Cytotoxic activity of gemcitabine and correlation with expression profile of drug-related genes in human lymphoid cells.
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.
Giovannetti
Elisa
E
Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, 55 Via Roma, 56126 Pisa, Italy. e.giovannetti@med.unipi.it
Mey
Valentina
V
Loni
Lucia
L
Nannizzi
Sara
S
Barsanti
Gemma
G
Savarino
Grazia
G
Ricciardi
Simona
S
Del Tacca
Mario
M
Danesi
Romano
R
D015720:5'-Nucleotidase / Q000235:genetics / Q000378:metabolism; D000368:Aged; D015703:Antigens, CD / Q000032:analysis; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D003842:Deoxycytidine Kinase / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016130:Immunophenotyping; D019169:Jurkat Cells; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D018579:Patient Selection; D011237:Predictive Value of Tests; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics / Q000378:metabolism; D016896:Treatment Outcome; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D000093542:Gemcitabine
D015703:Antigens, CD; D000964:Antimetabolites, Antineoplastic; D033721:Equilibrative Nucleoside Transporter 1; D012333:RNA, Messenger; C438649:SLC29A1 protein, human; D025521:Tumor Suppressor Proteins; D003841:Deoxycytidine; C093713:ribonucleotide reductase M2; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D003842:Deoxycytidine Kinase; D015720:5'-Nucleotidase; D000093542:Gemcitabine