persisted for at least 96 h. dFdCyd (10 nmol/L) did not radiosensitize MMR-proficient HCT116 or A549 cells, but following small interfering RNA -mediated suppression of hMLH1, this concentration produced excellent radiosensitization (radiation enhancement ratios = 1.6 F 0.1 and 1.5 F 0.1, respectively; P < 0.05) and a 2.5-fold increase in mutation frequency in A549 cells. Cytosine arabinoside (1-B-D-arabinofuranosylcytosine), which can be incorporated into DNA but does not inhibit RR, failed to radiosensitize MMR-deficient cells or increase mutation frequency in the MMR-deficient and MMR-proficient cells. However, the RR inhibitor hydroxyurea radiosensitized MMR-deficient cells and increased nucleotide misincorporations (z5-fold increase; P < 0.05), thus further implicating the inhibition of RR as the mechanism underlying radiosensitization by dFdCyd. These data showed that the presence and persistence of mismatched nucleotides is integral to radiosensitization by dFdCyd and suggest a role for hMLH1 deficiency in eliciting the radiosensitizing effect.

MLH1 Deficiency Enhances Radiosensitization with 5-Fluorodeoxyuridine by Increasing DNA Mismatches

“…These studies support our previous findings with dFdCyd and hydroxyurea, radiosensitizers that produce an imbalance in dNTP pools (primarily a decrease in dATP) due to inhibition of ribonucleotide reductase, where mismatches in DNA occurred only under radiosensitizing conditions, and MLH1 deficiency enhanced radiosensitization (Flanagan et al, 2007). Together, these studies strongly support errors of replication as a general mechanism of radiosensitization for drugs that produce imbalances in dNTPs.…”

“…A single mutation at nearly any site in the coding sequence for the supF gene sequence prevents the expression of ␤-galactosidase (Seidman et al, 1985). The assay was performed as described previously (Flanagan et al, 2007). In brief, cells were transfected with the pSP189 plasmid overnight, incubated with FdUrd for 24 h, and plasmid extracted 24 h later.…”

“…Together, these studies strongly support errors of replication as a general mechanism of radiosensitization for drugs that produce imbalances in dNTPs. We have demonstrated that a decrease in dTTP produces different replication errors than drugs that produce decreases primarily in dATP (Flanagan et al, 2007), yet a strong relationship between DNA errors and radiosensitization still exists. These results demonstrate an important role for MLH1 and further implicate insufficient MMR in radiosensitization with drugs that produce dNTP imbalances.…”

“…Although FdUrd has been demonstrated to enhance cytotoxicity when administered with radiation by inhibiting the repair of radiation-induced DNA damage, radiosensitization can also occur in the absence of detectable DNA dsbs, suggesting another mechanism exists to explain the radiosensitizing effect of these drugs (Bruso et al, 1990;Davis et al, 1995). Our recent studies with another antimetabolite radiosensitizer, gemcitabine (dFdCyd), demonstrated that, at radiosensitizing concentrations, the dFdCyd-mediated depletion in dATP pools via inhibition of ribonucleotide reductase produced DNA mismatches that, if left unrepaired, resulted in radiosensitization (Flanagan et al, 2007). These studies also demonstrated a role for the mismatch repair (MMR) protein MLH1 whereby cells deficient in MLH1 were unable to repair drug-induced DNA mismatches and were more easily radiosensitized than MLH1-expressing, presumably MMR-proficient cells.…”

MGMT Is a Molecular Determinant for Potency of the DNA-EGFR–Combi-Molecule ZRS1

“…The mechanism of DNA repair by MGMT is based on its ability to remove the methyl group of O6-methylguanine by transferring onto its own cysteine residue. In the absence of MGMT, the O6-methylguanine lesion persists and attempt to repair it by the mismatch repair (MMR) is known to be responsible for triggering apoptosis (13,36). The A549 and A427 are MMR-proficient cells (37,38).…”