Role of deoxycytidine kinase (dCK) activity in gemcitabine's radioenhancement in mice and human cell lines in vitro.
BACKGROUND
Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is a deoxycytidine nucleoside analog which has a marked effect on several enzymes involved in DNA synthesis and repair. Gemcitabine has been tested as a radiosensitizer in various biological models, and radiation dose modification factors (DMF) have been reported in the range between 1.1 and 2.4. Gemcitabine is a prodrug that requires intracellular activation by phosphorylation into its active triphosphate dFdCTP form. Deoxycytidine kinase (dCK) is the enzyme involved in the first phosphorylation cascade, and several observations have suggested that dCK was a limiting factor for the cytotoxic activity of gemcitabine.


OBJECTIVE
In the present article, we investigated the relationship between dCK activity and gemcitabine's radiosensitization in four mice and two human cell lines.


METHODS
Four mice and two human tumor cell lines were investigated. Radiosensitization was assessed on confluent cell incubated with 5 microM gemcitabine for 3 h prior to a single radiation dose. Enzymatic activity was assessed using deoxycytidine as substrate with (specific activity) or without (total activity) inhibition of thymidine kinase 2 activity. dCK protein level was assessed by immunoblotting using a rabbit anti-human dCK antibody. mRNA expression was assessed with Northern blot using beta-actin as internal control.


RESULTS
Gemcitabine's radiosensitization was heterogeneous with DMF ranging from 0.8 to 1.5. A good correlation was observed between the specific dCK activity and the protein level or the mRNA expression indicating that in our cell systems no post-transcriptional or post-translational activation occurred. An excellent correlation (r = 0.99) was observed between the specific enzymatic activity and gemcitabine's radiosensitization. Cell lines that expressed a high enzymatic activity were the more radiosensitized by gemcitabine. This correlation holds when radiosensitization was plotted against the dCK mRNA expression and protein level.


CONCLUSIONS
The present study has suggested the role of dCK activity in gemcitabine's radioenhancement in human and mice cell lines. The study suggests that determination of the enzymatic activity prior to a concurrent gemcitabine and radiotherapy treatment might represent a good predictive assay for tumor response. Such concept should deserve further testing in pre-clinical and clinical settings.
Department of Radiation Oncology and Laboratory of Radiobiology, Université Catholique de Louvain, St-Luc University Hospital, 10 Ave Hippocrate, 1200 Brussels, Belgium.
Vincent
V
Grégoire
Jean-François
JF
Rosier
Marc
M
De Bast
Monique
M
Bruniaux
Blanche
B
De Coster
Michelle
M
Octave-Prignot
Pierre
P
Scalliet
D000818:Animals; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003842:Deoxycytidine Kinase / Q000378:metabolism*; D005354:Fibrosarcoma; D006801:Humans; D051379:Mice; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D000093542:Gemcitabine
D011838:Radiation-Sensitizing Agents; D003841:Deoxycytidine; D003842:Deoxycytidine Kinase; D000093542:Gemcitabine