zd-9331 and Lung-Neoplasms

Uracil DNA glycosylase (UDG) is a base excision repair enzyme responsible for the removal of uracil present in DNA after cytosine deamination or misincorporation during replication. Inhibition of thymidylate synthase (TS), an important target for cancer chemotherapy, leads to deoxythymidine triphosphate (dTTP) pool depletion and elevation of deoxyuridine monophosphate (dUMP) pools which may also result in the accumulation of deoxyuridine triphosphate (dUTP). Large quantities of dUTP are believed to overwhelm the pyrophosphatase dUTPase, leading to misincorporation of uracil into DNA. Uracil is removed from DNA by uracil DNA glycosylase (UDG) resulting in an abasic site, but since the ratio dUTP:dTTP may remain high during continuing TS inhibition uracil can become re-incorporated into DNA causing a futile cycle eventually leading to DNA damage and cell death. This study has used isogenic cell lines differing in their expression of UDG to investigate the role of this enzyme in sensitivity to the specific TS inhibitors, ZD9331 and raltitrexed. The study showed that although increased expression and activity of UDG may lead to increased cell growth inhibition after TS inhibition over the first 24 h of treatment (measured using 3-(4,5-dimethyl (thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), probably due to increased damage to single-stranded DNA, the level of enzyme expression does not affect cell viability or cell death (measured using clonogenic assay, cell counting of attached/detached cells and cleavage of both poly ADP-ribose polymerase (PARP) and caspase 3). Increased expression and activity of UDG did not affect sensitivity to TS inhibition at later time points (up to 72 h treatment). Therefore UDG does not appear to play a major role in the response to TS inhibition, at least in the model used, and the results suggest that other determinants of response previously investigated, such as TS and dUTPase, may be more important for the response to TS inhibition.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Cell Survival; Comet Assay; DNA Damage; DNA Glycosylases; DNA Repair; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Inhibitory Concentration 50; Lung Neoplasms; N-Glycosyl Hydrolases; Quinazolines; Thiophenes; Thymidylate Synthase; Transfection; Tumor Cells, Cultured; Uracil-DNA Glycosidase

Uracil DNA misincorporation and misrepair of DNA have been recognized as important events accompanying thymidylate synthase (TS) inhibition. dUTPase catalyses the hydrolysis of dUTP to dUMP, thereby maintaining low intracellular dUTP. We have addressed the relationship between dUTPase expression and cellular sensitivity to TS inhibition in four human lung tumour cell lines. Sensitivity (5-day MTT assay) to the growth inhibitory effects of the non-polyglutamatable, specific quinazoline TS inhibitor ZD9331, varied up to 20-fold (IC(50)3-70 nM). TS protein expression correlated with TS activity (r(2)= 0.88, P = 0.05). Intracellular concentrations of drug following exposure to ZD9331 (1 microM, 24 h) varied by approximately 2-fold and dTTP pools decreased by > 80% in all cell lines. No clear associations across the cell lines between intracellular drug concentrations, TS activity/expression, or TTP depletion could be made. dUTPase activity varied 17-fold and correlated with dUTPase protein expression (r(2)= 0.94, P = 0.03). There was a striking variation in the amount of dUTP formed following exposure to ZD9331 (between 1.3 and 57 pmole 10(-6)cells) and was in general inversely associated with dUTPase activity. A large expansion in the dUTP pool was associated with increased sensitivity to a 24-h exposure to ZD9331 in A549 cells that have low dUTPase activity/expression. dUTPase expression and activity were elevated (approximately 3-fold) in two variants of a human lymphoblastoid cell line with acquired resistance to TS inhibitors, further suggesting an important role for this enzyme in TS inhibited cells.

Topics: Antineoplastic Agents; Apoptosis; DNA Repair; Humans; Lung Neoplasms; Pyrophosphatases; Quinazolines; Thymidylate Synthase; Tumor Cells, Cultured