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Role of the Alternative INK4A Proteins in Human Keratinocyte Senescence: Evidence for the Specific Inactivation of p16^INK4A upon Immortalization¹

Beatson Institute for Cancer Research, Cancer Research Campaign Beatson Laboratories, Glasgow G61 1BD, United Kingdom [J. M., E. K. P.]; Imperial Cancer Research Fund Laboratories, London WC2A 3PX, United Kingdom [F. J. S., G. P.]; and ABL Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201 [K. H. V.]

	ABSTRACT

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The INK4A locus on human chromosome 9p21 encodes two genes that have been implicated in replicative senescence and tumor suppression, p16^INK4A and p14^ARF. In contrast to p16^INK4A, which is up-regulated to high levels, we were unable to detect p14^ARF protein in senescent human keratinocytes. Also, p53, an established target of p14^ARF, did not increase, suggesting that p14^ARF is not instrumental in human keratinocyte senescence. In neoplastic keratinocyte cultures, p16^INK4A inactivation was invariably associated with the immortal phenotype, and there was evidence for the inactivation of p16^INK4A, independent of p14^ARF, in 6 of 10 lines that lacked large homozygous deletions. In contrast, we failed to detect exon 1ß mutations or p16^INK4A-independent deletions. These results emphasize the previously proposed role for p16^INK4A in human keratinocyte senescence but do not rule out a supporting role for p14^ARF inactivation.

	Introduction

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All human somatic cells, including keratinocytes, exhibit a limited replicative life span that culminates in senescence, but squamous cell carcinoma keratinocytes are often immortal, especially in advanced tumors (Refs. 1 and 2 and references therein). We previously reported that loss of heterozygosity at 9p21 and homozygous deletions of the INK4A locus at that site were associated with keratinocyte immortality in squamous neoplasia (1) . We later showed that p16^INK4A accumulated in keratinocytes as they approached senescence and was invariably inactivated in cultures of immortal neoplastic keratinocytes but generally not in the mortal cells (2) . However, the INK4A locus on human chromosome 9p21 encodes another gene, p14^ARF, that, like p16^INK4A, has been implicated in replicative senescence and squamous cell carcinoma development (3, 4, 5, 6, 7) . The coding regions of these genes overlap, so that homozygous deletions of p16^INK4A, in addition, frequently disrupt p14^ARF. Furthermore, many of the properties of the INK4A locus, including replicative senescence, could be attributed to p14^ARF rather than p16^INK4A (4 , 7 , 8) . p14^ARF requires a functional p53 gene to exert its cell cycle-inhibitory effects and is likely to work by affecting p53 stability, thereby causing it to accumulate and cause cell cycle arrest in G₁ and G₂ (9) . ARF accumulates strikingly as rodent fibroblasts approach senescence (10) , but its accumulation is much less striking in human fibroblasts, if the increased protein content of senescent cells is taken into account (8) . ARF also directs the accumulation of p53 in response to ras (11 , 12) and myc (10) oncogene activation, which results in senescence (11 , 12) and apoptosis (10) , respectively. In the light of these more recent observations, we have examined the role of p14^ARF in keratinocyte senescence and its loss in squamous neoplasia and keratinocyte immortalization. Human squamous carcinoma is an excellent system in which to address this question, because both p14^ARF and p16^INK4A have been shown to inhibit keratinocyte proliferation when they are ectopically expressed at high levels (7) , and INK4A homozygous deletions are frequently detected in squamous cell carcinoma lines (1 , 2) and tumors (5) .

	Materials and Methods

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Cell Culture.
The normal and neoplastic keratinocyte cultures and their genetic properties have been described previously (13) . The cells were generally cultured using 3T3 feeder layers in DMEM, as described previously (1 , 2) . Where stated, normal keratinocytes were sometimes cultured in serum-free keratinocyte growth medium (Clonetics, San Diego, CA), as described (2) . HaCat and SaOS-2 cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, and normal diploid fibroblasts were cultured in DMEM supplemented with 20% (v/v) fetal bovine serum.

