Role of deoxycytidine kinase (dCK) activity in gemcitabine’s radioenhancement in mice and human cell lines in vitro Vincent Gre ´goire a, * , Jean-Franc ¸ois Rosier a,b , Marc De Bast a , Monique Bruniaux a,b , Blanche De Coster a , Michelle Octave-Prignot a , Pierre Scalliet a a Department of Radiation Oncology and Laboratory of Radiobiology, Universite ´ Catholique de Louvain, St-Luc University Hospital, 10 Ave Hippocrate, 1200 Brussels, Belgium b Tumoral Metabolism Research Unit, C.H. Jolimont-Lobbes, 7161 Haine-St-Paul, Belgium Received 10 December 2001; received in revised form 8 April 2002; accepted 7 May 2002 Abstract Background: Gemcitabine (dFdC, 2 0 ,2 0 -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. Materials and methods: Four mice and two human tumor cell lines were investigated. Radiosensitization was assessed on confluent cell incubated with 5 mM 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 b-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 ðr ¼ 0:99Þ was observed between the specific enzymatic activity and gemcitabi- ne’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. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Radioenhencement; Deoxycytidine kinase; Cell lines; Gemcitabine 1. Introduction Gemcitabine (dFdC, 2 0 ,2 0 -difluorodeoxycytidine) is a deoxycytidine nucleoside analog which has a marked effect on several enzymes involved in DNA synthesis and repair [32,34]. Like other nucleoside analogs, gemcitabine is a prodrug that requires intracellular activation by phosphory- lation into its active triphosphate dFdCTP form. dFdCTP is incorporated into DNA at the penultimate position and blocks further elongation of DNA strand. An array of self- potentiation mechanisms have been identified and they likely contribute to both the high accumulation and the low elimination of the intracellular dFdCTP [17]. Gemcita- bine can also be incorporated into RNA [42] and can also induce apoptosis [4,14,20]. Gemcitabine has been tested in various phase I–II trials and promising clinical activity has been reported in non small cell lung cancer, as in pancreatic, ovarian, breast, bladder and head and neck tumors [1,5]. Gemcitabine has been tested as a radiosensitizer in various biological models. In vitro, radioenhancement was observed in several human and mice tumor cell lines with radiation dose modification factors (DMF) ranging from 1.1 to 2.4, depending on both drug incubation time and concen- Radiotherapy and Oncology 63 (2002) 329–338 0167-8140/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-8140(02)00106-8 www.elsevier.com/locate/radonline * Corresponding author.