Cell viability was quantified 48 h later by double-staining with annexin V conjugated to fluorescein isothiocyanate and propidium iodide (BenderMedsystems)

Cell viability was quantified 48 h later by double-staining with annexin V conjugated to fluorescein isothiocyanate and propidium iodide (BenderMedsystems). factor-1 and hCNT3 messenger RNA were analyzed BIO-5192 by real-time polymerase chain reaction. The effect of all-trans-retinoic acid on hCNT3 subcellular localization was analyzed by confocal microscopy and its effect on fludarabine-induced apoptosis was evaluated by flow cytometry analysis using annexin V staining. == Results == Chronic lymphocytic leukemia cases showing higherex vivobasal sensitivity to fludarabine also had a greater basal hCNT3-associated fludarabine uptake capacity compared to the subset of patients showingex vivoresistance to the drug. hCNT3 transporter activity in chronic lymphocytic leukemia cells from the latter patients was either negligible or absent. Treatment of the fludarabine-resistant subset of chronic lymphocytic leukemia cells with all-trans-retinoic acid induced increased fludarabine transport via hCNT3 which was associated with a significant increase in fludarabine sensitivity. == Conclusions == Improvement ofex vivofludarabine sensitivity in chronic lymphocytic leukemia cells is associated with increased hCNT3 activity after all-trans-retinoic acid treatment. Keywords:chronic lymphocytic leukemia, hCNT3, fludarabine, treatment, nucleoside == Introduction == Chronic lymphocytic leukemia (CLL) is a common type of leukemia in adults and is characterized by the persistent accumulation of CD5+B lymphocytes,1primarily due to defects in apoptosis. The disease has a highly variable BIO-5192 clinical course and treatment is usually restricted to patients with advanced and symptomatic disease. Fludarabine, a purine nucleoside analog, has long been a major choice for CLL chemotherapy. This drug is cytotoxic both against dividing and resting cells.2,3In dividing cells, fludarabine inhibits ribonucleotide reductase and DNA synthesis,4,5whereas in quiescent cells the main mechanism of cytotoxicity appears to be inhibition of cellular DNA repair processes leading to the induction of apoptosis.6,7Fludarabine monotherapy is associated with higher rates of long-lasting complete remission and improved overall response when compared with treatment with alkylating agents.811However, since the late 1990s, combination chemotherapies have become the recognized BIO-5192 gold standards of care. Purine analogs and alkylating agents have different mechanisms of action and, therefore, have partially non-overlapping toxicity profiles. Moreover, synergistic effects are evident when therapy with two such compounds is prescribed.1215More recent reports suggest that the administration of monoclonal antibodies, such as rituximab, can significantly improve the course of CLL. Indeed, the latest results show that chemoimmunotherapy, such as fludarabine, cyclophosphamide, and rituximab, is the optimal first-line treatment; however, some patients do not respond to this regimen.1618Resistance to fludarabine is a major problem in CLL treatment. As a DNA-damaging agent, fludarabine increases P53 levels by promoting post-translational stabilization of the protein, thereby inducing P53-dependent cell BIO-5192 death. Although mutations in theP53gene have been correlated with resistance to fludarabine and reduced survival of patients,1921the nucleoside analog can also induce apoptosis of CLL cellsin vitroin a P53-independent manner.22Furthermore, the cellular microenvironment likely influences chemoresistance. Bone marrow stromal cells protect CLL cells from fludarabine, dexamethasone, and cyclophosphamide by a mechanism requiring cell-cell contact.23Indeed, several mechanisms may contribute to fludarabine resistance in patients wild-type for P53. Before entering cells, fludarabine is rapidly dephosphorylated by membrane ectonucleotidases (CD73) and then transported inside the cell via nucleoside-selective plasma membrane transporters. Once inside the cell, the drug is phosphorylated by deoxycytidine kinase before its cytotoxic activity can be exerted.5,24Nucleoside uptake into cells is mediated by specific nucleoside transporter (NT) proteins belonging to two unrelated gene families, SLC28 and SLC29, encoding CNT (concentrative nucleoside transporter) and ENT (equilibrative nucleoside transporter) proteins, respectively.25,26Previous work by our group showed that primary CLL cells co-express hENT1, hENT2, hCNT2, and hCNT3, but accumulation of fludarabine in CLL cells is mediated mostly, if not exclusively, by ENT-type transporters.27hCNT3 but not hCNT2 can also transport fludarabine.2830Indeed, hCNT3 is even more efficient than is hENT2 in this regard; the former transporter has a higher affinity for the drug and, more importantly, it is a CNT, thus concentrating nucleoside analogs inside cells. Fludarabine-resistant CLL cells express high but variable levels of hCNT3 mRNA and cytosolic hCNT3 protein,31thus suggesting that hCNT3 is localized mainly in intracellular compartments of such CLL cells. We have recently reported that all-trans-retinoic acid (ATRA), a natural vitamin A derivative currently used in the treatment of acute promyelocytic leukemia,32can increase hCNT3 activity in MEC1 cells via a mechanism mediated by transforming growth factor 1 (TGF1).33 We hypothesized BIO-5192 that P53 wild-type cases showing resistance to fludarabine, might have low plasma membrane hCNT3 activity, thus compromising efficient fludarabine uptake into CLL cells, probably by retaining hCNT3 intracellularly. Moreover, we sought to determine whether the effect of ATRA on hCNT3, described to date only in a cell line,33was evident in primary CLL cells. This would provide a basis for Splenopentin Acetate modulating fludarabine-based therapies. == Design and Methods == == Chemical substances and reagents == ATRA.