aphidicolin and Lymphoma

Patients with lymphomas were shown to have authentically a higher frequency of fragile sites, both spontaneous and induced by aphidikoline expression, in comparison with the control group. Differential sensitivity of chromosomes caused by the action of aphidikolin was revealed. The fragility of chromosomes 9 and 11 in patients with lymphomas on sites 9q31-32 and 11p13-14 was authentically higher (alpha = 0.05) than that in healthy individuals. Site 9q31-32 appeared to be strictly specific for lymphoma disease.

Topics: Adolescent; Adult; Aphidicolin; Chromosome Fragility; Chromosomes, Human; Female; Humans; Lymphocytes; Lymphoma; Male; Middle Aged

In this report, we describe a new flow cytometry technique termed flow cytometric high-content screening (FC-HCS) which involves semi-automated processing and analysis of multiparameter flow cytometry samples. As a first test of the FC-HCS technique, we used it to screen a 2000-compound library, called the National Cancer Institute (NCI) Diversity Set, to identify agents that would enhance the anti-lymphoma activity of the therapeutic monoclonal antibody rituximab. FC-HCS identified 15 compounds from the Diversity Set that significantly enhanced the ability of rituximab to inhibit cell cycle progression and induce apoptosis in lymphoma cells. The validity of the screening results was confirmed for several compounds using additional assays of cell proliferation, apoptosis and cell growth. The FC-HCS technique was relatively simple and reliable and could process up to 1000 samples/day on a single flow cytometer. The FC-HCS technique may be useful for a variety of applications including drug discovery, immunologic monitoring of patients, functional genomics studies and tissue engineering efforts.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Aphidicolin; Apoptosis; Cell Cycle; Cell Line, Tumor; Drug Screening Assays, Antitumor; Flow Cytometry; Humans; Lymphoma; Phenanthrolines; Rituximab; Topotecan

Transforming growth factor beta 1 (TGF beta 1) has been shown to inhibit growth stimulation in normal human B cells as well as in Epstein Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines. The mechanisms for this potent growth inhibition are not completely defined. Here we show that a number of EBV-negative lymphoma B cell lines (BL-41, Ramos and CAPA-2), when exposed in vitro to TGF beta 1, undergo apoptosis. Maximum apoptosis was observed at 48 h following TGF beta 1 treatment, with no apparent effect on the expression of c-myc and bcl-2 proteins. Similar induction of apoptosis was observed when these lymphoma cell lines were treated with aphidicolin, a DNA synthesis inhibitor. In contrast, various preparations (14 out of 17) of normal human tonsilar B cells showed no significant apoptosis, although both TGF beta 1 and aphidicolin inhibited anti-mu/IL-4 induced DNA synthesis in all preparations. Furthermore, another TGF beta 1 sensitive EBV-negative BL cell line, CA46, exhibited no apoptosis in response to TGF beta 1 and aphidicolin, corroborating the findings in normal human B cells. Taken together, these data support the hypothesis that exposure to TGF beta 1, which results in cell cycle arrest and DNA synthesis inhibition, may not be obligatory or sufficient for the induction of apoptosis. Rather, induction of apoptosis or lack of it may be intrinsically determined by an interplay between extracellular and intracellular regulators of cellular growth.

Topics: Aphidicolin; Apoptosis; B-Lymphocytes; Cell Cycle; Cell Division; Chromatin; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, myc; Growth Inhibitors; Humans; Lymphoma; Nucleic Acid Synthesis Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

We investigated the temporal regulation of cyclin A- and B1-dependent kinases in human lymphoma cells treated with nitrogen mustard (HN2) and pentoxifylline, to determine whether the activity of these complexes correlated with cell cycle arrest induced by DNA damage. Cells were synchronized in G1/S, treated with HN2, and then postincubated with pentoxifylline. HN2-induced a protracted delay in G2 phase. This delay correlated with suppression of cyclin B1- and cdc2-kinase activities, and stabilization of hyperphosphorylated-cdc2 in the presence of similar cyclin B1 levels to those found in mitosis. HN2 had no discernible effect on the S phase activity of cyclin A- or cdk2-immune complexes. Entry of control cells into mitosis correlated with destruction of cyclin A, disappearance of cyclin A-bound cdk2 and decreased cdk2 kinase activity. G2 delay induced by HN2 was associated with stabilization of cyclin A, increased abundance of cyclin A-bound cdk2, and increased cdk2 activity. Cyclin A was also associated with cdc2, which, contrary to complexes containing cdk2, were only activated upon entry into mitosis. Pentoxifylline abrogated cell cycle arrest induced by aphidicolin and HN2 in human lymphoma cells. Pentoxifylline also reverted the activity of cyclin A- and B1-kinases in HN2-treated cells to approximately that observed in controls. Our findings suggest that delayed entry into mitosis following DNA damage correlates with suppression of cyclin B1/cdc2 and cyclin A/cdc2 complexes, while maintaining cyclin A/cdc2 complexes in an active state. Furthermore, we found that pentoxifylline disrupts the signal transduction pathway that regulates these complexes when damaged DNA is present, resulting in abrogation of cell cycle arrest.

Topics: Antigen-Antibody Complex; Aphidicolin; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Flow Cytometry; G2 Phase; Humans; Kinetics; Lymphoma; Mechlorethamine; Mitotic Index; Pentoxifylline; Protein Binding; Protein Kinases; Protein Serine-Threonine Kinases; Time Factors; Tumor Cells, Cultured

The murine T lymphoma EL4 subline produces large amounts of interleukin 2 (IL-2) upon stimulation with phorbol myristate acetate (PMA). It was found to survive continuous PMA stimulation (10 ng/ml, renewed daily, at 10(6) cells/ml), and to maintain its IL-2 production for several days in a culture medium low in protein. IL-2 production varied from day to day with a peak within the first 2 days of stimulation. PMA stimulation was accompanied by morphologic changes (enlargement and adhesiveness), arrest in cell division and DNA replication, continued RNA synthesis and increased mitochondrial activity. These effects were reversible upon removal of PMA, with a decrease of IL-2 secretion to the background level and a progressive recovery of cell growth. The reverted EL4 cells were again able to produce large amounts of IL-2 upon restimulation with PMA. Growth of EL4 cells was shown not to depend on autologous production of IL-2, and their DNA synthesis could be arrested by aphidicolin without a concomitant accumulation of IL-2 in the supernatant. This indicates that the increase in the amount of free IL-2 is not simply due to reduced consumption by the EL4 cells themselves. PMA appears to act at the level of the expression of the IL-2 gene, since IL-2 mRNA was much more abundant in the IL-2-secreting PMA-treated cells than in untreated controls. Our observations show that considerable amounts of IL-2 can be obtained from a single EL4 cell inoculum and may represent an original model to study the effects of PMA on the differentiation and maturation processes of cells.

Topics: Animals; Aphidicolin; Cell Division; Cell Line; Cyclosporins; Diterpenes; Dose-Response Relationship, Drug; Drug Administration Schedule; Gene Expression Regulation; Interleukin-2; Lymphoma; Mice; Phorbols; RNA, Messenger; Tetradecanoylphorbol Acetate