Western Blotting and Antibodies.
Human keratinocyte pellets (1 x 10⁶-2 x 10⁶ cells/pellet) were lysed for 30 min in buffer containing 20 mM HEPES (pH 7.9), 5 mM EDTA, 10 mM EGTA, 5 mM NaF, 0.1 µg/ml okadaic acid, 10% glycerol, and 1 mM DTT, containing 0.4 M KCl, 0.4% Triton X-100 and protease inhibitors, as follows: 5 µg/ml each aprotonin and pepstatin A, 1 mM benzamidine, and 50 µg/ml phenylmethylsulfonyl fluoride. Following sonication, the extracts were cleared by centrifugation, and the supernatants were stored at -70°C. Protein (100 or 200 µg) was subjected to electrophoresis on 17% (p14^ARF, p16^INK4A, and p21^WAF) or 12% (p53) Tris-glycine SDS polyacrylamide gels. After semidry blotting onto Immobilon-P filters (Millipore, Watford, United Kingdom), nonspecific binding sites were blocked by incubating the membrane in Tris-buffered saline-5% nonfat dried milk. Primary antibody incubations were carried out overnight at 4°C in Tris-buffered saline-5% milk with the antibodies against the following human proteins: p14^ARF (see Ref. 10 ), p16^INK4A (N-20 or C-20; Santa Cruz Biotechnology, Santa Cruz, CA), p53 (DO-1; Santa Cruz Biotechnology), and p21^WAF (Affiniti; Transduction Laboratories, Becton Dickinson, Oxford, United Kingdom). After being washed, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham International, Amersham, United Kingdom) and then developed with enhanced chemiluminescence substrate (Amersham International). Normal human epidermal keratinocytes were used as controls in all experiments. The membranes were stained with Ponceau solution and reprobed with Cdk4 and ERK2 (Affiniti) to ensure even loading and transfer. Neither Cdk4 nor ERK2 changes during replicative senescence, and ERK2 is not altered when keratinocytes become immortal or cancerous.³ Negative controls were cell lines lacking the INK4A locus (p14^ARF and p16^INK4A), SaOS-2 osteosarcoma cells (p53), HaCat cells (p21^WAF low, because no p21^WAF-deficient cells were available), and lethally irradiated 3T3 cells alone (X3T3). Positive controls were SaOS-2 cells (p14^ARF and p16^INK4A) and human diploid fibroblasts 20 h postirradiation with 4 Gy of rays (p53 and p21^WAF). The exposed films were scanned using a PDI Inc. gel scanner (PDI Inc., Huntingdon Station, NY), and the images were quantitated using the Quantity One program (PDI Inc., Huntingdon Station, NY).

PCR and DNA Sequencing.
Exons 1, 1ß, 2, and 3 of the INK4A locus were amplified by PCR from the DNA of human keratinocytes using the primers described by Kubo et al. (14) . The PCR cycles were as follows: 94°C for 5 min, followed by 25 cycles comprising 94°C for 30 s, the annealing temperature (14) for 30 s, and 72°C for 30 s, with the exception of the exon 1, for which the cycles comprised 94°C for 15 s, 67°C for 15 s, and 72°C for 15 s. Reactions were performed using cloned Pfu DNA polymerase and buffer (Stratagene, Cambridge, United Kingdom), with the exception of exon 1, which required a lower MgCl₂ concentration (0.5 mM). After purification on microspin S-400 columns (Pharmacia), the products were subjected to BigDye terminator cycle sequencing (Perkin-Elmer/Applied Biosystems). Sequencing was performed on a PTC 100 (Genetic Research Instrumentation Ltd.) thermocycler using GeneAmp 9600 (Perkin-Elmer Corp.) terminator cycle sequencing conditions. Samples were then run on an ABI 377 sequencer. All products were isolated twice, from independent PCRs, and sequenced in both directions. Mutations were additionally confirmed using an independently isolated DNA sample. Sequences were analyzed using the Lasergene package program (DNASTAR Inc., Madison, WI).

