thioguanine-anhydrous and Leukemia--T-Cell

The antimetabolite 6-thioguanine (6-TG) has been used to treat both human and canine lymphoid malignancies. 6-TG has been shown to be epigenetically active as a demethylating agent in a human lymphoma cell line, causing downregulation of DNA methyltransferase 1 (DNMT1) through ubiquitin-targeted degradation. Zebularine (Zeb), a similar cytidine analog, also has demethylating activity as well as oral bioavailability. The hypothesis of the present study was that 6-TG and Zeb would cause downregulation of DNMT1 and globally demethylate the genomic DNA of canine lymphoma cells. The secondary hypothesis was that these agents would cause a dose-dependent decrease in cell proliferation in canine lymphoma cells. Canine CLGL-90 malignant T cells and CLL 17-7 cells were incubated in modified RPMI media. They were treated with 6-TG, Zeb, or control media at biologically relevant concentrations.. Following treatment with each agent, DNMT1 protein and global DNA methylation were significantly decreased. A dose-dependent decrease in cell survival was also observed, with apoptosis being the primary mode of cell death in the CLGL-90 cell line.. These results confirm the demethylating action of 6-TG and Zeb in canine cells which is similar to that shown in human cell lines. Confirmation of this mechanism supports the clinical application of these compounds as demethylating drugs in veterinary patients.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cytidine; DNA-Cytosine Methylases; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Down-Regulation; Leukemia, B-Cell; Leukemia, T-Cell; Lymphoma; Thioguanine

Sequence-specific combinations of purine analogs, such as fludarabine or 6-mercaptopurine (6-MP), administered prior to cytosine arabinoside (ara-C) have been shown to abrogate ara-C resistance in human leukemia cells in vitro and in patients with relapsed acute myeloid or lymphoblastic leukemias. The two-drug combination of 6-MP plus ara-C results in greater cytotoxicity than that achieved with either ara-C or 6-MP alone. Further preclinical investigations have shown that the addition of PEG-asparaginase (PEG-ASNase) to the combination of 6-MP plus ara-C (6-MP + ara-C + PEG-ASNase) results in 15.6-fold synergism over that achieved with the two-drug regimen. This is due to increased DNA damage leading to apoptotic cell death.. Since the intravenous preparation of 6-MP is no longer available and since oral 6-thioguanine (6-TG) provides higher levels of intracellular thioguanine nucleotides than an isotoxic dose of oral 6-MP, we investigated the potential drug synergism of 6-TG plus ara-C plus PEG-ASNase (TGAP) in myeloid (HL60/S, HL60/SN3, U937) and lymphoblastic (CEM/0, CEM/ ara-C/B, CEM/ara-C/I, MOLT-4) leukemia cell lines. The CEM clones, MOLT-4 and HL60/SN3 cell lines expressed functional or measurable p53 protein, while the other cell lines did not.. The MTT and trypan blue dye exclusion assays were used to determine drug cytotoxicity. In addition, cellular apoptosis and cellular p53, p21/waf-1 and bcl-2 protein concentrations were determined by FACS analysis and ELISA assays.. Sequential exposure to 6-TG (24 h) plus ara-C (24 h) plus PEG-ASNase (24 h) produced 1.3- to 18.3-fold drug synergism over the two-drug combination of 6-TG plus ara-C. The molecular mechanism of synergism was due to the fact that the three-drug combination was capable of downregulating bcl-2 oncoprotein levels in these cell lines even when p53 was absent.. These studies strongly demonstrate that the TGAP regimen is highly synergistic in p53-null and p53-expressing leukemia cell lines. We conclude that this combination regimen is collaterally sensitive with ara-C and further evaluation in an investigational phase I trial in relapsed leukemia patients is warranted.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Asparaginase; Cytarabine; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; HL-60 Cells; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, T-Cell; Polyethylene Glycols; Proto-Oncogene Proteins c-bcl-2; Thioguanine; Tumor Cells, Cultured; Tumor Suppressor Protein p53; U937 Cells

The difference in the current cure rates between adult and childhood acute lymphoblastic leukaemia (ALL) may be caused by differences in drug resistance. Earlier studies showed that in vitro cellular drug resistance is a strong independent adverse risk factor in childhood ALL. Knowledge about cellular drug resistance in adult ALL is still limited. The present study compared the in vitro drug resistance profiles of 23 adult ALL patients with that of 395 childhood ALL patients. The lymphoblasts were tested by the MTT assay. The group of adult ALL samples was significantly more resistant to cytosine arabinoside, L-asparaginase, daunorubicin, dexamethasone and prednisolone. The resistance ratio (RR) was highest for prednisolone (31.7-fold) followed by dexamethasone (6.9-fold), L-asparaginase (6. 1-fold), cytosine arabinoside (2.9-fold), daunorubicin (2.5-fold) and vincristine (2.2-fold). Lymphoblasts from adult patients were not more resistant to mercaptopurine, thioguanine, 4-HOO-ifosfamide, mitoxantrone and teniposide. There were no significant differences in drug resistance between adult T-cell (T-) ALL (n = 11) and adult common/pre-B-cell (B-) ALL (n = 10). Additionally, adult T-ALL did not differ from childhood T-ALL (n = 69). There were significant differences between adult common/pre-B-ALL and childhood common/pre-B-ALL (n = 310) for prednisolone (RR = 302, P = 0.008), dexamethasone (RR = 20.9, P = 0.017) and daunorubicin (RR = 2.7, P = 0.009). Lymphoblasts from adults proved to be relatively resistant to drugs commonly used in therapy. This might contribute to the difference in outcome between children and adults with ALL.

Topics: Adolescent; Adult; Age Factors; Antineoplastic Agents; Asparaginase; Bone Marrow Cells; Child; Child, Preschool; Cytarabine; Daunorubicin; Dexamethasone; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; Female; Humans; Idarubicin; Ifosfamide; Infant; Leukemia, Prolymphocytic; Leukemia, T-Cell; Male; Mercaptopurine; Middle Aged; Mitoxantrone; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prednisolone; Prognosis; Statistics, Nonparametric; Teniposide; Thioguanine; Vincristine