ARTICLE

p16/Cyclin-Dependent Kinase Inhibitor 2A Deficiency in Human Melanocyte Senescence, Apoptosis, and Immortalization: Possible Implications for Melanoma Progression

Elena V. Sviderskaya, Vanessa C. Gray-Schopfer, Simon P. Hill, Nico P. Smit, Tracy J. Evans-Whipp, Jane Bond, Lucy Hill, Veronique Bataille, Gordon Peters, David Kipling, David Wynford-Thomas, Dorothy C. Bennett

Affiliations of authors: E. V. Sviderskaya, V. C. Gray-Schopfer, S. P. Hill, T. J. Evans-Whipp, L. Hill, D. C. Bennett, Department of Basic Medical Sciences, St. George’s Hospital Medical School, London, UK; N. P. Smit, Leiden University Medical Center, Department of Dermatology, Leiden, The Netherlands; J. Bond, D. Kipling, D. Wynford-Thomas, Department of Pathology, University of Wales College of Medicine, Cardiff, UK; V. Bataille, Twin Research and Genetic Epidemiology Unit, St. Thomas’ Hospital, London; G. Peters, Cancer Research UK London Institute, London.

Correspondence to: Dorothy C. Bennett, Ph.D., Department of Basic Medical Sciences, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK (e-mail: dbennett{at}sghms.ac.uk).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The melanoma susceptibility locus cyclin-dependent kinase inhibitor 2A encodes two unrelated cell growth inhibitors, p16 and alternative reading frame (ARF). In fibroblasts, both proteins are implicated in cellular senescence, a key barrier to tumor development. The p16 coding sequence is more often mutated in melanoma families than is the ARF sequence. To investigate the role of p16 in melanocytes, we assessed aspects of growth, apoptosis, and immortalization in melanocytes cultured from two melanoma patients, both of whom had two inactive p16 alleles but functional ARF. Methods: Growth and senescence were evaluated by cumulative population-doubling curves, and apoptosis by terminal deoxytransferase labeling. Expression of p53 and p21, which are associated with fibroblast senescence, was assessed by immunoblotting. Amphotropic retroviruses were used to transfer exogenous gene sequences into the melanocytes. Results: Both melanocyte cultures showed high rates of apoptosis, which were reduced when the cells were grown in the presence of keratinocyte feeder cells or human stem cell factor plus endothelin 1. With these growth factors, both cultures proliferated for 45–55 net population doublings, markedly longer than the maximum of 10 net population doublings of normal adult human melanocytes in similar media, indicating impaired senescence. One of the cultures developed chromosomal aberrations, with numerous dicentric chromosomes at senescence, consistent with telomere dysfunction. p53 and p21 levels were not elevated in senescent normal melanocytes but were elevated in senescent p16-deficient melanocytes. Interference with p53 function by transfer of human papillomavirus 16-E6 further extended the lifespan of p16-deficient melanocytes. Human telomerase reverse transcriptase was sufficient to immortalize both these cell strains but not normal melanocytes. Conclusion: Normal senescence in human melanocytes requires p16 activity. p53 contributes to a delayed form of senescence that requires telomere shortening, in p16-deficient melanocytes. These findings provide some basis for the role of p16 in melanoma susceptibility.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cyclin-dependent kinase inhibitor 2A (CDKN2A) locus, also known as multiple tumor suppressor 1 (MTS1), inhibitor of (cyclin-dependent) kinase 4, A (INK4A), or INK4A-alternative reading frame (INK4A-ARF), is a major tumor suppressor locus that undergoes somatic mutation, deletion, or silencing in a wide variety of spontaneous human neoplasms, including pancreatic cancer, glioma, and melanoma (14). Germline mutations, by contrast, are associated fairly specifically with melanoma susceptibility, although occasionally with pancreatic cancer (1,2). This specificity is unexplained and suggests that CDKN2A function in melanocytes may be unusual.

The CDKN2A locus encodes two unrelated proteins, both cellular growth inhibitors, in different reading frames (5). One is p16INK4A, here called p16, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 and CDK6). These kinases phosphorylate retinoblastoma (RB)-family proteins, preventing their growth-inhibitory effects (2,6). Thus, p16 promotes the growth-inhibitory functions of RB. The second product is ARF, also called p19Arf (mouse) or p14ARF (human) (5,7). ARF indirectly activates p53 by preventing p53 inhibition by mouse double minute 2 (MDM2) (710). To date, nearly all reported CDKN2A mutations associated with familial melanoma affect p16 function, but few affect that of ARF (1,2,11). This indicates a primacy of p16 for melanoma susceptibility, supported by the increased susceptibility to melanoma induction of mice deficient for Ink4a and expressing wild-type Arf but not mice deficient for Arf alone (12,13).

Both p16 and ARF have known functions in cell senescence (2,4,14), the loss of normal somatic cells’ ability to proliferate after a limited number of divisions (15,16). In human fibroblasts, either the p16/RB pathway or the p53 pathway alone appears sufficient to permit senescence (15), including accelerated senescence following RAS expression (17). Restoration of a normal p16 sequence (with its own promoter) to immortal mouse, hamster (18), or human (19) fibroblasts can induce senescence. The evidence for a role of ARF (as distinct from p53) in senescence is equivocal for human cells (20), although clear for mouse cells (8,21).