	Results

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Expression of P14^ARF and P16^INK4A in Human Keratinocyte Senescence.
We investigated the expression of the p14^ARF protein as normal human epidermal keratinocytes cultured in serum-free medium approached replicative senescence (Fig. 1) and showed that it was undetectable, even when the cells had senesced (Fig. 1a) . A similar result was obtained when human oral keratinocytes cultured using serum containing medium and feeder layers were examined (data not shown). The p14^ARF protein was, however, readily detected in SV40-immortalized human fetal keratinocytes, in which p53 and pRb are likely to be functionally compromised, and in the p53/pRb-negative SaOS 2 line, which was used as a positive control (Fig. 1a) . Reprobing the same filters with antibodies against p16^INK4A and p21^WAF clearly showed a large accumulation of the former protein (Fig. 1b) , as reported previously (2) , although the p21^WAF levels showed only a small increase, which then declined (Fig. 1c) . These results show that there is unlikely to be sufficient accumulation of p14^ARF during keratinocyte senescence to inhibit keratinocyte proliferation (7 , 9) .

View larger version (33K): [in this window] [in a new window]   Fig. 1. The levels of the INK4A, WAF, and p53 proteins at different population doubling levels of normal human epidermal keratinocytes. The levels of p14ARF protein were examined by Western blotting as cultured human keratinocytes approached senescence, and no detectable increase in the levels of this protein were found (a), in contrast to the striking increase in p16INK4A (b). c, the levels of p21WAF rose only very slightly and then returned to normal. d, the levels of p53 remained normal until the cells reached around 43 population doublings and then fell to 65% of that of the early-passage cells. These results were not due to differences in loading or membrane transfer because both the ERK2 and Cdk4 (data not shown) signals and the amount of protein on the filter as assessed by Ponceau’s stain (data not shown, see also "Materials and Methods") were very consistent. The rightmost six lanes are controls (see "Materials and Methods" for further details). Importantly, all of the human epidermal keratinocyte samples (HEK) had at least as much protein loaded as the positive controls.

These observations argue against an instrumental role for p14^ARF in human keratinocyte replicative senescence, but a permissive role for the protein might be predicted, based on its known ability to positively regulate p53 (9) and the established importance of p53 to human fibroblast senescence (15 , 16) . We, therefore, examined the status of the p14^ARF gene and protein in a panel of well-characterized immortal human head and neck keratinocyte lines.

Deletion and Mutation Analysis of the INK4A Locus in Immortal Neoplastic Human Keratinocytes.
Homozygous deletions of the INK4A locus have been reported before in cells derived from squamous neoplasms (1 , 2 , 5) and have been associated with the immortal phenotype (1 , 2) . We tested for the presence of all four INK4A exons in our panel of keratinocyte lines (Table 1) and found that 11 of 20 had one or more of these exons deleted. However, only 9 of 20 had a deletion of exon 1ß, which encodes the functional and unique portion of p14^ARF (4) , and no line sustained a deletion of exon 1ß alone. No deletions of p15^INK4B were present in the panel of cell lines we used (2) . We sequenced all of the exons that were present and found no mutations in exon 1ß. Lines BICR3 and BICR19 possessed nonsense mutations in exon 1 that disrupt the coding sequence of p16^INK4A but not p14^ARF, and line BICR56 possessed a stop codon at Tyr-129 in exon 2 of p16^INK4A (Fig. 2a) , which also does not affect the coding sequence of p14^ARF. DOK and SCC-12 have mutations in exon 2 that would be predicted to alter the function of p16^INK4A but not p14^ARF because only the NH₂-terminal portion of p14^ARF, encoded by exon 1ß, is essential for its function (11 , 17 , 18) . SCC-12 possesses a D84N p16^INK4A mutation (Fig. 2b) , R98Q in p14^ARF, and DOK possesses a D74Y p16^INK4A mutation (Fig. 2c) , R88L in p14^ARF. SCC-4 was found to contain an intronic deletion that resulted in the loss of the exon 3 splice acceptor site and the consequent loss of a normal exon 3 of p16^INK4A (Fig. 2d) . This alteration has been reported previously, and it is presumed that it alters the function of p16^INK4A but is unlikely to alter the function of p14^ARF.

View larger version (30K): [in this window] [in a new window]   Fig. 2. The sequence alterations in the p16INK4A gene. a, codon Tyr-129Stop mutation in the BICR56 cell line. b, codon Asp-84Asn mutation in the SCC-12 cell line. c, codon Asp-74Tyr mutation in the DOK cell line. d, exon 2/3 splice acceptor site deletion in cell line SCC-4. The normal human epidermal keratinocyte sequence (HEK) is included in all of the figures for comparison; arrows, positions of the alterations.

