DNA damage and homologous recombination signaling induced by thymidylate deprivation.

DNA damage is accepted as a consequence of thymidylate deprivation induced by chemotherapeutic inhibitors of thymidylate synthase (TS), but the types of damage and signaling responses remain incompletely understood. Thymidylate deprivation increases dUTP and uracil in DNA, which is removed by base excision repair (BER). Because BER requires a synthesis step, strand break intermediates presumably accumulate. Thymidylate deprivation also induces cell cycle arrest during replication. Homologous recombination (HR) is a means of repairing persistent BER intermediates and collapsed replication forks. There are also intimate links between HR and S-phase checkpoint pathways. In this study, the goals were to determine the involvement of HR-associated proteins and DNA damage signaling responses to thymidylate deprivation. When RAD51, which is a central component of HR, was depleted by siRNA cells were sensitized to raltitrexed (RTX), which specifically inhibits TS. To our knowledge, this is the first demonstration in mammalian cells that depletion of RAD51 causes sensitivity to thymidylate deprivation. Activation of DNA damage signaling responses was examined following treatment with RTX. Phosphorylation of replication protein A (RPA2 subunit) and formation of damage-induced foci were strikingly evident following IC(50) doses of RTX. Induction was much more striking following RTX treatment than with hydroxyurea, which is commonly used to inhibit replication. RTX treatment also induced foci of RAD51, gamma-H2AX, phospho-Chk1, and phospho-NBS1, although the extent of co-localization with RPA2 foci varied. Collectively, the results suggest that HR and S-phase checkpoint signaling processes are invoked by thymidylate deprivation and influence cellular resistance to thymidylate deprivation.

Glutathione depletion-induced thymidylate insufficiency for DNA repair synthesis.

Dietary methionine (Met) deficiency is known to divert folate away from de novo biosyntheses of purines and the pyrimidine, thymidylate, to the resynthesis of Met resulting in deoxynucleoside triphosphate imbalance. We have recently shown that Met can easily be depleted and methylation can be impaired by exposure to a model glutathione (GSH)-depleting agent, bromobenzene (BB). GSH depletion-induced Met depletion, therefore, could cause thymidylate insufficiency for DNA repair synthesis. The administration of thymidine (Thy) should repair this impairment. When this hypothesis was examined in the present study, several interesting results were found. The administration of Thy labeled with [2-14C]Thy to BB-treated Syrian hamsters at either 1, 5, 7 or 9 h after BB resulted in an attenuation of liver toxicity. Intrahepatic hemorrhage, which is a typical characteristic of BB toxicity in the Syrian hamster, was decreased in the BB + Thy groups. The attenuation of liver toxicity was accompanied by a progressive increase of Thy incorporation into liver genomic DNA at 24 h after BB. With respect to the time points chosen for Thy administration, Thy incorporation found in the BB + Thy groups were 2-, 2-, 3- and 4-fold of the controls that received only Thy. The results provide evidence that BB causes a progressive increase of thymidylate insufficiency in liver cells. Thymidylate insufficiency is due to Met depletion, a depletion that occurs as a result of GSH depletion.

A specialized form of chromosomal DNA degradation induced by thymidylate stress in mouse FM3A cells.

Thymidylate synthase-negative mutant mouse cells starved of thymidine or their parental FM3A cells treated with 5-fluoro-2'-deoxyuridine produced DNA fragments ranging from 50 to 200 kilobase pairs with a peak at 100 kb in length as determined by pulsed-field agarose gel electrophoresis. Accumulation of the DNA fragments following such thymidylate stress was time-dependent but their size distribution did not change in either case. Regions of the chromosomal DNA breaks seemed to be restricted to those where DNA replication was in progress as shown by pulse-labeling of the DNA synthesis. Emetine, an inhibitor of protein synthesis, blocked the accumulation of the DNA fragments when present during thymidylate stress. Cell-free extracts prepared from the thymidylate-stressed cells derived by either of the above means were capable of degrading DNA in chromatins prepared from normally growing cells in vitro. The resulting DNA fragments were similar but with a somewhat broader size distribution compared to those produced in vivo. The broader distribution of the fragments produced in the in vitro reaction became closer to the pattern obtained in vivo when ATP and 4 deoxyribonucleotides were added to the reaction.