Molecular analyses of senescence in human melanocytes have been reported (2225), but the mechanism effecting senescence in these cells remains unclear. Increased expression of p16 with increasing passage level has been observed consistently, but variable or falling expression of p53-activated genes p21 and p27 has been noted (2225). Most studies (2225) have been of cells that underwent accelerated arrest in the presence of high levels of cholera toxin, an agent that promotes both melanocyte growth and differentiation by elevation of intracellular cyclic adenosine monophosphate (cAMP) levels. This agent has been used at high levels to accelerate melanocyte senescence (22,24), but it is also added at low levels to many melanocyte culture media, including ours, because it promotes growth. Maximum reported life spans of adult melanocytes vary widely and depend on culture conditions; they range between 2.4 and 10 population doublings in various growth media with cholera toxin or reach approximately 40 population doublings without cholera toxin (young adult donors) (22,2529), comparable to typical figures for adult human fibroblasts.

Analyses of melanoma-prone kindreds have identified two individuals who carry mutations in both copies of the CDKN2A locus (3032). Evaluation of the resulting gene products suggests that p16 activity is defective in both patients but that ARF functions are retained (3032). Both of these patients have shown normal development and physiology, apart from a tendency to develop multiple primary melanomas by age 30 and numerous melanocytic nevi (more than 200 each), some dysplastic. The patients have been described (30,32,33). In our study, we characterized epidermal melanocyte strains from both patients, with the aim of investigating functions of p16 in melanocytes, their senescence, and genetic requirements for their immortalization.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Terminology

In human fibroblasts, normal cell senescence is also termed mortality stage 1 or M1. Cells can bypass M1 arrest and divide further upon addition of viral oncogenes such as simian virus 40 large T antigen or human papilloma virus 16 (HPV16)-E6 and -E7, whose protein products abrogate both the p53 and RB pathways (15,16). Net growth later stops again, in a phase called crisis or M2. Cells at M2 appear to proliferate but die frequently. M2 is associated with extreme telomere shortening, which promotes telomere uncapping, and thence chromosomal fusions and breakages (15,16,34). M1 senescence also seems to depend on limited telomere shortening, because forced expression of human telomerase reverse transcriptase (hTERT) appears to immortalize several human cell types (35). Cells at M2 can spontaneously immortalize by expression of endogenous hTERT or can be immortalized by exogenous hTERT.

Not all human cell types exhibit comparable M1 and M2 phases of arrest (15). For example, in mammary epithelial cells and keratinocytes, arrest occurred after fewer population doublings than in fibroblasts and required only the p16/RB1 pathway; both hTERT addition and p16/RB deficiency were needed for immortalization (36). The term "M0" was suggested for this type of arrest. However, the same cell types grew for further doublings in the presence of feeder cells and then senesced in an M1-like pattern, suggesting that M0 arrest was an artifact of inadequate growth conditions or stress (37).

"Cell senescence" refers herein to the cessation of growth seen in normal cells, generally M1 or M0 senescence, but not to M2 nor to forms of growth arrest in abnormal cells (for example, p16-deficient cells or cells after oncogene addition) for which the mechanism of arrest is unknown.

Cell Culture and Growth Curves

Biopsy specimens were taken from both patients after obtaining written informed consent and with approval from the Medical Ethics Commission, Leiden University Medical Center, and the East London and City Health Authority Research Ethics Committee. The Hmel-p16-1 melanocytes (human melanocytes lacking p16) were obtained from a patient homozygous (30) for a deletion in exon 2, resulting in the production of two frameshift proteins, neither of which shows any p16-related function (31). However, one of the products retains the amino-terminal domain and all of the known activities of ARF (31). The cells were explanted in Leiden from a 4-mm punch biopsy specimen. After an overnight incubation in trypsin (2.5 mg/mL), EDTA (0.5 mg/mL), and glucose (1 mg/mL) in phosphate-buffered saline (PBS) (pH 7.5) at 4 °C, the epidermis was separated from the dermis. The epidermal cell suspension was plated into a 6-cm dish in medium-1 (Ham’s F10 medium with 1% Ultroser-G [Life Technologies, Breda, The Netherlands], 16 nM 12-O-tetradecanoylphorbol-13-acetate, 0.1 mM isobutyl methylxanthine, and 2.5 nM cholera toxin [all obtained from Sigma Chemical Co., Zwijndrecht, The Netherlands]). The primary cell culture was treated twice with the antibiotic G418 (100 µM for 3 days) to remove fibroblasts and passaged four times in medium-1, as described (38). The cells were then transferred to St. George’s Hospital Medical School (London, UK) and grown as described (39) in medium-2 (RPMI-1640 medium with 10% fetal calf serum [FCS], 200 nM 12-O-tetradecanoylphorbol-13-acetate, and 200 pM cholera toxin), with 10% CO2 (which gives pH 6.9–7.0). Except where specified, all human melanocytes were grown in medium-3 (medium-2 supplemented with human stem cell factor [SCF; R&D Systems, Abingdon, UK] at 10 ng/mL and endothelin 1 [Edn1; Sigma Chemical Co., Poole, UK] at 10 nM).

Hmel-p16-2 melanocytes were explanted from an unrelated patient, with a different point mutation in each copy of INK4A. Both mutations impair p16 function, although to different degrees, but only one of them alters the amino acid sequence of ARF, resulting in no discernible effect on the known functions of the protein (32). At the Royal London School of Medicine, the culture was explanted from a punch biopsy specimen using a similar method, but initially the primary culture was grown in a keratinocyte growth medium (40). After 5 days, the cells were subcultured. After an additional 7 days, the cells were transferred to St. George’s Hospital Medical School, where they were grown in medium-3. Some early passages of Hmel-p16-1 and Hmel-p16-2 cells were grown with mitomycin C-treated XB2 immortal mouse keratinocyte feeder cells (XB2 feeder cells), as described (41).

