Abstract
Oncogene induced senescence (OIS) is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR), senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on RasV12-induced senescence in human diploid fibroblasts (HDFs). We showed here that hypoxia prevents execution of oncogene induced senescence (OIS), through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21CIP1 and p16INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α). In addition, hypoxia also decreased marks of H-RasV12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21CIP1 but not p16INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.
Highlights
Healthy, normal mammalian cells are characterised by a finite replicative potential, limiting their lifespan to a finite number of divisions, finding first described by Hayflick and Moorhead in 1961 [1]
We tested whether H-RasV12 overexpression results in generation of senescence-associated heterochromatic foci (SAHF), and here we showed that SAHF formation takes place only under normoxic conditions but not when the cells were cultured under hypoxic conditions (Figure 1C)
Our results suggest that H-RasV12-induced senescence is blocked under low oxygen conditions, and this inhibition of senescence resulted in restoration of cell proliferative capacity of human diploid fibroblasts (HDFs) (Figures 1B and D) as evidenced by Ki67 positivity and increased incorporation of BrdU, as well as decreased senescence markers SA-b-gal, H3K9me3 and SAHFs (Figures 1A and C)
Summary
Normal mammalian cells are characterised by a finite replicative potential, limiting their lifespan to a finite number of divisions, finding first described by Hayflick and Moorhead in 1961 [1]. Known as ‘‘Hayflicks limit’’ it is a state of ceased cellular proliferation, where cells still retain their metabolic activity, showing changes into a more flattened morphology when compared to normally proliferating cells. This phenomenon was named cellular senescence, and can be caused by different factors such as telomere attrition, DNA damage, oncogenes, oxidative stress [2]
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