P88

Abstract

Purpose

DNA minor groove is the main target of small molecules, which noncovalently and to a certain extent site-specifically bind to appropriate nucleotide sequences. Study of these substances can give rise to understanding the mechanistic relationship between sites of interaction and activity of appropriate enzymes with “houskeeping” function including helicases, topoisomerases, methyltransferases, demethylases and DNA/RNA-polymerases.

Results

We revealed for the first time that AT-specific minor groove binding ligands (MGBLs), in particular bisbenzimidazoles (Hoechst33258 and its derivatives), widely used in molecular and cell biology for DNA-staining, induce loss of heterozygosity at high frequency while point mutations and chromosome deletions at insignificant levels. Moreover, we demonstrated that the agents realized their genotoxic blastomogenic effects via homologous recombination mechanism exclusively. Lately the same mechanism of genotoxicity has been shown for MGBL carbazole derivative Curaxin, which is toxic for a broad range of tumor cell lines in vitro and inhibit tumor growth in different mouse models of cancer in vivo. Moreover, powerful antitumor activity has been demonstrated for Trabectedin, which binds to the DNA’s minor groove and alkylate guanine residues. All this provided a framework for wide-ranging investigation of cell response to MGBLs exposure, molecular mechanisms of their recombinogenic as also their anticancer activity. A special interest is paid to epigenetic mechanisms of MGBs action.

Our study aimed to examine the epigenetic effects of recombinogenic (Hoechst33342, Hoechst33258) and non-recombinogenic (DAPI, Diminazene, Pentamidine and Netropsin) MGBLs.

After we unmasked MGBLs’ recombinogenic activity, we hypothesized that their molecular mechanism of indirect DNA damage involves poly(ADP-ribose)polymerase-1 (PARP-1) activation. Surprisingly, we found that all AT-specific MGBLs preventing PARP-1 interaction with DNA inhibit its activation, and hence, the DNA-dependent pathway of PARP-1 activation function. These inhibitors effectively block PARP-1 activity in vivo, as it was demonstrated in a Drosophila experimental system and in human breast cancer-derived BT474 cell line.

Further epigenetic effects of these indirect genotoxic carcinogens were analyzed using HeLa cell population with epigenetically suppressed GFP-reporter gene as a model. All compounds had strong GFP- reactivation effect. The obtained results confirm scarce data of previous publications on the ability of DNA minor groove ligands to influence gene transcription process. Statistically significant results of changes of DNA methylation level were detected under 5-azaC and Hoechst 33258 treatment, but it was absent after Hoechst 33342 treatment. For the rest compounds significant loss of promoter region methylation was not observed. The common epigenetic marks of transcription include histone H3 trimethylation in lysine 4 (H3K4me3) and histone H3 and H4 acetylation (acH3/acH4) on promoter regions of genes. We showed that TSA, Hoechst 33342 and DAPI treatment of HeLa-TI cells lead to increased level of histone H3 trimethylation of lysines 4 (H3K4me3), but for all that the level of histone H4 acetylation remains without significant changes. On the contrary, histone H3 acetylation level was stably increased at all samples. The modifications are typical for silent genes and decrease of their amount suggests the transcriptional reactivation. Loss of H4K20me3 mark in comparison with untreated control was demonstrated for MGBLs.

Conclusion

Taking together, findings of the study will lay the fundamental groundwork for the development of novel anticancer strategies and new chemotherapy effects of small molecules. The mechanism of PARP-1 inhibition by MGBLs and its epigenetic influence on silent genes, via DNA methylation and histone modifications make reasonable further study of these compounds in three prospective directions: (1) as self-acting cytotoxic agents; (2) as a component of combined chemotherapy targeting DNA repair by PARP-1, thereby facilitating DNA damage caused by other anticancer drugs; (3) as an agents reactivating epigenetically repressed genes which silencing occurs during the earliest stages of neoplasia and accumulates with progression toward malignancy.