Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination.

PURPOSE: We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-ß-D-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-ß-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-ß-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG. METHODS: Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines. RESULTS: The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase. CONCLUSION: CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.

Synthesis and Structural Revision of Cyslabdan.

Cyslabdan was isolated from the culture broth of Streptomyces sp. K04-0144 as a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus. We accomplished the synthesis of cyslabdan according to a previously reported structure. However, we subsequently found that this structure was incorrect; our analysis of natural cyslabdan showed that it possessed R stereochemistry at the C8 position, not S, as had previously been reported. Thus, we completed the protecting-group-free synthesis of the correct structure of cyslabdan, which is described herein.

Potentiation of bleomycin in Jurkat cells by fungal pycnidione.

Most cancer cells have mutations in genes at the G1 checkpoint and repair DNA only in the G2 phase; therefore, the G2 checkpoint is a potential target to develop novel therapy. In the course of screening, a known compound, pycnidione, was isolated from the fungal culture broth of Gloeotinia sp. FKI-3416. Pycnidione irreversibly abrogated bleomycin-induced G2 arrest in Jurkat cells and synergically potentiated the cytotoxicity of bleomycin. To elucidate the mechanism of action, the effect of pycnidione on the signal transduction of the G2 checkpoint was analyzed, showing that the increased phospho-cyclin dependent kinase-1 (CDK1) level caused by bleomycin was abrogated in the presence of pycnidione, indicating that cells did not arrest at the G2 phase. Moreover, under these conditions, Chk1 and Chk2 levels were markedly down-regulated. Thus, we concluded that pycnidione abrogated bleomycin-induced G2 arrest by decreasing Chk1 and Chk2.

9-(2-C-Cyano-2-deoxy-beta-D-arabino-pentofuranosyl)guanine, a potential antitumor agent against B-lymphoma infected with Kaposi's sarcoma-associated herpesvirus.

Several 9-(2-C-cyano-2-deoxy-l-beta-d-arabino-pentofuranosyl)purine derivatives were tested against Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cells. The guanine derivative (3, CNDAG), as well as the 2-amino-6-substituted-purine derivatives 4, 5, and 6, exhibited cell growth inhibitory activity against KSHV-infected cells, but showed no cytotoxicity against KSHV-negative cells at >15 microM concentrations. Therefore, it was found that compounds 3, 4, 5, and 6 showed selective cytotoxicity against PEL cells infected with KSHV.