View larger version (52K): [in this window] [in a new window]   Fig. 3. The expression of the p14ARF, p16INK4A, and p53 proteins in immortal human keratinocyte lines. A large panel of neoplastic keratinocyte lines were examined for the expression of the INK4A proteins and p53 by Western blot. a, expression of p14ARF and p16INK4A in normal and immortal human keratinocytes. The expression of p16INK4A was detected in only five of the lines, and in the case of lines SCC-12, DOK, and SCC-4 these were mutant proteins; only very low levels were detected in SCC-13, and BICR68 expressed low levels upon immortalization (see Fig. 2 and Table 1 for details). In contrast, wild-type p14ARF was detected at very variable levels in six lines and was probably functional in lines SCC-12 and DOK (see text for details). The variation in the expression of ARF could not be attributed to differential loading as assessed by Ponceau staining (data not shown) or reprobing the membranes with ERK2 (third and sixth panels) and Cdk4 antibodies (data not shown, see also "Materials and Methods"). SaOS-2 is the positive control, and X3T3 is an additional negative control. All of the samples showed at least as strong an ERK2 signal as SaOS-2. b, the expression of p53 in normal keratinocytes and lines BICR68, BICR78, and BICR82 shows that the line BICR68 over-expresses a wild type p53 protein (see also Table 1 and Ref. 13 ), which is inconsistent with the inactivation of p14ARF. BICR78, which expresses a stable, mutated p53 protein (see Table 1 and Ref. 13 for details) was included as a positive control, BICR82 was included as a negative control, and SaOS-2 and X3T3 were included as additional negative controls. The ERK2 levels were constant throughout, showing that the loading and transfer of the samples was consistent.

	Discussion

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Our previous results, together with the report that targeted disruption of the INK4A locus rendered mouse fibroblasts susceptible to immortalization and mice to squamous tumor development (Ref. 3 ; see also Ref. 11 ), encouraged the belief that p16^INK4A might act as a squamous tumor suppresser gene by suppressing the immortal phenotype. Recently, however, the situation has been complicated by the discovery that deletions of the INK4A locus also disrupt another gene, p14^ARF, and the targeted disruption of this gene alone is now known to predispose mouse cells to immortalization and neoplasia (4) by compromising the function of p53 (4 , 9) .

These results stimulated us to evaluate the role of p14^ARF in human keratinocyte senescence and to reevaluate the role of p16^INK4A. We found no evidence for the up-regulation of p14^ARF in human keratinocyte senescence, nor did we find any evidence of an increase in its activity as assessed by p53 accumulation. Therefore, although we cannot rule out an effect of p14^ARF that is too subtle to be detected by the methods we have used, our data suggest that there may be key differences between human and mouse cells in the execution of their senescence program because our results are at variance with those reported for mouse fibroblasts (10) . Our findings are, however, completely consistent with a number of reports, which fail to detect p53 accumulation in senescent human fibroblasts (see, for example, Ref. 15 ). Furthermore, there is a key difference between human and mouse fibroblast senescence in vitro, which could reconcile these discrepancies. Human fibroblast senescence (19) is dependent on telomeric attrition and the consequent DNA damage-like cell cycle checkpoint that this likely induces (15) . Significantly, there is no evidence to implicate ARF in DNA damage signaling to the cell cycle (4 , 9) . In contrast, because the telomeres of mouse chromosomes are much longer than those of human cells (20) , mouse embryo fibroblast senescence is unlikely to be triggered by a critically short telomere, and it could well be mediated by ARF.