The WM melanoma cell lines were obtained from M. Herlyn (Wistar Institute, Philadelphia, PA), the SKMEL23 and A375P melanoma cell lines from I. R. Hart (St. Thomas’ Hospital, London, UK), the COLO 858 melanoma cell line from G. Moore (Denver General Hospital, Denver, CO), and the MCF7 breast carcinoma cell line from M. Clemens (St. George’s Hospital Medical School). Melanoma cell lines and MCF7 breast cancer cells were grown in RPMI-1640 medium supplemented with 10% FCS. Nohm-1 normal human neonatal melanocytes (39) were grown in medium-3. FBR2 human fibroblasts were provided by R. F. Newbold (Brunel University, Uxbridge, UK), and Hfib cells were derived by Dorothy C. Bennett from neonatal human foreskin. All human fibroblasts were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FCS. All cultures were grown under 10% v/v CO2, except that 5% CO2 was used with medium-1.

To generate growth curves, cells were detached with trypsin, counted in triplicate using a hemocytometer, and replated at a recorded density. The relative population increase was calculated and converted to number of population doublings.

Assay for Apoptosis

Cells (104 in 0.5 mL) were seeded on sterile glass coverslips in 16-mm wells and grown as specified. The cells were then fixed by a gentle infiltration of a 37% w/v formaldehyde solution into the medium (to preserve floating cells) to a final 3.7% w/v concentration. After 10 minutes, the cells were washed twice carefully in Dulbecco’s PBS (containing CaCl2 and MgCl2) and labeled with terminal deoxytransferase from an ApopTag peroxidase in situ kit (Intergen, Oxford, UK), and the reaction products were detected using the same kit, according to the manufacturer’s recommended protocol.

Immunoblotting

Nearly confluent 10-mL cell cultures were washed in PBS and lysed in 0.5 mL of cold RIPA buffer (PBS with 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) containing the protease and phosphatase inhibitors phenylmethylsulfonyl fluoride (2 mM), aprotinin (2 µg/mL), EDTA (2 mM), and sodium orthovanadate (1 mM). Protein concentrations in the cell lysates were determined by the bicinchoninic acid method. Cell lysates (30 µg) were subjected to electrophoresis through 12% polyacrylamide gels containing SDS and transferred to Immobilon-P membranes (Millipore, Bedford, UK). The membranes were blocked in 5% dried skim milk for at least 1 hour at 37 °C, with shaking, and probed overnight at 4 °C with mouse anti-human monoclonal antibodies against p53 (0.1 µg/mL; Oncogene Research Products, Nottingham, UK) or p21 (1 : 500; Pharmingen, Heidelberg, Germany) in PBS with 5% dried skim milk and 0.1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G antibodies (1 : 2000; Amersham, Little Chalfont, UK), and the labeled proteins were visualized with an enhanced chemiluminescence (ECL) kit (Amersham), according to the manufacturer’s instructions. To confirm that equivalent protein concentrations were loaded, membranes were stripped with 2% w/v SDS and 100 mM 2-mercaptoethanol in 62.5 mM Tris–HCl (pH 6.7) for 30 minutes at 50 °C and reprobed with a mouse anti-actin antibody (1 : 1000; Chemicon International, Harrow, UK). Films were scanned with a Molecular Dynamics (Sunnyvale, CA) laser densitometer, using ImageQuant software for Windows (Amersham). Reported experiments were done three times with similar results.

Retroviral Gene Transfer Into Melanocytes

A full-length complementary DNA sequence for hTERT (obtained from Geron, Menlo Park, CA) was cloned into the pBABEpuro vector (originally from Dr. H. Land, then at the Imperial Cancer Research Fund Laboratories, London, UK), which contains a selectable marker for resistance to puromycin, and transfected by the calcium phosphate method into the Omega E ecotropic packaging line (42). Supernatant containing virus was used to infect {Psi}CRIP amphotropic packaging cells (43), which were grown in DMEM supplemented with 10% calf serum. PA317 amphotropic packaging cells containing HPV16-E6 and -E7 in pBABEneo vectors (44), which carry a selectable marker for G418 resistance, were obtained from Dr. Denise Galloway (Fred Hutchinson Cancer Research Center, Seattle, WA) and grown in DMEM supplemented with 10% FCS. Producer cells were selected in puromycin (2.5 µg/mL) or G418 (400 µg/mL) as appropriate, except when harvesting virus. To harvest virus, producer cells were incubated in fresh medium for 18 hours, and then the medium was removed, snap-frozen, and stored at –70 °C until use.

Melanocytes were infected 1 day after plating at 4 x 104/mL to 5 x 104/mL, using appropriate containment procedures. A typical infection protocol was as follows: After thawing the supernatant containing the viral particles, the medium was passed through a 0.45-µm filter and supplemented with polybrene (8 µg/mL; 5 or 3.5 µg/mL in later experiments). This medium was then incubated with melanocytes that had been pretreated with polybrene at the same concentration for 1 hour. After 4 hours, the virus-containing medium was removed and replaced with fresh medium-3. Melanocyte cultures were grown with XB2 feeder cells (initially 3 x 104/mL) where cells became very sparse following infection, and some cultures were selected in 1.5 µg/mL puromycin from day 6 to day 20 and/or in 1 mM G418 from day 4 to day 20 after infection. Drug selection was not generally continued beyond day 20. Uninfected cells were killed by either drug in this time.

Statistical Analysis

For apoptosis assays, results are presented as mean and 95% confidence interval of at least four measurements from two or three independent experiments. Differences among groups were compared by using a two-tailed Student’s t test. P values less than .05 were considered statistically significant.


