Id-1 Delays Senescence But Does Not Immortalize Keratinocytes

The packing cells were cultured in Dulbecco’s modified Eagle’s and transfected with Id-1 containing vector or linker (without Id-1) vector by using CaC1 2 and 2 x HBSS. After overnight incubation, the cells were fed with fresh medium and incubated at 32 0 C for an additional 24-48 h. The supernatants were collected for subsequent infection of primary cultures of keratinocytes.

-3-While studies examining replicative senescence have traditionally involved dermal fibroblasts as pioneered by Hayflick (1), more recently a growing interest in human keratinocyte senescent pathways has emerged (2)(3)(4). Immortalization of keratinocytes requires a geneticallydefined program distinct from other cell types. For example, to achieve immortalization of keratinocytes requires inactivation of Rb/p16 function plus activation of telomerase, whereas in human fibroblasts, telomerase activation suffices to significantly extend their life span (5).
Recently, it has been suggested that overexpression of the helix-loop-helix protein Id-1, was sufficient to immortalize primary human keratinocytes (6). It was concluded that Id-1 expression lead to activation of telomerase activity with inactivation of Rb and p16 in keratinocytes (ibid).
Since the methodology employed in this report involved a selection step following transfection of the Id-1 cDNA, it was possible that the selected clones of immortalized keratinocytes had acquired other mutations that were contributing to bypassing of replicative senescence (4).
To further investigate the role of Id-1 in an unselected population of cultured keratinocytes, a retroviral vector containing the Id-1 cDNA was used to examine the relationship between Id-1 expression and the resultant phenotype in neonatal foreskin-derived human keratinocytes. Retrovirus-mediated gene-transfer facilitates introduction of target genes with high efficiency, so that effects of the transduced gene can be examined in whole cell populations (7). In contrast to the initial report (6), the overexpression of Id-1 by keratinocytes using this different experimental approach delayed the onset of replicative senescence, but did not prevent senescence. The Id-1 expressing keratinocytes with delayed senescence in later passaged cells resembled spontaneously senescent keratinocytes in that there was no increase in telomerase, but there were shortened telomeres (approximately 6kb in length), and increased p16, and active Rb levels. The importance of these findings is discussed in terms of normal epidermal homeostasis and cutaneous oncology (8)(9).

Cell Culture and Treatment
Keratinocyte cultures were initiated from discarded neonatal foreskins, in which the epidermis was initially separated from dermis by exposure to dispase. The subsequent epidermal sheet was treated with trypsin / EDTA to produce a single cell suspension of keratinocytes that were placed in plastic petri dishes (Corning, NY) as previously described (3).
Keratinocytes were fed with a low calcium (0.07 mm), serum-free medium (KGM, Clonetics Corp., San Diego, CA). From each foreskin, the initial epidermal suspension was seeded into 3 different 35 mm dishes, and then transferred to a 6-well cluster plate for retroviral infection.
After infection and washing, keratinocytes were routinely cultured in 10 cm dishes, and passaged at 60-70% confluency with feeding using fresh KGM every other day. Cell counts to determine population doublings was performed manually using a hemocytometer and estimated based on the log 2 (number of keratinocytes at subculture/ number of cells initially plated) as primarily described (4). For induction of p16 and p21, representative keratinocytes from various passages were incubated with phorbol ester (TPA;100 nM; Sigma Chemical Co., St. Louis, MO) for 3 days, and then protein extracts performed as described below. For growth control and TPA response controls, neonatal foreskin-derived keratinocytes were derived exactly as above except they were either; never exposed to the retroviral infection, or were exposed to retroviral preparations containing only linker, but not containing the Id-1 cDNA.

Retroviral vectors and transduction of normal keratinocytes.
Id-1 cDNA was obtained from BamH1 and NOT1 of LZRS and MGF-based retroviral expression vector. The LZRS vector was kindly provided by Dr. Paul A. Khavari (Stanford University School of Medicine, Stanford CA) and used as previously described (10). The Phoenix-Ampo retroviral packing cells were obtained from American Type Culture Collection  The normal keratinocytes were seeded into 6-well plates and infected with 300 ul of viral supernatant in the presence of 4 ug/ml hexadimethrine bromide (Polybrene) (Sigma H-9268) for 1 h at 32 0 C, then the supernatant was removed and replaced with fresh medium, incubated at 37 0 C in 5% CO 2 overnight. After being washed with PBS, the infected cells were propagated and treated with TPA. The overexpression of the Id-1 protein was detected with Western blot and immunocytochemistry. Immunostaining was performed using the SC-488 anti-Id-1 ab (Santa Cruz) combined with an avidin-biotin peroxidase detection system (Vectastain, Vector Labs, Burlingame, CA). Positive staining was accomplished using 3-amino-4-ethylcarbazole as the chromagen producing a positive red reaction with 1% hematoxylin as the counter-stain as described previously (11).

