Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

The novel cyclic dinucleotide 3'-5' cyclic diguanylic acid binds to p21ras, induces the expression of the CD4 receptor and causes cell cycle arrest in lymphohemopoietic cell lines

D Amikam DSc 1,2, O Steinberger MSc 1, Z Lapidot DSc 1, Z Ben-Ishai PhD 1

1 Molecular Oncology Laboratory, Rambam Medical Center, Haifa, Israel, 2 Biotechnology in Medicine Section, Tel-Hai Academic College, Upper Galillee, Israel,

AIMS: Studying the effect of cyclic diguanylic acid (c-di-GMP) on lymphohemopoietic cell lines. METHODS: [ˆ3H] Methyl-thymidine incorporation into cells was quantified by liquid scintillation counting. Immunoprecipitation was done using anti-p21ˆrˆaˆs monoclonal Y13-259 antibody. Immunofluorescent staining and flow cytometric analysis was done using anti-CD4 leu3A, anti-CD8 leu2A and fluorescein isothiocyanate conjugated rabbit anti-mouse IgG. RESULTS: C-di-GMP is a novel cyclic regulatory nucleotide identified in prokaryotic systems and found to be active in eukaryotic systems. When exposed to 50 m c-di-GMP, Jurkat, Molt 4 and K562 cells exhibited a marked increase in [ˆ3H] thymidine incorporation over controls of up to 43-,113- and 90-fold, respectively. C-di-GMP's natural entrance into these cells was experimentally verified. SDS/PAGE analysis of [ˆ3ˆ2P] c-di-GMP-exposed cells followed by autoradiography revealed the labeling of three proteins at 18-27 kDa. One of the c-di-GMP-binding proteins was found to be the p21ˆrˆaˆs, whose binding is specific and apparently irreversible. When exposed to 50 m c-di-GMP, a markedly elevated expression of the CD4 receptor over controls of up to 6.3-, 2.5- and 2-fold was exhibited by Jurkat, Molt 4 and K562 cells, respectively. C-di-GMP also caused in these cell lines blockage of the cell cycle at S-phase, characterized by increased cellular thymidine uptake, reduced G2/M-phase cells, increased S-phase cells and decreased cell division. All effects described appear to be unique for c-di-GMP. CONCLUSIONS: Taken together, we feel that the novel nucleotide c-di-GMP might offer a potentially powerful tool for studying signals involved in T cell regulation.

KEY WORDS: 3'-5' cyclic diguanylic acid, p21ˆ, , , s, CD4 receptor.

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on cell cycle inhibitors.