    RESULTS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cellular Phenotypes, Growth Kinetics, and Apoptosis of p16-Deficient Melanocytes

Unexpectedly, for cells deficient in a cell cycle inhibitor, Hmel-p16-1 melanocytes grew very slowly in a standard melanocyte medium, medium-1, undergoing only an estimated four net population doublings in approximately 4 months. They were transferred to another standard melanocyte medium, medium-2, but then grew even less, with no net increase in cell number during approximately 70 days and three subcultures (Fig. 1Go, beginning of curve A). Although the cells had been in culture for approximately 7 months by that point, dividing cells could be seen by microscopy, and the cells did not look senescent (Fig. 2Go, A and B). By contrast, normal adult melanocytes would generally senesce within approximately 3 months under similar culture conditions (28). To account for the low net growth rate, we surmised that the cultures had a high rate of cell death, as suggested by the presence of visible debris and dead cells (Fig. 2Go, A).



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Fig. 1. Growth rates and life spans of p16-deficient melanocytes under different culture conditions. To generate growth curves, cells were detached with trypsin, counted in triplicate using a hemocytometer, and replated at a recorded density. The relative population increase was calculated and converted to number of population doublings. Doublings are shown relative to the first cell count. Each point represents one subculture. AC) Hmel-p16-1 cells grown in medium-1 (Ham’s F10 medium with 1% Ultroser-G, 16 nM 12-O-tetradecanoylphorbol-13-acetate, 0.1 mM isobutyl methylxanthine, and 2.5 nM cholera toxin in 5% CO2) for 4, 4, or 9 passages respectively, and then in medium-2 (RPMI-1640 medium with 10% fetal calf serum, 200 nM 12-O-tetradecanoylphorbol-13-acetate, and 200 pM cholera toxin in 10% CO2), with the additional supplements indicated. XB2/–XB2 = XB2 keratinocyte feeder cells added/removed. SCF, Edn1 = stem cell factor and/or endothelin 1 added from this time onward. D) Hmel-p16-2 cells grown in medium-3 (medium-2 with SCF and Edn1) from passage 1.

 


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Fig. 2. Characteristics of Hmel-p16-1 cells at low and high passage levels. Phase-contrast (a and c) and matched bright-field (b and d) images to show melanin pigment. Scale bars = 100 µm. a and b) At passage 11 in medium-2 (RPMI-1640 medium with 10% fetal calf serum, 200 nM 12-O-tetradecanoylphorbol-13-acetate, and 200 pM cholera toxin in 10% CO2). Cells have nonsenescent appearance (bipolar, no or very little pigment), despite lack of net growth in this medium. A dividing cell is identified by arrows, and floating debris from dead cells is identified by arrowheads. c and d) Cells at high passage level grown in medium-3 (medium-2 with stem cell factor and endothelin 1) at growth arrest, showing highly aberrant appearance (arrow) and pigmentation of most cells (d). e) Chromosome spread for Hmel-p16-1 at high passage level (passage 20, {approx}40 population doublings) showing several dicentric chromosomes (arrows) and other rearrangements (stars).

 
Hmel-p16-1 cell growth improved dramatically after XB2 feeder cells were added to the cultures. The cultures grew exponentially, with a doubling time of approximately 2.5 days (Fig. 1Go, curve A), which is unusually short for human melanocytes (2530). Growth was similarly rapid in medium-3, which is medium-2 supplemented with two known keratinocyte products, Edn1 and SCF (Fig. 1Go, A) (45,46). Moreover, when new Hmel-p16-1 cultures from stocks frozen at passage 4 were grown solely in medium-3, they grew well immediately (Fig. 1Go, curve B) but eventually slowed and stopped growing after 40 population doublings from the first cell count at 120 days, approximately 44 estimated net population doublings in total. Similar results were obtained with an independent culture derived from a second biopsy from the same patient (data not shown). By comparison, 10 population doublings is the longest life span reported, to our knowledge, for human adult melanocytes in a similar medium containing cholera toxin (28). Hmel-p16-1 cells grown in medium-1 for approximately 470 days grew for an additional 10 population doublings when cultured in medium-3, with net growth stopping at more than 600 days (Fig. 1Go, curve C), showing that life span in days was dependent on the culture conditions. The net proliferation rate of Hmel-p16-1 cells was much slower in medium-1 than in medium-3, with a population-doubling time of approximately 1 month versus 2.5 days, respectively.

We also measured life span in a second p16-deficient melanocyte strain, Hmel-p16-2, grown in medium-3. Although growth of Hmel-p16-2 cells was slower than that of Hmel-p16-1 cells, cells from the second strain also had an exceptionally long life span, growing for 50 population doublings from the first count (at day 20), before ceasing growth at approximately 500 days (Fig. 1Go, curve D).

We next tested whether the poor growth of Hmel-p16-1 cells in medium-2 was associated with a high rate of apoptosis. As expected, there was little apoptosis in control normal human melanocytes cultured in medium-2 (Fig. 3Go, A), because this is a standard melanocyte medium. The rate of apoptosis was substantially higher in Hmel-p16-1 cells cultured in medium-2, but not in medium-3, or in medium-2 supplemented with SCF or Edn1 alone (Fig. 3Go, B). Similar results were obtained with Hmel-p16-2 cells (Fig. 3Go, C). Thus, high apoptotic rates were associated with p16-deficient genotypes in melanocytes. This finding may not extend to all cell types, because no excessive cell death or impaired growth was noted in dermal fibroblasts grown from the same two patients in a standard fibroblast medium (DMEM and 10% FCS) (31,32).



