Resistance to gemcitabine in a human follicular lymphoma cell line is due to partial deletion of the deoxycytidine kinase gene.
BACKGROUND
Gemcitabine is an analogue of deoxycytidine with activity against several solid tumors. In order to elucidate the mechanisms by which tumor cells become resistant to gemcitabine, we developed the resistant subline RL-G from the human follicular lymphoma cell line RL-7 by prolonged exposure of parental cells to increasing concentrations of gemcitabine.


RESULTS
In vitro, the IC50 increased from 0.015 microM in parental RL-7 cells to 25 microM in the resistant variant, RL-G. Xenografts of both cell lines developed in nude mice were treated with repeated injections of gemcitabine. Under conditions of gemcitabine treatment which totally inhibited the development of RL-7 tumors, RL-G derived tumors grew similarly to those of untreated animals, demonstrating the in vivo resistance of RL-G cells to gemcitabine. HPLC experiments showed that RL-G cells accumulated and incorporated less gemcitabine metabolites into DNA and RNA than RL-7 cells. Gemcitabine induced an S-phase arrest in RL-7 cells but not in RL-G cells. Exposure to gemcitabine induced a higher degree of apoptosis in RL-7 than in RL-G cells, with poly-(ADP-ribose) polymerase cleavage in RL-7 cells. No modifications of Bcl-2 nor of Bax expression were observed in RL-7 or RL-G cells exposed to gemcitabine. These alterations were associated with the absence of the deoxycytidine kinase mRNA expression observed by quantitative RT-PCR in RL-G cells. PCR amplification of désoxycytidine kinase gene exons showed a partial deletion of the dCK gene in RL-G cells.


CONCLUSIONS
These results suggest that partial deletion of the dCK gene observed after selection in the presence of gemcitabine is involved with resistance to this agent both in vitro and in vivo.
Galmarini
Carlos María
CM
INSERM 590 - Laboratoire de Cytologie Analytique, Faculté de Médecine Rockefeller, Université Claude Bernard Lyon 1, Lyon, France. fgalma@rockefeller.univ-lyon1.fr
Clarke
Marilyn L
ML
Jordheim
Lars
L
Santos
Cheryl L
CL
Cros
Emeline
E
Mackey
John R
JR
Dumontet
Charles
C
D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000493:pharmacokinetics; D003842:Deoxycytidine Kinase / Q000172:deficiency* / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D005260:Female; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D008224:Lymphoma, Follicular / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation / Q000379:methods; D009696:Nucleic Acids / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014183:Transplantation, Heterologous; D014316:Tritium / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured; D000093542:Gemcitabine
D009696:Nucleic Acids; D003841:Deoxycytidine; D014316:Tritium; D003842:Deoxycytidine Kinase; D000093542:Gemcitabine