aphidicolin and Burkitt-Lymphoma

G2 arrest induced by nitrogen mustard in human lymphoma CA46 cells is associated with a failure to activate hyperphosphorylated cdc2/cyclin B1 complexes. We investigated the possibility that this might be due to a suppression of cdc25C phosphatase activity. cdc25C from interphase cells migrated as a 54- to 57-kDa doublet in SDS gels and exhibited basal phosphatase activity. cdc25C from mitotic cells migrated as a 66-kDa hyperphosphorylated species and exhibited elevated phosphatase activity. cdc25C hyperphosphorylation and activation were mediated by cdc2, supporting the view of a cdc2-cdc25C autocatalytic feedback loop. Immunofluorescence and cell fractionation studies suggested cdc2-cdc25C interaction occurred within the cytoplasm. Cells arrested in G2 phase following nitrogen mustard treatment or cells arrested in S phase with aphidicolin failed to dephosphorylate and activate cdc2, and this correlated with failure to convert cdc25C into the most active hyperphosphorylated species. Our findings suggest that checkpoints guarding against mitotic entry in the presence of unreplicated or damaged DNA suppress formation of the cdc2-cdc25C autocatalytic feedback loop that normally brings about rapid activation of cdc2.

Topics: Aphidicolin; Burkitt Lymphoma; CDC2 Protein Kinase; cdc25 Phosphatases; Cell Cycle Proteins; Cell Line; G2 Phase; HeLa Cells; Humans; Interphase; Kinetics; Mechlorethamine; Nocodazole; Proteins; Time Factors; Tumor Cells, Cultured

We have determined the levels of cellular DNA polymerases and Epstein-Barr virus specific DNA polymerase in three Burkitt's lymphoma cell lines producing varying amounts of EBV, one of which was induced by 12-0-tetra-decanoylphorbol-13-acetate (TPA). There was a proportional increase in the level of EBV-DNA polymerase with an increase in the percent of virus-producing cells. However, there was a reciprocal relationship between the levels of EBV-DNA polymerase and DNA polymerase alpha i.e., in cell line containing the highest level of EBV-DNA polymerase, activity of DNA polymerase alpha, but not of DNA polymerase beta, was reduced to an insignificantly low level. TPA does not have any direct effect on activities of either EBV-DNA polymerase or DNA polymerase alpha. EBV-DNA polymerases isolated from cells grown with or without TPA are indistinguishable in their properties such as elution position on phosphocellulose column, molecular weight, mono and divalent cation requirements, pH optimum, and other requirements for optimum activity. Addition of crude extracts of cells grown in presence of TPA to the purified DNA polymerase alpha did not inhibit its activity indicating that the observed loss was not due to any specific inhibitor present in TPA treated cells. Raji, a nonproducer cell line, did not contain EBV-DNA polymerase. There was no induction of EBV-DNA polymerase when Raji cells were grown in presence of TPA. The phenomenon of reduction in the levels of DNA polymerase alpha in cells induced to produce EBV may represent a mechanism by which the host DNA replication is shut off following virus infection.

Topics: Aphidicolin; Burkitt Lymphoma; Cell Line; Diterpenes; DNA Polymerase II; DNA-Directed DNA Polymerase; Herpesvirus 4, Human; Humans