Comparative Effects of Hypoxia on Normal and Immortalized Human Diploid Fibroblasts

Abstract

Hypoxia, a condition of reduced oxygen concentration, is observed in many physiological and pathophysiological states. However, there is rather limited information regarding hypoxic effects in the processes of immortalization and senescence of human cells. Here, the effects of hypoxia induced by either 1.5% O₂, or by a hypoxia-mimetic agent, CoCl₂, on the protein expression of normal human diploid fibroblasts (HDFs) undergoing replicative senescence, and in their Simian Virus 40 (SV40) T antigen immortalized counterparts are described. The data demonstrated that, in all cell types assayed, either hypoxia or CoCl₂ induced the main regulator of transcriptional responses to reduced oxygen tension, namely the hypoxia inducible factor-1α (HIF-1α), in a dose- and time-dependent manner. In the immortalized HDFs, the transcriptional activity of HIF-1α was also evident by the accumulation of its main downstream gene targets, namely erythropoietin (EPO) and the vascular endothelial growth factor (VEGF). Interestingly, the immortalized HDFs were found to exhibit higher HIF-1α endogenous levels and induction, following cell exposure to hypoxic conditions, as compared to either young or senescent cells. Subsequent analysis of the expression levels of two pro-survival proteins, bcl-2 and clusterin/apolipoprotein J (CLU), in cells exposed to hypoxic conditions, revealed that although bcl-2 was up-regulated independently of the cell type, CLU was induced only in the CoCl₂-treated immortalized HDFs. These findings indicate that the distinct cellular contexts of normal and immortalized HDFs may induce differential responses to hypoxia.