For many researchers, irreversible cell cycle
arrest is the canonical trait of senescent cells. Such growth arrest can be induced
experimentally by the up-regulation or over-expression of cyclin dependent
kinase inhibitors (CDKi). Thus valuable
models are, at least potentially, available in which to study the physiological
effect of growth arrest distinct from the DDR or any other upstream response. Unfortunately there has been little
characterization of the phenotype of cells rendered ‘senescent’ by this means.
Blagosklonny and co-workers (Korotchkina et al. 2009) used an
isopropyl-thio-galactosidase (IPTG)-inducible p21 expression construct to
induce a senescence-like state in an HT1080-derived cell line (HT-p21-9). Characterisation of the phenotype of these
cells does not appear to have been attempted beyond observing irreversible
growth arrest and the presence of increased SA-β-Gal
activity. Given that HT1080 is a highly
tumorigenic fibrosarcoma carrying an activated N-ras oncogene (Benedict et al. 1984), it probably represents a
poor genetic background in which to assess whether markers of immunogenic
conversion or resistance to cell death can be induced by CDKi overexpression
alone. However, the basic principle of using
such a construct for that purpose is sound.
Tokarsky-Amiel et al (2013) showed that overexpression of p14ARF in the
epidermis of the skin of mice (using a tetracyclin-inducible construct) resulted
in mass apoptosis and cell cycle arrest.
As measured by SA-β-Gal
activity, the p14ARF transgene drove senescence in up to 8% of the
surviving cells in the epithelium by a p53-dependent mechanism (demonstrated by
ablation of p53 through co-expression of a specific shRNA directed against it). These senescent cells were viable within the
epidermis for several weeks consistent with lack of clearance. Unfortunately, minimal analysis of their
phenotype was conducted (beyond assessment of the message levels for the
senescence-associated genes Pai-1 and Dcr2).
Thus, the immune state of the p14ARF-senescent cells is currently
unclear and the picture is complicated by the fact that senescent rodent cells
do not display a senescent secretome under some conditions. However, given that alopecia and follical
stem cell dysfunction were observed in the animals, it is clear that cells
rendered ‘senescent’ in this manner can exert phenotypic effects. Thus, there is some evidence that cell cycle
arrest alone may be sufficient to cause problems in highly mitotic tissues such
as the epidermis, but large amounts of work remain to be done.
CDKi overexpression systems clearly have the
potential to be valuable tools. However the
extent to which these are physiologically reflective can legitimately be challenged. This can be understood in two ways (i) the
mechanism by which the growth arrest is induced has not been reported in vivo and (ii) cells do not become
senescent en mass but gradually as a
result of tissue turnover throughout life.
Thus, findings made with these systems could be considered ‘artefactual’
By way of addressing these concerns, it is worth
remembering that for many years replicative senescence was dismissed as a
‘tissue culture artefact’ because senescent cells had not been observed in vivo (evidence for their existence in
tissue remained severely limited until the late 1990s). By the same token, elevation of CDKi alone in
cells in vivo is not impossible. Absence of evidence is never evidence of
absence. Similarly, many
over-expression systems model systems can be said to be non-physiological. However, valuable data is routinely gathered
using them and in this instance could allow researchers to gage the maximum
physiological impact that irreversible growth arrest can have on tissue
function. Thus, if these limits are
recognized, such models are potentially utile, especially when combined with
detailed analysis of phenotypes known to exist in other ‘senescent cells’ (e.g.
apoptosis resistance, immune ligand presentation and the secretory response)