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Fig. 3. High rates of apoptosis in p16-deficient melanocytes cultured without extra growth factors. Melanocytes of various strains, as in the panel headings, were grown for 14 days in RPMI-1640 medium with fetal calf serum, 12-O-tetradecanoylphorbol-13-acetate, and cholera toxin, with the additional supplements indicated. Apoptosis was assessed by labeling with terminal deoxytransferase from an ApopTag digoxygenin-peroxidase in situ kit. Open bars = stem cell factor (SCF) + endothelin 1 (Edn1). Square hatching = SCF. Diagonal hatching = Edn1. Closed bars = no additional supplement. Growth in either 5% or 10% fetal calf serum gave no difference in rates of apoptosis (data not shown), and so these data were pooled. Each bar shows the mean and 95% confidence interval of at least four measurements derived from two or three independent experiments. Hmel-p16-E6 indicates Hmel-p16-1 cells after infection with HPV16-E6 (before variant cells emerged). Differences of means were tested for statistical significance using a two-tailed Student’s t test. Apoptotic rates were statistically significantly lower in cells supplemented with SCF + Edn1 than in those not supplemented, in Hmel-p16-1 (P = .004) and Hmel-p16-2 cells (P<.001), but not in normal Nohm-1 or Hmel-p16-1E6 cells. Apoptotic rates in cells cultured without SCF or Edn1 were statistically significantly higher in Hmel-p16-1, Hmel-p16-2, and Hmel-p16-1E6 than in Nohm-1 cells (P = .015, P<.001, P = .015, respectively). Apoptotic rates in cells cultured in the presence of SCF and Edn1 were not statistically significantly higher in any of the p16-deficient cultures than in Nohm-1 cells.

 
Chromosomal Analysis and Passage Level

We karyotyped Hmel-p16-1 and Hmel-p16-2 cells at various passage levels (Table 1Go). This was in part to test for evidence of arrest through M2 crisis or severe telomere deficiency, where cells show chromosomal aberrations, including dicentric chromosomes which are normally rare (16,34). In Hmel-p16-1 cells, abnormalities were found at all passage levels tested, even around 11 net population doublings. The Hmel-p16-1 culture grown for several months in medium-2 (Fig. 1Go, culture A, and Table 1Go) showed nonclonal abnormalities, including monosomy for chromosome 17p in nearly all cells examined, through various abnormalities of 17p. Other Hmel-p16-1 cultures (Fig. 1Go, cultures B and C, and Table 1Go) showed various abnormalities in a proportion of cells, with no obvious pattern or consistent 17p abnormalities but indicating general chromosomal instability. At high passage levels (approximately 44 population doublings), all abnormal karyotypes from culture B of Hmel-p16-1 cells included one or more dicentric chromosomes (Fig. 2Go, E), supporting the hypothesis of telomere deficiency or dysfunction. Conversely, all tested Hmel-p16-2 cells had normal diploid karyotypes up to 50 population doublings when growth had almost ceased. A normal neonatal human melanocyte strain grown in medium-2 had a normal diploid karyotype at passage 28 (39), as did two such strains grown in medium-3 at passages 21 or higher (data not shown).


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Table 1. Karyotypes of p16-deficient human melanocyte strains and immortal lines
 
Thus, diploid Hmel-p16-2 melanocytes showed relatively normal, albeit delayed, senescence without evidence of telomeric crisis. By contrast, the aneuploid Hmel-p16-1 cells may have reached a crisis or M2-like state rather than normal senescence. It should be noted that Hmel-p16-1 cells would have undergone more mitoses than the diploid strain would have, given their higher death rate for some months and their similar number of net population doublings (because net population growth equals proliferation minus death).

p53 and p21 Expression in Growing and Senescent p16-Deficient Melanocytes

Abnormal apoptosis occurs in various tissues of Rb1-nullizygous mice (47) and is p53-dependent in some, although not all, of these tissues (48). Accordingly, we examined whether the apoptosis in p16-deficient melanocytes cultured in medium-2 was p53-dependent and also whether p53 might have a role in the eventual growth arrest of these cells. We also analyzed expression of the p53-activated CDK inhibitor p21, partly to assess p53 activity and partly because p21 apparently suppressed apoptosis in p53-positive melanoma cells (49).

We first compared p53 protein levels in Hmel-p16-2 cells at different passage levels. p53 expression levels were relatively low in Hmel-p16-2 cells at passage 7 and rose sequentially at passages 13 and 20 (Fig. 4Go, A). At passage 20, growth was ceasing (approximately 47 population doublings; Fig. 1Go, curve D). p21 expression levels were likewise high in Hmel-p16-2 cells at passage 20, when cells were senescent but had not risen by passage 13 (Fig. 4Go, B). These data are consistent with the possibility that apoptosis in medium-2 was associated with p53 expression in the absence of p21 expression. p53 levels did not change with the presence or absence of SCF or Edn1 in the culture medium (data not shown), indicating that their suppression of apoptosis was not connected with p53. The rises in p53 and p21 levels as these cells senesced were consistent with M1-like senescence, requiring p53 and p21 as in fibroblasts (15,50,51).



