INTRACELLULAR REACTIVE OXYGEN SPECIES AS APPARENT MODULATORS OF HEAT-SHOCK-PROTEIN-27 (HSP27) STRUCTURAL ORGANIZATION AND PHOSPHORYLATION IN BASAL AND TUMOR-NECROSIS-FACTOR-ALPHA-TREATED T47D HUMAN CARCINOMA-CELLS

The small stress protein heat-shock protein 27 (hsp27) is an oligomeri c phosphoprotein, constitutively expressed in most human cells, which enhances cellular resistance to tumour necrosis factor alpha (TNF alph a). This phenomenon correlates with dramatic changes in hsp27 cellular location, structural organization and phosphorylation. To gain a bett er understanding of the molecular mechanisms regulating these properti es of hsp27, we investigated whether they were a consequence of the in tracellular production of reactive oxygen species (ROS) generated by T NF alpha. Here, we report that, in T47D carcinoma cell lines, the rapi d burst of intracellular ROS production and changes in hsp27 locale, s tructural organization and phospho-isoform composition induced by TNF alpha were abolished by the overexpression of the antioxidant enzyme s eleno-glutathione peroxidase (GSHPx). These effects were greatly dimin ished when GSHPx-expressing cells were grown in the absence of seleniu m, a cofactor that is essential for seleno-GSHPx activity, indicating that they are directly linked to the increased GSHPx activity. Moreove r, in growing T47D cells, GSHPx expression induced intracellular redis tribution of hsp27 and decreased the phosphorylation of this protein w ithout altering its pattern of oligomerization. In contrast, the heat- mediated phosphorylation of hsp27 was not altered by decreased intrace llular ROS levels. Hence, in growing and TNF-treated cells, several hs p27 properties appear to be modulated by fluctuations in intracellular ROS levels.