Yoshinobu Matsuo

Structure, expression, and chromosomal localization of the human gene encoding a germinal center-associated nuclear protein (GANP) that associates with MCM3 involved in the initiation of DNA replication

A 210kDa protein named GANP is upregulated in germinal center (GC)-B cells in the spleen of antigen-immunized mouse. We studied a human ganp gene (hganp) encoding a putative polypeptide of 1980 amino acids. The carboxyl-terminal 721-amino-acid sequence of hGANP is identical to Map80, that is presumably generated by alternative splicing of hganp/Map80 gene. The genomic segment carrying hganp and Map80 genes was isolated, and the chromosomal location was determined on 21q22.3. Northern blot analysis with RNAs from various organs demonstrated a single band of 7kb hganp mRNA, which suggests a preferential transcription of hganp gene from the hganp/Map80 locus. The hGANP expression was upregulated in GCs of the tonsil, as demonstrated by in-situ RNA hybridization and immunohistochemical analyses. The hGANP, with the domain (Map-box) capable of binding to MCM3 in B cells, might be involved in regulation of cell-cycle progression and DNA replication of GC-B cells.

CD28 molecule as a receptor-like function for accessory signals in cell-mediated augmentation of IL-2 production

IL-2 production by PHA-stimulated MOLT 14 cells (a TcR gamma/delta-bearing human leukemic T cell line) and MOLT 16 cells (a TcR alpha/beta-bearing human leukemic T cell line) was markedly augmented by coculturing with BALL-1 cells ( a human leukemic B cell line), or with recombinant human interleukin-1 alpha (rhIL-1 alpha). We have previously shown that the augmentation of IL-2 production, induced by BALL-1 cells, requires cell to cell contact and is an IL-1-independent pathway. In this report, the expression of the CD28 molecule on MOLT 14 cells and MOLT 16 cells was examined for its role in IL-2 production augmented by BALL-1 cells. A 1-hr preincubation of MOLT 14 cells and MOLT 16 cells with anti-CD28 mAb resulted in the inhibition of BALL-1 cell-induced augmentation of IL-2 production (90 and 62% inhibition of control, respectively). The inhibition was observed in a dose-dependent manner of anti-CD28 mAb added and reached a plateau level at concentrations of 0.05 micrograms/ml of anti-CD28 mAb. This was sufficient to cover all the CD28 molecules expressed on the surface of both T cells as detected by flow cytometric analysis. Flow cytometric analysis also showed that the inhibition was not due to a modulation of CD28 molecules. In contrast, the treatment with anti-CD28 mAb did not inhibit IL-2 production which was augmented by rhIL-1 alpha costimulator. These results suggest that the CD28 molecule on the T cells is important for the interaction with BALL-1 cells which causes the augmentation of IL-2 production and further imply that the CD28 molecule is a receptor for an accessory signal provided by BALL-1 cells.

Modulation of Methotrexate Cytotoxicity with Natural Interferon upon Human Leukemia Cell Line HL-60

The effect of alpha- and gamma-interferon on methotrexate cytotoxicity against human promyelocytic cell line HL-60 has been evaluated. Synergistic inhibition of proliferation is observed with the combination of methotrexate and gamma-interferon. Enhanced cytotoxic effect of methotrexate with alpha- or gamma-interferon is removed by adding thymidine to the growth medium. The depletion of folate pools caused by methotrexate is enhanced in presence of interferons, this depletion is also removed by adding thymidine to the growth media. Synchronization of HL-60 cells in S phase of cell cycle caused by methotrexate is enhanced in the presence of interferons. These results suggest that specially gamma-interferon changes the cellular environment of HL-60 cells in such a way as to make methotrexate more potent cytotoxic agent to HL-60 cells causing cell death. This study also clearly indicates the biochemical role of thymidine in modulating the activity of methotrexate in combination with interferons.