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Fig. 4. Expression patterns of p53 and p21 in p16-null melanocytes. a) Immunoblot showing p53 expression in Hmel-p16-2 at various passage levels as indicated. The blot was stripped and reprobed with actin as a loading control. The band intensities were quantified by scanning densitometry, and the graph shows p53 levels relative to actin levels. b) Immunoblots and graph showing p21 expression in Hmel-p16-2 cells at different passage levels. c) Immunoblots and histogram of p53 expression in various growing and senescent cells. MCF7 breast cancer cells were used as a positive control. FBR2 and Hfib were human dermal fibroblasts, and Nohm1 cells were normal human neonatal melanocytes. Passage levels of growing cells were as follows: MCF7, unknown (high); FBR2, 24; Hmel-p16-1, 15; Hmel-p16-2, 13; and for senescent cells: Hfib, 29; Nohm-1, 22; Hmel-p16-1, 20; Hmel-p16-2, 20; Hmel-p16-1E6; 20 (this denotes Hmel-p16-1 cells expressing E6, assayed before variant cells appeared). d) Immunoblots and graph of p21 expression in the same set of cultures as in c. e and f) Graphs of p53 and p21 expression (immunoblots not shown) in melanocytes compared with cell lines from an early primary melanoma (WM1650) and metastatic melanomas (WM793, SKMEL23, A375P, and COLO858). Melanoma lines SKMEL23 and A375P are known to have a normal p53 sequence (52). All immunoblot results are representative data from one of three experiments.

 
We next compared p53 and p21 levels in various growing and senescent cell types, including these melanocytes (Fig. 4Go, C and D). p53 and p21 levels were lower in growing Hmel-p16-2 cells and higher in growing Hmel-p16-1 cells than in growing human fibroblasts. This finding suggested that the apoptosis in both strains was not totally p53-dependent (Hmel-p16-2 cells had some excessive apoptosis but a relatively low p53 level) but may have been partly so, because Hmel-p16-1 cells had higher levels of both apoptosis and p53. Compared with levels in growing Hmel-p16-1 cells, p53 and p21 levels remained high in senescent cells, with a possible increase in p21 level. This did not show a clear role for p21 in the arrest of Hmel-p16-1 cell growth. Further evidence of the role of p53 in the observed apoptosis was provided by Hmel-p16-1 cells infected with an exogenous HPV16-E6 sequence (see next section for more details). These cells expressed little p53 or p21 (Fig. 4Go, C and D), as expected, because E6 destabilizes p53. Infection with the HPV16-E6 sequence did not fully suppress the excessive apoptosis seen in medium-2 (Fig. 3Go, D, filled bar), further indicating that the apoptosis was partially p53-independent.

Senescent normal human melanocytes (strain Nohm-1) (39) expressed low levels of p53 and p21 (Fig. 4Go, C and D), consistent with previous reports that p53-activated genes are not elevated in senescent human melanocytes (2225). Elevated p53 and p21 levels at or before growth arrest were seen in melanocytes only when they were deficient in p16 expression.

p53 and p21 levels in some of the lines were then compared with levels in several p53-expressing immortal human melanoma cell lines including line WM1650 from an early (radial growth phase) primary melanoma (Fig. 4Go, E and F). Most melanoma lines, including SKMEL23 and A375P, have a wild-type p53 sequence (52,53). p53 and p21 levels were higher in all the metastatic melanoma lines than in any growing melanocyte cultures and were intermediate in WM1650 cells. Thus, like growing Hmel-p16-1 melanocytes in medium-3, human melanoma cells can proliferate rather than senesce while expressing both p53 and p21.

Immortalization by Exogenous hTERT but not HPV16-E6 or HPV16-E7

Immortalization of human melanocyte cultures using only HPV16-E6 and -E7 genes has been reported (54) but may have involved additional, spontaneous genetic changes (such as hTERT activation), because it was not seen with simian virus 40 large T antigen alone (55). We investigated whether p16-deficient melanocytes could be immortalized by the forced expression of hTERT, the catalytic subunit of human telomerase. Amphotropic retroviral vectors were used to express hTERT or HPV16 oncogenes E6 and E7, which inactivate p53 and the RB family, respectively (15). Expression of E6 or E7 proteins had minimal effects on the lifespan of Hmel-p16-1 (Fig. 5Go, A), whereas expression of hTERT (or hTERT plus E6 in Hmel-p16-1 cells) immortalized both cultures (Fig. 5Go, A and B), as judged by growth for at least an additional 50 population doublings. Cell-doubling times were approximately 2 days.



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Fig. 5. Growth kinetics of p16-null melanocytes with and without expression of exogenous genes. a) Hmel-p16-1 cells, showing growth curve for untreated cells for reference (closed triangle), and for cultures infected at the time indicated with a control pBABEpuro vector only (inverted open triangle) or a vector containing human papillomavirus 16 (HPV16)-E7 (multiplication sign), HPV16-E6 (diamond), human telomerase reverse transcriptase (hTERT) (culture Hermes 1) (circle), or hTERT plus HPV16-E6 (open triangle). Number of population doublings in infected cultures is relative to the first count after infection, to exclude effect of initial cell death during the infection procedure, which was variable. Cells in the vector control culture declined in number after three counts and were lost. b) Hmel-p16-2 cells. Uninfected cultures showing senescence (diamond) and three cultures infected at the indicated time with hTERT, designated Hermes 2A (square), Hermes 2B (inverted open triangle), and Hermes 2C (circle). c) Further growth of cultures from panel a: Hermes 1 culture, showing stable and constant growth rate (circle), and Hmel-p16-1 with HPV16-E6 (diamond), showing initial poor growth followed by acceleration to a rate similar to that seen with hTERT. Doublings are relative to the start of the curves. Representative data from a single experiment are shown.

