Functional proteomic analysis of promyelocytic leukaemia nuclear bodies in irradiation-induced MCF-7 cells.

It is well established that promyelocytic leukaemia nuclear bodies (PML NBs) play important roles in DNA damage responses (DDR). After irradiation, PML NBs dynamically recruit or release important proteins involved in cell-cycle regulation, DNA repair and apoptosis. As PML protein is the key molecule of PML NBs' dynamic assembling, we aimed to characterize the PML-interacting proteins in (60)Co-irradiated MCF-7 cells. A proteomic approach using CoIP, mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 124 proteins that may associate with PML after irradiation. Bioinformatic analysis of the identified proteins showed that most of them were related to characterized PML functions, such as transcriptional regulation, cell-cycle regulation, cell-death regulation and response to stress. Four proteins, B23, MVP, G3BP1 and DHX9, were verified to co-localize with PML differentially before and after ionizing radiation (IR) treatment. The proteins identified in this study will significantly improve our understanding of the dynamic organization and multiple functions of PML NBs in DDR.

FLASH degradation in response to UV-C results in histone locus bodies disruption and cell-cycle arrest.

Eucaryotic cell nuclei contain a number of different organelles that are highly dynamic structures and respond to a variety of stimuli. Here we investigated the effect of UV irradiation on a recently identified group of organelles, Histone Locus Bodies. Histone Locus Bodies contain at least two main proteins, FLASH and NPAT, and have been shown to be involved in replication-dependent histone gene transcription. We show that these organelles are disrupted after sublethal irradiation and both FLASH and NPAT are degraded, which in turn results in cell-cycle arrest at the S/G2 transition. The effect on the cell cycle is due to reduced transcription of histone genes and restoring normal histone protein levels by stabilizing histone mRNA allows cells to progress through the cell cycle. This provides a novel mechanism of S-phase arrest in response to DNA damage that potentially allows DNA repair before cells continue into mitosis, and thus prevents transmission of genomic alterations.

Light-independent phytochrome signaling mediated by dominant GAF domain tyrosine mutants of Arabidopsis phytochromes in transgenic plants.

The photoreversibility of plant phytochromes enables continuous surveillance of the ambient light environment. Through expression of profluorescent, photoinsensitive Tyr-to-His mutant alleles of Arabidopsis thaliana phytochrome B (PHYB(Y276H)) and Arabidopsis phytochrome A (PHYA(Y242H)) in transgenic Arabidopsis plants, we demonstrate that photoconversion is not a prerequisite for phytochrome signaling. PHYB(Y276H)-expressing plants exhibit chromophore-dependent constitutive photomorphogenesis, light-independent phyB(Y276H) nuclear localization, constitutive activation of genes normally repressed in darkness, and light-insensitive seed germination. Fluence rate analyses of transgenic plants expressing PHYB(Y276H), PHYA(Y242H), and other Y(GAF) mutant alleles of PHYB demonstrate that a range of altered light-signaling activities are associated with mutation of this residue. We conclude that the universally conserved GAF domain Tyr residue, with which the bilin chromophore is intimately associated, performs a critical role in coupling light perception to signal transduction by plant phytochromes.

[Structural rearrangements of the chromatin during the adaptive response in two genetically close djungarian hamster fibroblast cell lines with different radiosensitivity].

The capacity of cell for the adaptive response (AR) induction after gamma-irradiation using micronuclear test was investigated. Our model consists of the parental djungarian hamster embryonic fibroblast cell line DH-TK- and its radioresistant progeny (PIC-20). We demonstrated that AR for the more radiosensitive parental cell line was shifted to the lower adaptive and to the challenge doses. The maximal AR for DH-TK- cells was induced at 0.3 Gy adaptive dose and 1.5 Gy challenge dose (adaptive response coefficient (ARC) was 0.4+/- 0.1), whereas for PIC-20 cells these means were 0.5 Gy and 3.0 Gy correspondingly (ARC = 0.45+/-0.1). Using the method of anomalous viscosity time dependence (AVTD) we demonstrated the chromatin rearrangements in both cell lines during 3-5 h after adaptive dose application. The rearrangement degree evaluated by the relative maximal reduced viscosity was considerably higher in PIC-20 cell line than that in DH-TK cells (2.4+/-0.3 vs 1.4+/-0. 1). Interestingly, the time of chromatin rearrangement did not depend neither on the dose nor on the cell type and was similar in both cell lines after 5 h of adaptive dose application. It was also shown that during the AR chromatin relaxation was lower after exposure to both the adaptive and challenge doses than after challenge dose only. In contrast, in the degree of AR chromatin relaxation was higher for both cell lines.