Although our data suggest that p14^ARF does not play an active role in the induction or maintenance of the senescent phenotype of human keratinocytes, they do not exclude a permissive role for p14^ARF in senescence. We, therefore, tested whether there was any evidence for the inactivation of p14^ARF that was independent of p16^INK4A or vice versa. Nine of 20 immortal lines studied had large homozygous deletions that eliminated all of the exons of both p14^ARF and p16^INK4A, and one line expressed low levels of both p14^ARF and p16^INK4A protein. The remaining 10 either had partial deletions of the INK4A locus or retained the INK4A locus grossly intact. Three cell lines had p16^INK4A mutations, which do not affect the coding sequence of p14^ARF, and a fourth had specifically silenced the p16^INK4A gene (see also Ref. 2 ). Because all these cell lines expressed the elevated levels of wild-type p14^ARF, consistent with cells harboring mutant p53 genes (9) , these data support a role for p16^INK4A inactivation in keratinocyte immortalization, as suggested previously (1 , 2 , 13) . A further two lines harbored missense mutations in the shared exon 2 of both the p14^ARF and the p16^INK4A genes, but such mutations would not be anticipated to alter the function of p14^ARF, only p16^INK4A (11 , 17 , 18) . Similarly, another two lines had deleted exons 1, 2, and 3 of the INK4A locus but had left the p14^ARF-specific exon 1ß intact, and depending on the exact nature of these deletions, these lines too may have retained some p14^ARF activity but no p16^INK4A (11 , 18) . In summary, at least 4, probably 6, and possibly 8 of the 10 lines without large deletions of the INK4A locus have inactivated p16^INK4A and not p14^ARF.

In contrast, we found no evidence of mutation or deletion of exon 1ß, and only two lines expressed a p16^INK4A protein and no p14^ARF; however, one cell line did show evidence of p14^ARF inactivation that had apparently occurred by an independent mechanism to that of p16^INK4A.

The data we have presented here add further support to the idea that the p16^INK4A gene is involved in the human keratinocyte senescence program and that its inactivation contributes to immortality (1 , 2 , 13) , but we were unable to find any evidence for p14^ARF inactivation alone. One argument that could be put forward is that all of the lines we studied except five had sustained homozygous p53 mutations, and because p53 is required for p14^ARF function (9) , this would remove the requirement for p14^ARF inactivation in most of the lines. Clearly, a larger series of squamous lines with wild-type p53 sequence needs to be examined to address this question.

It should also be stressed that our results do not rule out a transient role for p14^ARF inactivation in concert with that of p16^INK4A during the generation of immortal human head and neck keratinocytes. Indeed, the high frequency of homozygous deletions of the INK4A locus rather than specific alterations of the p16^INK4A gene does argue for the presence of another tightly linked gene, the inactivation of which can cooperate with that of p16^INK4A in the pathogenesis of human squamous tumors, including their immortalization. The further selection for dysfunctional p53 genes could be explained on the grounds that p53 participates in many functions that p14^ARF does not. Most notably, the loss of p53 but not p14^ARF renders cells genetically unstable, and this would be necessary in neoplastic human keratinocytes to promote tumor progression as well as the immortal phenotype, which requires the inactivation of several genetic pathways (13) . Furthermore, cells with p53 mutations can still obtain a selective advantage in mouse cancers where ARF has been experimentally deleted first (4) .

The data described here emphasize the importance of p16^INK4A to human keratinocyte senescence and its inactivation to immortality. The selection against p16^INK4A and the INK4A locus in human head and neck cancer may be connected with an escape from either replicative senescence (3) or senescence provoked by an activated oncogene (11 , 12) . Further work is clearly required to resolve this issue, but whatever senescence mechanism is dominant in the suppression of human squamous cancer, our data support a role for p16^INK4A in its execution.

	ACKNOWLEDGMENTS

 
We thank Professor John Wyke for critical review of the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by grants from the Cancer Research Campaign and the Association for International Cancer Research (to E. K. P. and J. M.) and from the Imperial Cancer Research Fund (to F. J. S. and G. P.). K. H. V. was supported, in part, by the National Cancer Institute, Department of Health and Human Services, under contract with ABL.

² To whom requests for reprints should be addressed, at Beatson Institute for Cancer Research, Cancer Research Campaign Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, United Kingdom. Phone: 44-141-330-3653; Fax: 44-141-942-6521; E-mail: ekp1n{at}beatson.gla.ac.uk

³ M. Agochiya, V. G. Brunton, D. W. Owens, E. K. Parkinson, C. Paraskeva, N. W. Keith, and M. C. Frame. Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells, submitted for publication.

⁴ I. Ganly and E. K. Parkinson, unpublished data.

Received 1/18/99. Accepted 4/19/99.

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