Μελέτη σε επίπεδο ηλεκτρονικής μικροσκοπίας της πρωτεΐνης H2AX που αναγνωρίζει βλάβες του DNA στον ανθρώπινο καρκίνο

Abstract

Eukaryotic DNA is organized into noucleosomes and high order chromatin structure, which plays an important role in the regulation of many nuclear processes including DNA repair. The DNA within our cells is continually being exposed to DNA-damaging agents. These include ultraviolet light, natural and man-made mutagenic chemicals and reactive oxygen species generated by ionizing radiation. Of the various forms of damage that are inflicted by these mutagens, probably the most dangerous is the DNA double strand breaks (DSBs). These are generated when the two complementary strands of the DNA double helix are broken simultaneously at sites that are sufficiently close to one another that base-pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. DSBs pose a serious threat to cell viability and genome stability and they are also generated when replication forks encounter blocking lesions. The failure to repair DSBs or misrepair can result in cell death or large-scale chromosome changes including deletions, translocations and chromosome fusions that enhance genome instability and are hallmarks of cancer cells. Cells have evolved groups of proteins that function in signaling networks that sense DSBs, arrest the cell cycle and activate DNA repair pathways. Histone H2AX is a member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionary conserved C-terminal motif. The serine residue in this motif becomes rapidly phosphorylated in cells when DSBs are introduced (γ-H2AX) forming foci. These γ-H2AX foci may play an essential role in the efficient recruitment of proteins involved in the repair of the DNA DSBs. This role may be to mark the site of the damage. It is also possible that the H2AX phosphorylation alters chromatin structure to facilitate repair or to stabilize the break region so that the DNA ends remain in proximity. Given the above, the main purpose of this study was to ultrastructurally localize using immunogold, γ-H2AX foci in human lung fibroblasts irradiated with specific doses of ionizing radiation in order to create DSBs and to examine if a similar reaction takes place in cancer lung cells. The results indicate that when fibroblasts are experimentally exposed to increasing doses of radiation, aggregates of gold particles are observed indicating localization of γ-H2AX foci. In a series of similar experiments using cancer lung tissue, the same pattern of gold particle localization is observed suggesting that in these cells the formation of γ-H2AX foci is triggered. It is the first time ever that γ-H2AX foci formation is ultrastructurally identified with electron microscopy and this is very important since it confirms the existence of foci in chromatin and indicates the sites of DNA double strand breaks.