guanosine-triphosphate and Leukemia--Lymphocytic--Chronic--B-Cell

To test whether nelarabine is an effective agent for indolent leukemias and to evaluate whether there is a relationship between cellular pharmacokinetics of the analog triphosphate and clinical responses.. Thirty-five patients with relapsed/refractory leukemias (n = 24, B-cell chronic lymphocytic leukemia and n = 11, T-cell prolymphocytic leukemia) were entered onto three different protocols. For schedule A, patient received nelarabine daily for 5 days, whereas for schedule B, nelarabine was administered on days 1, 3, and 5. Schedule C was similar to schedule B except that fludarabine was also infused. Plasma and cellular pharmacokinetics were studied during the first cycle.. Responses were achieved in 20%, 15%, and 63% of patients receiving schedule A, B, and C, respectively. Histologic category, number of prior therapies, and fludarabine refractoriness did not influence the response rate. The most common nonhematologic toxicity was peripheral neuropathy. Grade 4 neutropenia and thrombocytopenia complicated 23% and 26% of courses respectively, and were significantly more frequent among patients with pre-existing marrow failure. Pharmacokinetics of plasma nelarabine and arabinosylguanine (ara-G) and of cellular ara-G triphosphate (ara-GTP) were similar in the two groups of diagnoses, and the elimination of ara-GTP from leukemia cells was slow (median, > 24 hours). The median peak intracellular concentrations of ara-GTP were significantly different (P = .0003) between responders (440 micromol/L; range, 35 to 1,438 micromol/L; n = 10) and nonresponders (50 micromol/L; range, 22 to 178 micromol/L; n = 15).. Nelarabine is an effective regimen against indolent leukemias, and combining it with fludarabine was most promising. Determination of tumor cell ara-GTP levels may provide a predictive test for response to nelarabine.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Arabinonucleosides; Arabinonucleotides; DNA (Cytosine-5-)-Methyltransferases; Drug Therapy, Combination; Female; Guanosine Triphosphate; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Prolymphocytic, T-Cell; Male; Middle Aged; Tumor Cells, Cultured; Vidarabine

The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42.. Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA-mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL-overexpressing cells). All tested cells remained sensitive to MPA-mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers.. These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells.

Topics: Apoptosis; Blotting, Western; cdc42 GTP-Binding Protein; Cell Line; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Green Fluorescent Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Jurkat Cells; K562 Cells; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Microscopy, Electron; Mutation; Mycophenolic Acid; Necrosis; RNA, Small Interfering; Signal Transduction; Transfection; Tumor Cells, Cultured

Chemokine-induced activation of alpha4beta1 and alphaLbeta2 integrins (by conformational change and clustering) is required for lymphocyte transendothelial migration (TEM) and entry into lymph nodes. We have previously reported that chemokine-induced TEM is defective in chronic lymphocytic leukemia (CLL) and that this defect is a result of failure of the chemokine to induce polar clustering of alphaLbeta2; engagement of alpha4beta1 and autocrine vascular endothelial growth factor (VEGF) restore clustering and TEM. The aim of the present study was to characterize the nature of this defect in alphaLbeta2 activation and determine how it is corrected. We show here that the alphaLbeta2 of CLL cells is already in variably activated conformations, which are not further altered by chemokine treatment. Importantly, such treatment usually does not cause an increase in the GTP-loading of Rap1, a GTPase central to chemokine-induced activation of integrins. Furthermore, we show that this defect in Rap1 GTP-loading is at the level of the GTPase and is corrected in CLL cells cultured in the absence of exogenous stimuli, suggesting that the defect is the result of in vivo stimulation. Finally, we show that, because Rap1-induced activation of both alpha4beta1 and alphaLbeta2 is defective, autocrine VEGF and chemokine are necessary to activate alpha4beta1 for ligand binding. Subsequently, this binding and both VEGF and chemokine stimulation are all needed for alphaLbeta2 activation for motility and TEM. The present study not only clarifies the nature of the alphaLbeta2 defect of CLL cells but is the first to implicate activation of Rap1 in the pathophysiology of CLL.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Movement; Chemokine CXCL12; Chemokines; Guanosine Triphosphate; Humans; Intercellular Adhesion Molecule-1; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Function-Associated Antigen-1; Lymphocytes; Shelterin Complex; Telomere-Binding Proteins; Vascular Endothelial Growth Factor A

2-Chloro-2'-deoxyadenosine (CldAdo) and 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A) have shown marked activity in the treatment of indolent lymphoid malignancies. Based on the susceptibility of various lymphocyte populations to apoptosis, we investigated whether CldAdo or F-ara-A would induce this process in lymphocytes from patients with chronic lymphocytic leukemia (CLL). In vitro exposure of leukemic lymphocytes to CldAdo or F-ara-A for 24 to 72 hours elicited features of apoptosis visible by light and electron microscopy. Analysis of DNA integrity showed DNA cleavage into nucleosomal-sized multimers. Using a quantitative assay, drug-induced DNA fragmentation was both time and dose dependent. Inhibition of active macromolecular synthesis did not prevent drug-induced fragmentation; however, both drug-induced and spontaneous DNA fragmentation were prevented by intracellular calcium chelation. In vitro culture with phorbol ester generally decreased drug-induced DNA cleavage. After prolonged incubation, CLL cells exhibited spontaneous cleavage; albeit, at significantly lower rates than drug-treated cells. Heterogeneity was observed for spontaneous and drug-induced DNA fragmentation and was significantly lower in B-leukemic cells obtained from patients with high-risk and refractory disease. We conclude that CldAdo and F-ara-A are potent inducers of apoptotic death in CLL and that this feature correlates with the disease status.

Topics: Adenosine Triphosphate; Apoptosis; Cladribine; Cycloheximide; Dactinomycin; DNA, Neoplasm; Egtazic Acid; Guanosine Triphosphate; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Microscopy, Electron; Puromycin; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uridine Triphosphate; Vidarabine