 
An interesting effect of E7 on Hmel-p16-1 cells was a loss of visible pigmentation, although the growth rate was not increased. Pigmentation normally increased at the time of growth retardation, when cells often became large and ramified (Fig. 2Go, C and D). However, pigmentation was lost rapidly in the entire culture infected with E7, even at the time of growth retardation (Fig. 6Go, A and B). By contrast, cells immortalized with hTERT were lightly pigmented and similar in pigmentation level (data not shown) and cell shape (Fig. 6Go, C) to the growing parental line at a lower passage level. Cells expressing E6 alone were well pigmented as growth slowed. The slight extensions of life span seen in cells expressing E6 (approximately 15 population doublings) or E7 (approximately 10 population doublings) indicated that both p53 and residual RB activity contributed to the growth arrest of uninfected Hmel-p16-1 cells.



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Fig. 6. Appearance of high-passage-level p16-deficient melanocytes infected with retroviruses expressing human papillomavirus 16 (HPV16)-E6 or HPV16-E7 oncogenes or human telomerase reverse transcriptase. a and b) Hmel-p16 cells infected with HPV16-E7, at stage of growth retardation. Phase contrast (a) and matched bright-field (b) image showing lack of pigmentation, even in larger stellate cells. c) Hmel-p16-2 cells infected with human telomerase reverse transcriptase. Morphology is similar to that of the parental line. The cells were slightly pigmented and formed brown pellets when harvested (data not shown). The subline shown proved to be diploid (Hermes 2C). d) Variant cells in culture of Hmel-p16-1 infected with HPV16-E6, phase contrast. Growth had been slowing and many large, abnormal, pigmented cells were present (arrow), but colonies of small, rapidly growing, unpigmented cells (*) appeared and took over the culture. Scale bars = 100 µm. a, b, and c are at the same magnification.

 
After the growth rate of one culture infected with E6 had slowed, colonies of small, unpigmented, rapidly-growing cells appeared among the others (Fig. 6Go, D). These cells were found to be immortal (Fig. 5Go, C). These variant cells had a population-doubling time of approximately 2 days, compared with 12 days for the culture before they appeared.

Hmel-p16-1 cells immortalized with hTERT showed small chromosomal aberrations, whereas two of three clones of Hmel-p16-2 cells immortalized with hTERT initially had normal diploid karyotypes (Table 1Go). These immortal lines were designated Hermes 1, 2A, 2B, and 2C (Table 1Go) for designating human extended-replication melanocytes. Additional chromosomal abnormalities were observed in the spontaneously immortalized cells that arose from the E6-infected culture (Table 1Go), supporting the biologic observations that these were not typical of the original E6-infected culture.

We made at least five attempts to immortalize three separate normal human melanocyte strains (i.e., without p16 lesions) with hTERT. Our attempts were unsuccessful, despite the use of enriched growth medium, feeder cells, and drug selection indicating viral infection (data not shown). Expression of hTERT alone thus appears insufficient for immortalization of normal human melanocytes.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our findings provide three main lines of evidence that normal human melanocyte senescence is p16-dependent and does not involve p53 and p21, unlike that of human fibroblasts (15,17) or of epithelial cells grown with feeder cells (37). First, two strains of human p16-deficient melanocytes showed a markedly extended culture life span of 45–55 population doublings, compared with a reported maximum of approximately 10 population doublings for normal adult melanocytes in any culture medium containing cholera toxin (22,2629). Second, p53 and p21 levels were not increased in normal senescent melanocytes under any culture condition tested [(24) and herein], whereas levels of both were elevated in both p16-deficient strains at growth arrest. Third, p16-deficient melanocytes could be immortalized by hTERT, whereas three strains of normal melanocytes could not (even when grown with feeder cells).

This behavior is comparable to the M0 senescence reported for some human epithelial cells (36) and later attributed to stressful culture conditions (37). However, the medium used in the present study was a growth-factor-rich medium in which melanocytes grew as well as with feeder cells or better (Fig. 1Go). It is possible that all known culture conditions for melanocytes to date are "stressful." However, the alternative idea, that p16-mediated senescence is normal in melanocytes irrespective of their environment, is attractive as a potential explanation of why INK4A and CDK4 are susceptibility genes specific for melanoma. The present study and that of Brookes et al. (31), based on genetic deficiency in p16, provide direct evidence for p16/RB-mediated senescence of human cells. By contrast, previous evidence came from delay of senescence by the HPV16-E7 oncogene, which has targets besides RB-family proteins (15,17,36).

It is possible that p16-mediated senescence in melanocytes is connected with the presence of the cAMP agonist cholera toxin, because cAMP agonists are reported to reduce melanocyte life span (2225). cAMP agonists might evoke some form of stress, although (at concentrations used for routine culture) they increase melanocyte proliferation rate (39). Alternatively, it has been proposed (22,23,29) that this shortening of lifespan is associated with cell differentiation, also promoted in melanocytes by cAMP (2225,39).

Diploid p16-deficient melanocytes (Hmel-p16-2) eventually did show M1-like arrest, with elevated p21 levels, no chromosomal aberrations, and no evidence of excessive cell death. These observations suggest that the p53/p21 pathway is able to provide a secondary form of melanocyte senescence when p16 is dysfunctional, just as the RB pathway or the p53 pathway alone each permits senescence in fibroblasts when the other pathway is disrupted (15,17,56), this senescence being delayed relative to normal M1 senescence (56). This apparent secondary arrest also recalls the secondary p53-mediated senescence seen in epithelial cells when M0 senescence was disrupted (36). Because Hmel-p16-1 cultures contained aneuploid cells from an early stage, they might have developed unknown genetic aberrations affecting their growth properties. Indeed, the frequent dicentric chromosomes seen when this cell strain ceased growth suggest that it spontaneously underwent severe telomere attrition and reached M2 (crisis). It seems likely that this strain developed a genetic aberration, perhaps in the p53 pathway, which enabled the bypass of M1 as well as M0. These interpretations for the two strains are consistent with the finding that hTERT was sufficient for immortalization of both p16-deficient cultures, although not normal melanocytes. Bandyopadhyay et al. (25) reported the isolation of melanocyte clones with greatly extended life spans, possibly immortal, following expression of hTERT in normal melanocytes; however, the authors noted other alterations in gene expression, including reduced p16 expression, in these potentially immortal cultures. It was not known whether the cells were diploid, and the clones were selected after rather than before infection, so possibly only cells with low p16 levels formed sizeable clones. These findings thus do not conflict with the conclusion that both deficiency of the p16/RB pathway and telomere maintenance are required for immortalization of human melanocytes.

