mercaptopurine and thiazolyl-blue

This study was designed to assess the predictive value of an MTT (3-[4,5-dimethylthiazol-2,5-diphenyl] tetrazolium bromide) in vitro assay for the evaluation of leukemic cell resistance/sensitivity to cytotoxic drugs, and to compare these results with clinical and laboratory parameters in cases of childhood acute lymphoblastic leukemia (ALL).The chemoresistance of leukemic cells was ascertained by means of an MTT assay in 32 previously untreated children with ALL. The children were treated using the protocol ALL-BFM 90. Statistical correlations were made between in vitro drug resistance to anti-cancer drugs: prednisolone (PRED), vincristine (VCR),daunorubicin (DNR), etoposide (VP-16) and cytosine arabinoside (ARA-C) and in vivo clinical and laboratory parameters: age, sex, risk factor (RF), leukocytes (WBC)and absolute number of blast cells (BC) at diagnosis (BC0), BC at day 8 (BC8), the percentage of blast cells in bone marrow at day 15 (BM15) and at day 33 (BM33),and leukocyte surface antigens CD3, CD4, CD5, CD8, CD10, CD19, CD20, HLADR.

Topics: Adolescent; Age Factors; Antineoplastic Combined Chemotherapy Protocols; Asparaginase; Child; Child, Preschool; Coloring Agents; Cyclophosphamide; Cytarabine; Daunorubicin; Drug Resistance, Neoplasm; Female; Humans; In Vitro Techniques; Male; Mercaptopurine; Methotrexate; Multivariate Analysis; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Predictive Value of Tests; Prednisone; Remission Induction; Sex Factors; Tetrazolium Salts; Thiazoles; Vincristine

Azathioprine (AZ) is a prodrug of 6-mercaptopurine (6-MP), but little is known about the relative suppressive efficacy of these agents against blastogenesis of human peripheral blood mononuclear cells (PBMCs) in-vitro. We compared the pharmacological efficacy of AZ and 6-MP against T cell mitogen-induced blastogenesis of PBMCs in-vitro. PBMCs were obtained from seven healthy subjects. The potency of AZ and 6-MP to suppress PBMC-blastogenesis in-vitro was compared using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay procedures. Production of 6-MP from AZ in PBMC culture was examined by high-performance liquid chromatography. Both AZ and 6-MP dose-dependently suppressed PBMC blastogenesis. Mean +/- s.d. IC50 (concentration of drug that gave 50% inhibition) values for AZ and 6-MP were 230.4 +/- 231.3 and 149.5 +/- 124.9 nM, respectively. Thus, the potencies of AZ and 6-MP to suppress PBMC blastogenesis were not significantly different. A significant correlation was observed between the IC50 values of AZ and 6-MP (P < 0.01, n = 6). After incubation of PBMCs with 100 microM AZ for 4 days, 35.3-92.5 microM 6-MP was liberated into the culture medium. The ratio of 6-MP liberation from AZ was influenced by the presence of PBMCs, but not by the medium or fetal bovine serum. The results suggest that the suppressive potency of the prodrug AZ and its converted form 6-MP against blastogenesis of human PBMCs in-vitro is similar, although PBMCs appeared to convert AZ to 6-MP. AZ is suggested to be effective after conversion to 6-MP to express immunosuppressive efficacy in-vitro.

Topics: Adult; Azathioprine; Cell Separation; Female; Humans; Immunosuppressive Agents; In Vitro Techniques; Lymphocyte Activation; Male; Mercaptopurine; Mitogens; Monocytes; T-Lymphocytes; Tetrazolium Salts; Thiazoles

The knowledge about drug resistance in childhood leukemias and acute lymphoblastic leukemia (ALL) in general is limited. This is because of the lack of a suitable in vitro drug sensitivity assay, which is in part due to low in vitro ALL cell survival. We recently adapted the highly efficient 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay to test cells from ALL patients and showed that its results were comparable with those of the DiSC assay, up to now the most valid but laborious assay. In this study, in vitro drug sensitivity was assessed in cells from 82 children with leukemia, 79 of whom had ALL, with the MTT assay. Dose response curves were obtained for 6-mercaptopurine, 6-thioguanine (6-TG), prednisolone (Pred), daunorubicin (DNR), vincristine (VCR), cytosine arabinoside (Ara-C), L-asparaginase (L-Asp), mafosfamide, and mustine. A cytotoxic effect of methotrexate could be detected in only a few cases. Large interindividual differences in drug sensitivity were detected. Compared with leukemia cells from newly diagnosed patients, leukemia cells from relapsed patients were significantly more in vitro resistant to 6-TG, Pred, Ara-C, mafosfamide and mustine but not to DNR, VCR, and L-Asp. Improvements of culture medium and methods to increase MTT reduction were studied. From 10 components tested, addition of insulin and bovine serum albumin to serum-containing medium improved ALL cell survival. Addition of succinate did not increase the amount of MTT reduction. We conclude that the in vitro MTT assay highly facilitates large-scale studies on drug resistance of ALL patients that can lead to rational improvements in existing treatment protocols.

Topics: Asparaginase; Cells, Cultured; Child; Coloring Agents; Culture Media; Cyclophosphamide; Cytarabine; Daunorubicin; Dose-Response Relationship, Drug; Drug Resistance; Drug Screening Assays, Antitumor; Humans; Mechlorethamine; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prednisolone; Tetrazolium Salts; Thiazoles; Thioguanine; Vincristine