doi:10.1016/j.ijrobp.2005.04.012 BIOLOGY CONTRIBUTION MISMATCH REPAIR PROFICIENCY IS NOT REQUIRED FOR RADIOENHANCEMENT BY GEMCITABINE CHRIS VAN BREE,PH.D., HANS M. RODERMOND, B.SC., JUDITH DE VOS, B.SC., JAAP HAVEMAN,PH.D., AND NICOLAAS A. P. FRANKEN,PH.D. Department of Radiotherapy, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Purpose: Mismatch repair (MMR) proficiency has been reported to either increase or decrease radioenhance- ment by 24-h incubations with gemcitabine. This study aimed to establish the importance of MMR for radioenhancement by gemcitabine after short-exposure, high-dose treatment and long-exposure, low-dose treat- ment. Methods and Materials: Survival of MMR-deficient HCT116 and MMR-proficient HCT116  3 cells was analyzed by clonogenic assays. Mild, equitoxic gemcitabine treatments (4 h, 0.1 M vs. 24 h, 6 nM) were combined with -irradiation to determine the radioenhancement with or without recovery. Gemcitabine metab- olism and cell-cycle effects were evaluated by high-performance liquid chromatography analysis and bivariate flow cytometry. Results: Radioenhancement after 4 h of 0.1 M of gemcitabine was similar in both cell lines, but the radioen- hancement after 24 h of 6 nM of gemcitabine was reduced in MMR-proficient cells. No significant differences between both cell lines were observed in the gemcitabine metabolism or cell-cycle effects after these treatments. Gemcitabine radioenhancement after recovery was also lower in MMR-proficient cells than in MMR-deficient cells. Conclusion: Mismatch repair proficiency decreases radioenhancement by long incubations of gemcitabine but does not affect radioenhancement by short exposures to a clinically relevant gemcitabine dose. Our data suggest that MMR contributes to the recovery from gemcitabine treatment. © 2005 Elsevier Inc. Mismatch repair, Gemcitabine, Ionizing radiation. INTRODUCTION Gemcitabine (difluorodeoxycytidine, dFdC) is a deoxycyti- dine analog with clinical applications in non–small-cell lung cancer and pancreatic cancer (1, 2). Gemcitabine is predom- inantly administered to patients as a 30-min i.v. infusion with tolerated serum levels of 10 – 40 M, after which gemcitabine is rapidly eliminated (3). For activation, gem- citabine requires phosphorylation, with the initial phosphor- ylation by deoxycytidine kinase being the rate-limiting step, to gemcitabine triphosphate, which has a longer intracellu- lar half life (4). Pretreatment with a high dose for a short period (micromolar range for 2– 6 h), as well as a lower dose for a longer period (nanomolar range for 24 h), has shown that gemcitabine is a potent enhancer of radiosensitivity that is persistent for up to 72 h after the end of drug exposure (3–6). Studies in vivo have indicated that an optimal ther- apeutic ratio is observed when gemcitabine is administered 24 h before radiotherapy, primarily a result of the more rapid recovery of normal tissues from gemcitabine treat- ment (7–10). These studies have suggested that clinical radioenhancement of gemcitabine may be the result of an interaction with several fractions of the radiotherapy sched- ule. DNA repair pathways using short DNA patches, such as nonhomologous end joining and base excision repair, are thought not to play an important role in gemcitabine ra- dioenhancement (11, 12). Instead, homologous recombina- tion, a long-patch DNA repair pathway, has been argued to be the target for gemcitabine to enhance cellular radiosen- Reprint requests to: Chris van Bree, Ph.D., Department of Radiotherapy, Academic Medical Center, University of Amster- dam, Room F0-205, P.O. Box 22700, Amsterdam 1100 DE, The Netherlands. Tel: (+31) 20-566-8769; Fax: (+31) 20-609-1278; E-mail: c.vanbree@amc.uva.nl Acknowledgments—The authors thank C. H. van Oven (Center for Microscopic Research, AMC) for technical assistance with flow cytometry and Dr. A. B. P. van Kuilenburg and R. Leen (Labora- tory of Genetic Metabolic Disease, AMC) for measuring the gem- citabine metabolites. Dr. Boland (Division of Gastroenterology, University of California, San Diego) is acknowledged for supply- ing the HCT116 + 3 cells, Dr. J. B. Reitsma (Department of Clinical Epidemiology, AMC) for support with statistical analysis, and Prof. H. H. Kampinga (Department for Stress and Radiation Biology, University of Groningen) for helpful discussions and critical reading of the manuscript. Maurits and Anna de Cock are gratefully acknowledged for financial support of laboratory equip- ment. Received Sept 13, 2004, and in revised form Jan 28, 2005. Accepted for publication April 4, 2005. Int. J. Radiation Oncology Biol. Phys., Vol. 62, No. 5, pp. 1504 –1509, 2005 Copyright © 2005 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/05/$–see front matter 1504