INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence

Abstract

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16 INK4a and p14 ARF , both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary ®broblasts from a member of a melanomaprone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16 INK4a de®cient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14 ARF . Although they have a ®nite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We ®nd that in human ®broblasts, ARF is not induced demonstrably by RAS, pointing to signi®cant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.

CBX7 and miR‐9 are part of an autoregulatory loop controlling p16INK4a

“…Next, we took advantage of the Leiden strain of human fibroblasts that carry a mutation that functionally inactivates both copies of p16 INK4a (Brookes et al ., 2002). Immunostaining revealed that the levels of the altered protein in Leiden fibroblasts are lower than those of wild‐type p16 INK4a in IMR90 cells (Fig.…”

“…Leiden human fibroblasts have been previously described (Brookes et al ., 2002). Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Paisley, UK) with 10% foetal bovine serum (PAA, Amersham, UK) and 1% antibiotic–antimycotic solution (Invitrogen, Paisley, UK).…”

Remodeling of chromatin structure in senescent cells and its potential impact on tumor suppression and aging

“…In mouse cells, activated oncogenes also activate p53 by upregulation of p14ARF. However, this pathway is apparently not conserved in human cells (17,42,132). Activation of the p53 pathway contributes to activation of the pRB pathway.…”

Immortalized cells as experimental models to study cancer

“…A complementary approach to understanding the role of p16 INK4A in suppressing transformation is the use of p16 INK4A -deficient fibroblasts (Leiden cells) (Brookes et al 2002;Drayton et al 2003). These human cells have a homozygous deletion in INK4A that renders them 16 INK4A deficient, yet they translate a protein that retains some of the known functions of p14 ARF (Brookes et al 2002).…”

“…These human cells have a homozygous deletion in INK4A that renders them 16 INK4A deficient, yet they translate a protein that retains some of the known functions of p14 ARF (Brookes et al 2002). Co-expression of hTERT, c-Myc, and RAS in Leiden cells, but not in control p16 INK4A -expressing fibroblasts, allows for tumor formation in immunocompromised mice, but at long latency and low frequency (Drayton et al 2003).…”