Apoptotic and antiproliferative effects of gemcitabine and gemcitabine plus Ara-C on blast cells from patients with blast crisis chronic myeloproliferative disorders

Department of Hematology, University of Florence-Azienda Ospedaliera Careggi, Firenze, Italy. santini@cesit1.unfi.it

BACKGROUND: Blast phase of chronic myeloid leukemia (CML) as well as the rare acute transformation of other chronic myeloproliferative disorders constitute forms of leukemia that are particularly refractory, even to aggressive chemotherapy. Many attempts have thus been made to identify new drugs that could be active in these diseases. We wanted to evaluate whether gemcitabine (dFdC), a pyrimidine analogue widely employed in lung cancer chemotherapy, was able to block in vitro proliferation of bcr/abl-positive and bcr/abl-negative blast cells in primary culture. We already showed that gemcitabine is active in inhibiting proliferation and inducing apoptosis of HL60 cells. METHODS: We studied the influence of dFdC on the proliferative potential of blasts by means of tritiated thymidine uptake, colony formation in semisolid medium and cell cycle parameters at flow cytometry. The efficacy of dFdC in inducing apoptosis was evaluated by flow cytometry (A0 peak) and by DNA agarose gel electrophoresis. RESULTS: We demonstrated that dFdC already inhibits tritiated thymidine uptake at doses of 10 microM after 72 hours of culture, and that this effect is dose dependent. The addition of Ara-C 5 microM in the culture medium of dFdC provoked a synergistic inhibitory effect. Consistent results were obtained when cell cycle distribution was studied. In fact, cell incubation in the presence of dFdC resulted in a significant decrease of cells in S phase, although with a certain heterogeneity among cases. The antileukemic activity of dFdC appeared to be specific since it was mediated through apoptosis. We could demonstrate the appearance of the pre-G1 apoptotic peak at cytofluorimetric analysis, and the characteristic DNA fragmentation pattern at agarose electrophoresis in all 10 cases after treatment with different doses of dFdC. Induction of apoptosis was maximal for the highest doses of dFdC (100 mM) and for the combination of dFdC and Ara-C. INTERPRETATION AND CONCLUSIONS: Following incubation with Gemcitabine leukemic blasts from chronic myeloproliferative disorders are induced to accumulate intracytoplasmatic and nuclear Ara-C and undergo apoptosis. These observations suggest that gemcitabine could be considered a candidate drug, capable of being used in polychemotherapy of refractory acute phase chronic myeloproliferative disorders.