Western Blot Analysis.
Whole cell lysates were prepared to detect different proteins. In brief, keratinocytes were

Telomerase Assay and Telomere Length Determination
The assay for telomerase activity utilized the PCR-based, telomerase repeat amplification protocol (TRAP assay); (12). The estimation of telomere length was performed by hybridizing a

I. Overexpression of Id-1 Extends Keratinocyte Lifespan
Neonatal foreskin-derived keratinocytes normally proliferate rapidly in KGM (0.07 mM calcium), dividing every 1-2 days for 3-4 passages (approximately 20 population doublings; PD), before undergoing irreversible growth arrest (i.e. replicative senescence). Using unselected cell populations infected with an empty (only linker cDNA) retroviral vector, similar kinetics and number of PD was observed. However, unselected keratinocyte populations infected with an Id-1 containing retrovirus had a significantly greater number of population doublings (greater than 60 PD in two of the three independently generated Id-1 overexpressing cell lines), compared to uninfected or linker-control keratinocyte populations. Id-1 overexpressing keratinocytes also proliferated rapidly (dividing every 1-2 days), but in contrast to the earlier report (6)

II. Molecular Phenotype of Senescent Keratinocytes
While overexpression of Id-1 by proliferating retroviral infected keratinocyte cell lines in early (PD 6 and PD 9) was expected (Figure 2), the relative levels of Id-1 in these transduced keratinocytes at the onset of replicative senescence, was of interest. When non-transduced keratinocytes undergo spontaneous senescence either due to confluency or replicative exhaustion, Id-1 levels are barely detectable compared to early passage proliferating normal keratinocytes ( Figure 1; lanes 1 and 2). Interestingly, compared to these growth arrested non-transduced keratinocytes, the senescent Id-1 transduced keratinocyte lines continue to overexpress Id-1, although the relative levels decreased slightly compared to the proliferating Id-1 transduced keratinocytes examined at earlier passages ( Figure 1 ; lanes 6 and 7). There was either no change, or minimal change, in the levels of either Bcl-x or β-actin ( Figure 1). Examination of Bcl-x levels was selected because of the ability of Bcl-x to influence resistance of keratinocytes to apoptosis (14).
Since we previously established that keratinocytes undergoing spontaneous replicative senescence strongly induced p16 levels (3) Figure 3; lane 1), and early and mid-passage keratinocytes infected to overexpress Id-1 ( Figure   3). However, upon onset of replicative senescence; Id-1 expressing keratinocyte cell lines expressed higher levels of the active (hypophosphorylated) form of Rb (Figure 3; lanes 6 and 7).

III. Telomerase Activity and Telomere Length
As the initial report suggested a link between Id-1 and telomerase activity (6)

IV. Id-1 Overexpression Modulates p16 Levels
To begin to probe the potential mechanism by which Id-1 could delay senescence in our keratinocyte populations, the influence of Id-1 levels on p16 induction was determined.
Exposure of proliferating keratinocytes to phorbol ester (i.e. TPA) induces irreversible growth arrest accompanied by increased p16 levels after 72 hours following TPA treatment (

Discussion
A growing body of evidence implicates Id proteins such as Id-1 as regulators of cell growth in numerous cell lineages (reviewed in 8). In many cell types, including the normal keratinocytes examined in this study, Id-1 expression is down-regulated upon withdrawal of growth factors or onset of senescence (16)(17)(18)(19). The forced overexpression of Id-1 in unselected keratinocyte populations using a retroviral vector significantly impacted the behavior of the cells by delaying the onset of replicative senescence. However, in contrast with the earlier report (6), we did not observe an immortalized phenotype, induction of telomerase, or inactivation of Rb in three different independently created cell lines overexpressing Id-1. The most likely explanation for this discrepancy is that the previous investigators isolated selected clones that included keratinocytes with elevated telomerase activity and inactivated Rb/p16 pathway. We suspect that our keratinocyte cell lines succumbed to the onset of replicative senescence secondary to shortened telomeres, since no induction of telomerase activity was detected. It would appear for some type of fibroblasts, retinal pigment epithelial cells, mesothelial cells as well as keratinocytes, that replicative senescence is a telomere length-sensitive response, with the primary difference reflected by the ability of hTERT overexpression, by itself, to overcome senescence in fibroblasts and retinal pigment cells (2,4,5). Taken together, the results in this report are consistent with the notion that immortalization of human keratinocytes requires not only inactivation of Rb/p16, but also activation of telomerase (5,11,20).
Another contributing factor to the difference between Alani et. al (6)  In conclusion, these results indicate that Id-1 has important growth-regulatory effects on keratinocyte populations, including a delay in the onset of replicative senescence. However, since telomerase is not directly activated by Id-1, telomeres continue shortening in these unselected keratinocyte lines triggering the onset of senescence, which is accompanied by elevated p16 levels and activated Rb. Further studies are indicated to more clearly define the link between Id-1 and regulation of p16 gene expression, as such a line of inquiry could lead to better understanding of the biology of epidermal stem cells (25), as well as cutaneous neoplasia (8,9,26,27). Thus, as an important component of normal tissue turnover and regenerative repair processes, the proliferative potential of transiently amplifying progenitor cells committed to the epithelial lineage must be tightly controlled. In human skin, disease states which include high epithelial turnover as seen following thermal injuries or in psoriasis (28)  in inflammatory bowel disease). Thus, the current results together with previous reports (2,4,5,6,8,9) suggest that the molecular underpinnings of keratinocyte immortalization are quite distinct. These data converge upon the p16/Rb regulatory complex as a crucial regulator of keratinocyte replicative senescence that must be inactivated for telomerase-dependent immortalization. It will also be of interest to determine the role of Id-1 in normal cutaneous homeostasis, and the target genes and binding partners for Id-1 in epidermal keratinocytes.