Normal Human Fibroblasts Are Resistant to RAS-Induced Senescence

Abstract

Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated ␤-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTertimmortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS.Normal cells have several mechanisms in place to protect against uncontrolled proliferation and tumorigenesis. One line of defense is cellular senescence, a permanent growth arrest that occurs after extended periods of cell division, exposure to oxidative stress, or expression of activated oncogenes (26,40). Senescent cells remain alive and metabolically active but are arrested in the G 1 phase of the cell cycle, are resistant to growth factor stimulation, and show common biochemical markers such as expression of an acidic senescence-associated ␤-galactosidase (SA-␤-Gal) activity (6). In addition, senescent cells show altered differentiation functions that are cell type specific. For example, human fibroblasts secrete growth factors and extracellular matrix-remodeling enzymes upon the induction of senescence (6). While senescence has been characterized primarily in cultured cells, there is also evidence that it occurs in vivo. Cells expressing markers of senescence such as SA-␤-Gal have been identified in normal tissues (7), and senescence can be induced following some types of chemotherapeutic treatments (38).The best-characterized example of oncogene-induced senescence is the response of normal fibroblasts to expression of an activated allele of H-ras (H-rasV12). Normal ras proteins are important for transducing mitogenic signals in the cell and are mutated to constitutively active forms in approximately 20% of human cancers (4). These activated alleles contribute to transformation by increasing proliferation, angiogenesis, and invasion of tumors, as well as desensitizing ce...

Enhanced elimination of oxidized guanine nucleotides inhibits oncogenic RAS-induced DNA damage and premature senescence

“…In contrast, the comparable cells overexpressing both MTH1 and oncogenic H-RAS did not display any of these senescent features (Figures 1a-c). Elevated p16INK4a levels have been reported to be essential for the induction of RAS-induced senescence (Benanti and Galloway, 2004). We found that introduction of oncogenic RAS led to elevated Figure 1 MTH1 overexpression prevents oncogenic RAS-induced senescence.…”

The DNA Damage Signaling Pathway Connects Oncogenic Stress to Cellular Senescence

“…In fact, certain isolates of normal human diploid fibroblasts are resistant to Ras-induced senescence and have very low levels of p16INK4a. 47 Again, it would be interesting to study whether oncogenes can induce ROS or the DDR in these cells. Another characteristic of cellular senescence that has been less well studied is the stability of the phenotype.…”

Real-time in vivo imaging of p16Ink4areveals cross talk with p53

“…gene expression (Benanti and Galloway, 2004). To explore this notion in a more physiological setting rather than using ectopic expression of oncogenic Ras in cultured cells, the p16-luc mice were subjected to a conventional chemically induced skin papilloma protocol with a single dose of DMBA followed by multiple treatments with TPA.…”

“…To date, much of our current knowledge of how human p16 Ink4a gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells (Serrano and Blasco, 2001;Gil and Peters, 2006;Kim and Sharpless, 2006). However, because human p16 Ink4a gene expression is also induced by tissue culture-imposed stress (Ramirez et al, 2001;Benanti and Galloway, 2004;Ince et al, 2007;Shay and Wright, 2007), it remains unclear whether the induction of human p16…”