Apurinic/apyrimidinic endonuclease-1 protein level is associated with the cytotoxicity of L-configuration deoxycytidine analogs (troxacitabine and beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine) but not D-configuration deoxycytidine analogs (gemcitabine and beta-D-arabinofuranosylcytosine).
Beta-L-dioxolane-cytidine (L-OddC, BCH-4556, Troxacitabine), a novel L-configuration deoxycytidine analog, is under phase III clinical trial for cancer treatment. We showed that human apurinic/apyrimidinic endonuclease (APE-1) has exonuclease activity for preferentially removing L-OddC and other L-configuration nucleosides over D-configuration nucleosides from the 3' terminus of DNA in vitro. In this study, we examined whether APE-1 protein plays a role in the cytotoxicity of L-OddC. We established RKO (human colorectal carcinoma) cell lines that can be induced by doxycycline to overexpress 4- to 5-fold either APE-1 wild type (wt), C65A (redox deficient), E96A (exonuclease deficient), or E96Q (exonuclease deficient) mutants and to down-regulate endogenous APE-1 by short hairpin RNA to 10% of the original level. Clonogenic results indicated that the induction of wt or C65A, but not E96A or E96Q, made cells approximately 2-fold resistant to L-OddC and beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (L-Fd4C), whereas the down-regulation of APE-1 sensitized cells by approximately 2-fold to L-OddC and L-Fd4C. The alteration of APE-1 in cells did not change the sensitivity of these cells to beta-D-2',2'-difluorodeoxycytidine (dFdC; gemcitabine) and beta-D-arabinofuranosylcytosine (AraC), both of which are D-configuration deoxycytidine analogs. The DNA incorporation of L-OddC, but not that of dFdC, was decreased by the induction of wt APE-1 but not E96A mutant and was increased by the down-regulation of APE-1. The rate of retention of L-OddC was inversely correlated to the level of APE-1 in isolated nuclei; however, this was not the case for dFdC. In conclusion, this study supports the hypothesis that APE-1 plays a critical role in the actions of L-configuration but not D-configuration nucleoside analogs.
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
Wing
W
Lam
Shin-Young
SY
Park
Chung-Hang
CH
Leung
Yung-Chi
YC
Cheng
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D015179:Colorectal Neoplasms; D003596:Cytosine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D004148:Dioxolanes / Q000493:pharmacokinetics / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D000093542:Gemcitabine
D004148:Dioxolanes; D034741:RNA, Small Interfering; D003841:Deoxycytidine; C074908:troxacitabine; D003596:Cytosine; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D000093542:Gemcitabine