Pharmacological Research 55 (2007) 343–349 Cytotoxic activity of gemcitabine and correlation with expression profile of drug-related genes in human lymphoid cells Elisa Giovannetti a,∗ , Valentina Mey a , Lucia Loni b , Sara Nannizzi a , Gemma Barsanti b , Grazia Savarino a , Simona Ricciardi a , Mario Del Tacca a , Romano Danesi a a Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, 55 Via Roma, 56126 Pisa, Italy b Campo di Marte General Hospital, Lucca, Italy Accepted 8 January 2007 Abstract Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA polymerization with promising activity in hematologic malignancies. Gemcitabine enters the cell mostly via the human equilibrative nucleoside transporter-1 (hENT1), while drug metabolism occurs by phosphorylation by deoxycytidine kinase (dCK), 5 ′ -nucleotidase (cN-II) and cytidine deaminase (CDA) are the main inactivating enzymes. The aim of this study was to investigate the role of these determinants in gemcitabine cytotoxicity and analyze their expression in lymphoid cells. Cytotoxicity was assessed by MTT, and modulated by simultaneous addition of 2 ′ -deoxycytidine (dCK natural substrate), tetrahydrouridine (CDA competitive inhibitor) and diethylpyrocarbonate (cN-II non-competitive inhibitor), while the expression of hENT1, dCK, cN-II, CDA and RR in WIL2-S, Jurkat and CCRF- CEM cells as well as in lymphoid cells from 25 chronic lymphocytic B-leukemia (B-CLL) patients was studied with quantitative-PCR. Cell cycle modulation and induction of apoptosis were analyzed by cytofluorimetry and bisbenzimide staining. Gemcitabine was highly cytotoxic, increased the cells in S-phase and significantly enhanced apoptosis. The crucial role of metabolism in gemcitabine activity was confirmed by the significant modulation of cytotoxicity by inhibitors of dCK, CDA and cN-II. Furthermore, PCR demonstrated a correlation between gemcitabine sensitivity and expression of its determinants, and that their values were within those observed in patients. These data indicate that gemcitabine is cytotoxic against lymphoid cells, affecting cell cycle and apoptosis. Furthermore, chemosensitivity may be predicted on the basis of gene expression profile of critical determinants involved in gemcitabine mechanism of action, suggesting the use of pharmacogenetic profiling for treatment optimization. © 2007 Elsevier Ltd. All rights reserved. Keywords: Gemcitabine; Human lymphoid cells; Cell cycle modulation; Apoptosis; Gene expression 1. Introduction The majority of adults affected by malignant lymphoprolifer- ative diseases eventually become chemoresistant and alternative treatments should be explored to improve their outcomes [1,2]. Gemcitabine (2 ′ ,2 ′ -difluorodeoxycytidine, dFdC) has a broad spectrum of antitumour activity against solid tumours; how- ever, its effectiveness in hematologic malignancies remains to be established [3]. Several deoxynucleoside analogs are essential components of the management of acute leukemias (cytarabine), hairy cell leukemia (pentostatin) and low-grade non-Hodgkin’s lymphomas (cladribine, fludarabine) [4]. There is substantial ∗ Corresponding author. Tel.: +39 050 830148; fax: +39 050 562020. E-mail address: e.giovannetti@med.unipi.it (E. Giovannetti). burden of evidence of the antiproliferative effect of gemcitabine in vitro against leukemic cell lines [5–7], and initial clinical studies showed a promising activity in refractory Hodgkin’s dis- ease, non-Hodgkin’s lymphoma, cutaneus and peripheral T-cell lymphomas [3,2,9] and relapsed or refractory acute leukaemia [2,8,10]. Because of its hydrophilicity, gemcitabine does not cross the membrane by diffusion and it is transported into the cells mostly by the human equilibrative nucleoside transporter-1 (hENT- 1) [11]. Previous studies reported that hENT1-related mRNA expression inversely correlated with in vitro resistance to ara- binosylcytosine in acute lymphoblastic leukaemia [12] and to cytarabine in childhood acute myeloid leukaemia [13]. Further- more, it has also been reported that pharmacological inhibition of hENT1 resulted in either increased retention and cytotoxi- city of cladribine in cells from CLL patients and in cultured human leukemic lymphoblasts [14,15]. Recently, a significant 1043-6618/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2007.01.003