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Enhancement of DNA Ligase I Level by Gemcitabine in Human Cancer Cells¹

Institute for Drug Development, Cancer Therapy and Research Center, San Antonio, Texas 78245

Purpose: DNA ligase I is an essential enzyme for completing DNA replication and DNA repair by ligating Okazaki fragments and by joining single-strand breaks formed either directly by DNA-damaging agents or indirectly by DNA repair enzymes, respectively. In this study, we examined whether the DNA ligase I level could be modulated in human tumor cell lines by treatment with gemcitabine (2', 2'-difluoro-2'-deoxycytidine), which is a nucleoside analogue of cytidine with proven antitumor activity against a broad spectrum of human cancers in clinical studies.

Experimental Design: To determine the effect of gemcitabine on DNA ligase I expression, Western blot analysis was used to measure the DNA ligase I levels in MiaPaCa, NGP, and SK-N-BE cells treated with different concentrations of gemcitabine and harvested at different time intervals. Cell cycle analysis was also performed to determine the underlying mechanism of DNA ligase I level enhancement in response to gemcitabine. In addition, other agents that share the same mechanism of action with gemcitabine were used to elucidate further details.

Results: When different types of tumor cell lines, including MiaPaCa, NGP, and SK-N-BE, were treated with gemcitabine, the level of DNA ligase I increased severalfold despite significant cell growth inhibition. In contrast, other DNA ligases (III and IV) either remained unchanged or decreased with treatment. Cell cycle analysis showed that arrest in S-phase corresponded to an increase of DNA ligase I levels in gemcitabine treated cells. Other agents, such as 1-ß-D-arabinofuranosylcytosine and hydroxyurea, which partly share mechanisms of action with gemcitabine by targeting DNA polymerases and ribonucleotide reductase, respectively, also caused an increase of DNA ligase I levels. However, 5-fluorouracil, which predominantly targets thymidylate synthase, did not cause an increase of DNA ligase I level.

Conclusions: Our results suggest that an arrest of DNA replication caused by gemcitabine treatment through incorporation of gemcitabine triphosphate into replicating DNA and inhibition of ribonucleotide reductase would trigger an increase in DNA ligase I levels in cancer cells. The elevated presence of DNA ligase I in S-phase-arrested cells leads us to speculate that DNA ligase I might have an important role in repairing DNA damage caused by stalled replication forks.

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