CELL CYCLE EFFECTS OF GEMCITABINE Paolo CAPPELLA, Daniela TOMASONI, Mario FARETTA, Monica LUPI, Francesco MONTALENTI , Federica VIALE, Fabio BANZATO, Maurizio D’INCALCI and Paolo UBEZIO * Department of Oncology, Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy Gemcitabine (2,2-difluoro-2-deoxycytidine, or dFdC) is a promising anticancer agent with demonstrated clinical activ- ity in solid tumours currently undergoing clinical trials. De- spite extensive studies on the biochemical mechanism of action, cell cycle perturbations induced by dFdC have not yet been thoroughly investigated, apart from the expected inhi- bition of DNA synthesis. The aim of our study was to clarify whether cell population kinetics is a vital factor in the cyto- toxicity of dFdC in single or repeated treatments and in the dFdC-cisplatin combination. Ovarian cancer cells growing in vitro were treated with dFdC for 1 hr in a range of concen- trations from 10 nM to 10 M. Cell kinetics was investigated by DNA-bromodeoxyuridine flow cytometry, using different experimental protocols to measure either the time course of DNA-synthesis inhibition or the fate of cells in G 1 , S or G 2 M at the time of dFdC treatment or 24 hr later. A modified sulforhodamine B test was used to assess the growth inhibi- tion caused by dFdC given alone or with cisplatin. Although dFdC promptly inhibited DNA synthesis, cytotoxicity on pro- liferating cells was not specific for cells initially in the S phase. DNA synthesis was restored after a G 1 block of variable, dose-dependent length, but recycling cells were intercepted at the subsequent checkpoints, resulting in delays in the G 2 M and G 1 phases. The activity of repeated treatment with dFdCdFdC or dFdCcisplatin was highly dependent on the interval length between them. These results suggest that the kinetics of cell recycling from a first dFdC treatment strongly affects the outcome of a second treatment with either dFdC itself or cisplatin. © 2001 Wiley-Liss, Inc. Key words: cell cycle; cell proliferation; chemotherapy; flow cytom- etry; gemcitabine; apoptosis Gemcitabine (2',2'-difluoro-2'-deoxycytidine, or dFdC) is an effective anticancer agent undergoing clinical trials that has dem- onstrated clinical activity in non-small-cell lung, ovarian, pancreas and breast cancer. 1,2 As a nucleoside analogue of cytidine, its activity compared favourably with that of cytosine arabinoside (ara-C) in the treatment of solid tumors. 3 dFdC is well tolerated and has a mild toxicity profile. 4 The drug is phosphorylated inside the cells leading to the diphosphate (dFdCDP) and triphosphate (dFdCTP) derivatives, which target DNA and RNA and are presumably responsible for the drug’s effects. dFdCTP is incorporated into DNA, allowing incorporation of a single subsequent nucleotide and then blocking DNA polymerase processing. In this way, the drug escapes the check of proofreading enzymes and its removal is impaired (masked chain termination). 5 dFdCDP interferes with the enzyme ribonucleotide reductase too, causing depletion of dCTP pool that potentiates the effects of the drug. In fact dCTP counteracts the effect of dFdC by multiple mechanisms: (1) competing with dFdC for DNA incorporation, (2) contributing to dFdC inactivation, (3) down-regulating deoxycytidine kinase required for dFdC phos- phorylation. 6 Despite extensive studies on the drug’s metabolism, cell cycle perturbations induced by dFdC have not been thoroughly investi- gated, apart from the expected inhibition of DNA synthesis. Our study describes the time course of the drug’s cell cycle effects, particularly the flux of cells through G 1 and G 2 M checkpoints. Cell kinetics was used as the basis for interpreting the time dependence of the effect of a second treatment with either dFdC or cisplatin to gain fresh insight on the schedule dependence of dFdC cytotoxic- ity. MATERIAL AND METHODS Cell culture and drug treatment Cells from a human ovarian carcinoma line (IgroV-1) were grown in RPMI 1640 (Gibco, Gaithersburg, MD), pH 7.4, 10% FBS (Mascia Brunelli, Milano, Italy), 2 mM L-glutamine (Gibco) and maintained in 5% CO 2 at 37°C with 96% relative humidity. Cells were grown as monolayers in T25 tissue flasks (Iwaki, Bibby Sterilin, Staffordshire, UK) and routinely subcultured twice weekly, detaching them using 1 ml 0.05% trypsin-0.02% EDTA (Mascia Brunelli) in PBS. Trypsin activity was stopped using culture medium. All detached cells were counted using a Coulter Counter ZM (Coulter Electronics, Harpenden, UK). Cells in their exponential phase of growth were treated for 1 hr with dFdC (generously provided by Lilly, Firenze, Italy) or cis- platin (Bristol Myers, New York, NY). The clinical formulation of dFdC was used, but true dFdC concentrations are reported. The absence of any effect of the eccipient (mannitol), in the concen- trations used in our study, had already been verified. After treat- ment, cells were washed twice using warm PBS and left in drug- free culture medium for the specified times. At the appropriate times, cells were detached, counted and fixed in cold 70% ethanol. Cell cycle effects were also investigated using 2'-Bromode- oxyuridine (BrdUrd) (Sigma, St. Louis, MO). Because BrdUrd replaces thymidine during DNA synthesis, BrdUrd pulse labeling (15 min labeling with 20 M BrdUrd) catches S-phase cells engaged in DNA synthesis at that time. 7 Analysis at 7 hr postla- beling detects their movement through the S phase and the outflow of unlabeled cells from G 1 and G 2 M. Later on the cell fluxes of BrdUrd-positive and BrdUrd-negative cells can be followed inde- pendently. 8,9 Reduced ability of BrdUrd-positive cells to complete the cell cycle after treatment were quantified by the time course of the fraction of the cells originally in S phase that still remained undivided at a given time (fSu), defined as fSu  t  = % BrdUrd + undivided  t  % BrdUrd + undivided  t  + % BrdUrd + divided  t  /2 Inhibition of DNA synthesis after 1 hr dFdC was evaluated by the absence of incorporated BrdUrd upon a BrdUrd pulse in the last 15 min of the treatment. Increase of the duration of S phase (i.e., reduction of the DNA synthesis rate) in a time interval after treatment was evaluated by the time course of the relative move- ment (RM) defined as the average DNA content of undivided BrdUrd-positive cells in a scale from 0 (DNA content of G 1 cells) to 1 (DNA content of G 2 M cells). The RM of control cells at t = 0 was 0.45. Grant sponsor: Progetto Strategico del CNR “Metodi e Modelli Mate- matici nello studio dei fenomeni biologici”. *Correspondence to: Istituto di Ricerche Farmacologiche “Mario Ne- gri”, Via Eritrea 62, 20157, Milano, Italy. Fax: +39-02-3546277. E-mail: ubezio@marionegri.it Received 1 August 2000; Revised 28 December 2000, 8 March 2001; Accepted 23 March 2001 Published online 17 May 2001 Int. J. Cancer: 93, 401– 408 (2001) © 2001 Wiley-Liss, Inc. Publication of the International Union Against Cancer