Our finding of the apparent ability of melanoma cells and some melanocytes to grow well while expressing the p53-activated growth inhibitor p21 may partially explain the curiously low incidence of p53 mutations in melanoma—only approximately 10%–20%. Melanomas often express normal p53 at high levels (52,53).

Infection with HPV16-E6 and HPV16-E7 oncogenes without hTERT did not lead to immortalization of p16-deficient melanocytes, although a small extension of life span was observed with either E6 (supporting a role for p53 in the senescence of p16-deficient melanocytes) or E7 (indicating some residual activity of the RB family without p16). It is interesting that immortal cells did arise, apparently clonally, after p53 blockade by E6; however, these had acquired further chromosomal changes. This occurrence reinforces the importance of monitoring culture growth and karyology and of distinguishing between direct effects of a genetic manipulation and secondary effects resulting from additional spontaneous genetic change. It was also interesting that pigmentation was lost in p16-deficient melanocytes expressing E7, presumably as a result of extreme reduction of RB family protein activity. This finding is consistent with the association between pigmentation and p16 expression in mouse melanocytes (57) and with the possibility that activity of RB family protein(s) is required for melanocyte differentiation.

A novel finding, not seen in fibroblasts, was the reduced viability of p16-deficient compared with normal melanocytes in a standard melanocyte medium. This observation was reminiscent of the excessive apoptosis seen in certain tissues of Rb1-null mice with intact p53 and Arf genes (47), and in Ink4a-Arf-null mouse melanocytes expressing exogenous Arf (57). The apoptosis in our cultures appeared to be partially p53-dependent. It is not clear why it was more marked in Hmel-p16-1 cells. One possible reason is that the donor for this strain also carries a glucose-6-phosphate dehydrogenase deficiency (33), which may decrease viability of some cell types [although no excessive cell death was observed in fibroblasts from the same patient (31)]. It is likewise not clear why spontaneous chromosomal abnormalities arose in Hmel-p16-1 but not in Hmel-p16-2 cells. The most abnormal cultures were those that were grown extensively in the serum-free medium-1 (Fig. 1Go, culture C) or in medium-2 (Fig. 1Go, culture A). Thus, high selection pressure through suboptimal growth conditions may have played a part. Alternatively, there may have been some genetic predisposition to chromosomal instability in this patient, at least in melanocytes. The patient’s fibroblasts remained diploid in culture (31).

The apoptosis of the p16-deficient melanocytes was suppressed by keratinocytes or their products, suggesting that it may not occur in intact skin. In other words, the excessive apoptosis of human melanocytes deficient in the p16/RB pathway may be cryptic in vivo, becoming overt only in the absence of the epidermis. In this case, p16/RB deficiency would confer no disadvantage on melanocytes while in the epidermis. It would instead confer an advantage for melanocytes that had activated the RB pathway, which (from the fibroblast model) would be expected following an oncogenic alteration such as activation of the BRAF gene (58), leading to clonal proliferation and premature senescence (17). Such a senescent clone might be a normal mole or nevus (29). To produce a progressively growing lesion—that is, a melanoma—further alterations overcoming senescence would be necessary, namely disruption of the p16/RB pathway and activation of telomerase.

Early melanomas might be expected to carry a p16/RB deficiency and therefore be dependent on keratinocytes or their products for survival. This predicted behavior is reminiscent of the early, radial growth phase of melanoma, which occurs only in or near the epidermis (59). If this growth pattern is indeed due to keratinocyte dependence, then loss of the dependence would be needed for further progression and invasion into the dermis—the nodular or vertical growth phase of melanoma, which is competent for metastasis (58). Any genetic lesion that inhibits apoptosis could achieve this loss of dependence. It is thus interesting that many of the consistent genetic alterations reported in invasive and metastatic melanoma do suppress apoptosis. Such alterations include RAS activation, p53 mutation (occasional), {beta}-catenin activation, activation of diverse tyrosine kinases, and phosphatase and tensin homolog (PTEN) loss, among others (60,61).

These results thus provide a number of testable hypotheses about the development and progression of melanoma. One important avenue for future research will be to analyze the unusual features of p53 and p21 function in melanoma cells and Hmel-p16-1 melanocytes.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Supported by Wellcome Trust grant 046038/Z/95/Z (E. V. Sviderskaya and T. J. Evans-Whipp), European Commission contract QLK4-1999-01084 (V. C. Gray-Schopfer), Dutch Cancer Society grant 98-1821 (N. P. Smit), and the Imperial Cancer Research Fund (V. Bataille and G. Peters).

We are indebted to Patricia Purkis for making the primary culture of Hmel-p16-2 cells and to Irene Leigh for her support.


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 Materials and Methods
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 Discussion
 References
 

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Manuscript received June 25, 2002; revised March 3, 2003; accepted March 25, 2003.


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