PubMed · 15652108
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Michael Bigby, Sam Gibbs, Ian Harvey, Jane Sterling. 2004. Warts.. https://pubmed.ncbi.nlm.nih.gov/15652108/
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The removal of histones from DNA and their subsequent replacement is likely to be necessary for all processes that require access to the DNA sequence in eukaryotic cells. The histone chaperone chromatin assembly factor 1 (CAF-1) mediates histone H3-H4 assembly during DNA replication and nucleotide excision repair in vitro. We have found that budding yeast deleted for the genes encoding CAF-1 are highly sensitive to double-strand DNA-damaging agents. Our genetic analyses indicate that CAF-1 plays a role in both homologous recombination and nonhomologous end-joining pathways and that the function of CAF-1 during double-strand repair is distinct from that of another histone H3-H4 chaperone, anti-silencing function 1 (ASF1). CAF-1 does not protect the genome by assembling it into a damage-resistant chromatin structure, because induction of CAF-1 after DNA damage is sufficient to restore viability. Furthermore, CAF-1 is not required for repair of the DNA per se or for DNA damage checkpoint function. CAF-1-mediated resistance to DNA damage is dependent on the ability of CAF-1 to bind PCNA, indicating that PCNA may recruit CAF-1 to sites of double-strand DNA repair. We propose that CAF-1 has an essential role in assembling chromatin during double-strand-DNA repair.
Formulations of ultra-deformable liposomes containing bleomycin (Bleosome) have previously been described and proposed for topical treatment of skin cancer [Lau, K.G., Chopra, S., Maitani, Y., 2003. Entrapment of bleomycin in ultra-deformable liposomes. S. T. P. Pharm. Sci. 13, 237-239]. In this study, the stability of various Bleosome formulations was characterised and a purification process was established to isolate Bleosome for testing on cultures of either human cutaneous keratinocytes (NEB-1) immortalised by human papilloma virus (HPV)-type 16, or a spontaneously immortalised human squamous cell carcinoma (SCC) from a primary tumour. Bleosome facilitated entrapment of high concentrations of active bleomycin and samples purified by gel-filtration chromatography remained stable during 7 days of storage at 4 degrees C or at room temperature. Serially-diluted samples of this purified, high-strength product, 'high dose' were applied onto keratinocyte cell cultures to elucidate Bleosome LD50 profiles. In vitro data revealed that the LD50 of bleomycin encapsulated in Bleosome was approximately three-fold higher than free bleomycin solution for SCC cells, and nearly 30 times higher for NEB-1 cells. However, Bleosome containing 30 microg/ml of active bleomycin killed more than twice as many SCC cells than NEB-1 cells. At that concentration, the potency of liposomal bleomycin on causing cell death of SCC cells was found to be similar to that of free bleomycin solution. This effect was not seen on NEB-1 cells. It seems that SCC cells were particularly susceptible to Bleosome containing high levels of bleomycin. Results from these experiments promote the development of a novel product for the topical treatment of skin cancer.
The intra-S-phase checkpoint is a signaling pathway that induces slow DNA replication in the presence of DNA damage. In humans, defects in this checkpoint pathway might account for phenotypes seen in autosomal recessive diseases including ataxia telangiectasia-like disorder and Nijmegen breakage syndrome, where MRN complex components,Mre11 and Nbs1, are mutated. Here we provide evidence that the equivalent budding yeast complex, MRX (Mre11/Rad50/Xrs2), is not required for the intra-S-phase checkpoint in response to DNA alkylation damage, but is required in the presence of double-stranded DNA breaks. These data indicate, at least in budding yeast, that alternate pathways enforce replication slowing depending on the particular DNA lesion.