ARTICLE

p16Ink4a in Melanocyte Senescence and Differentiation

Elena V. Sviderskaya, Simon P. Hill, Tracy J. Evans-Whipp, Lynda Chin, Seth J. Orlow, David J. Easty, Sok Ching Cheong, David Beach, Ronald A. DePinho, Dorothy C. Bennett

Affiliations of authors: Department of Anatomy and Developmental Biology, St. George's Hospital Medical School, London, U.K.; Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; New York University Medical Center, New York, NY; Wolfson Institute for Biomedical Research, University College, London.

Correspondence to: Dorothy C. Bennett, Ph.D., St. George's Hospital Medical School, Department of Anatomy and Developmental Biology, Cranmer Terrace, London SW17 ORE, U.K. (e-mail: dbennett{at}sghms.ac.uk).


    ABSTRACT
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The Ink4a-Arf tumor suppressor locus encodes two growth inhibitors, p16 and Arf, both of which are also implicated as effectors in cellular senescence. Because human germline defects in the INK4A-ARF locus are associated with familial melanoma, melanocytes may have unusual INK4A-ARF functions or controls of cell senescence. Because senescence is believed to be an anticancer mechanism, we investigated the role of Ink4a-Arf and its individual components in melanocyte senescence. Methods: Melanocytes were cultured from littermate mice with zero, one, or two functional copies of the Ink4a-Arf locus. Senescence was evaluated by cumulative population doubling curves and by the assessment of acidic {beta}-galactosidase (an indicator of senescence) expression. Pigmentation and cell size were evaluated by spectrophotometry and microscopy. p16 and Arf expression in primary and spontaneously immortalized melanocyte or melanocyte precursor cell lines were evaluated by immunoblotting. Retroviral vectors containing normal p16 and Arf complementary DNAs were used to restore expression of these genes in Ink4a-Arf–/– melanocytes. Results: Wild-type melanocytes (i.e., Ink4a-Arf+/+) senesced within 4–5 weeks of culture. Ink4a-Arf–/– melanocytes did not senesce and readily became immortal. Ink4a-Arf+/– melanocytes showed defective senescence. Senescent Ink4a-Arf+/+ melanocytes were heavily pigmented, but Ink4a-Arf+/– and Ink4a-Arf–/– melanocytes were less pigmented. All of six spontaneously immortalized melanocyte or melanocyte precursor lines from Ink4a-Arf+/+ mice lacked p16 protein expression, although most retained Arf protein expression. After restoration of p16 but not Arf expression, Ink4a-Arf–/– melanocytes stopped growing, became highly melanized, and expressed acidic {beta}-galactosidase. By contrast, restoration of Arf but not p16 expression led to cell death without evidence of senescence. Conclusion: Normal mouse melanocyte senescence and associated pigmentation require both copies of Ink4a-Arf and appear to depend more on p16 than on Arf function. Mutations of the INK4A-ARF locus may favor tumorigenesis from melanocytes by impairing senescence, cell differentiation, and (where ARF is disrupted) cell death.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The INK4A-ARF locus, also known as CDKN2 (cyclin-dependent kinase inhibitor 2) and MTS1 (multiple tumor suppressor 1), is deleted, mutated, or silenced in a wide variety of human malignancies (13). The genes within this locus have emerged as major tumor suppressor genes, ranking in importance with the retinoblastoma gene (RB1) and the p53 gene (TP53). The INK4A-ARF locus encodes two potent inhibitors of cellular growth, p16INK4A (inhibitor of cyclin-dependent kinase 4, a) and ARF (alternative reading frame relative to p16), whose structural sequences overlap in different reading frames (4). p16INK4A, here called p16, inhibits the phosphorylation of RB family proteins by CDK4 and CDK6, and thus promotes RB1-mediated growth suppression (3,5). By contrast, ARF (4), known as p19Arf in the mouse and p14ARF in the human, interacts with MDM2 ("mouse double minute 2") and activates p53, apparently by preventing the inhibition of p53 function and the targeting of p53 for proteolysis by MDM2 (69).

p16 and ARF are also involved in cellular senescence— the acquired inability of normal somatic cells to divide after a finite number of divisions (13,10). Fibroblasts cultured from mice with a homozygous deletion for the Ink4a-Arf locus (Ink4a-Arf–/– or nullizygous) do not senesce, whereas Ink4a-Arf hemizygous (Ink4a-Arf+/–, deletion of one allele) fibroblasts senesce with similar kinetics to wild-type fibroblasts (11). Fibroblasts nullizygous for Arf alone also do not senesce normally (6), showing that Arf is required for the senescence of mouse fibroblasts. Introduction of normal p16 genomic or complementary DNA (cDNA) sequences restores the ability of immortal mouse, hamster (12), and human fibroblasts (13) to senesce, showing that p16 expression can promote senescence in these cells. Similarly, in human fibroblasts, selective ablation of either pathway by viral oncogenes has shown that either the p16/RB1 pathway or the ARF/p53 pathway is sufficient for senescence (14). Human epithelial cells also show a rapid form of senescence (designated "M0"), which is controlled primarily by the p16/RB1 pathway (15). Furthermore, the finding that premature expression of both p16 and Arf proteins is associated with accelerated senescence in fibroblasts lacking Bmi-1, a mammalian homologue of Drosophila polycomb (16), suggests that p16 and Arf are linked with the mechanisms that control senescence. This observation is supported by the finding that the expression of p16 and Arf proteins is suppressed by overexpression of exogenous Bmi-1, which inhibits senescence (16). Bmi-1 acts with other Polycomb group proteins in a protein complex that silences transcription and is involved in the establishment of the embryonic body axis (16). This is another clocklike process, like senescence, since genes are sequentially activated over time (16).

Together, these findings (6,1016) suggest a potentially general mechanism for the control of senescence. However, there is also evidence of differences in the detail of this control among different cell types (15,17). Accordingly, and because human germline defects in the INK4A-ARF locus are associated with familial melanoma (and, less commonly, pancreatic adenocarcinoma), melanocytes may have unusual INK4A-ARF functions or controls of cell senescence. The germline defects in the INK4A-ARF locus include some that affect the p16 but not the ARF protein sequence (2,3,18). For example, 20 of 37 (54%) of the mutations from melanoma families listed by Ruas and Peters (2) affected p16 and not ARF protein. Moreover, quantitative changes in p16 expression (although usually without detectable mutations) are frequent in sporadic melanoma, even at the primary stage (19).

In this study, we used cultured melanocytes from littermate Ink4a-Arf nullizygous, hemizygous, and wild-type mice to investigate the roles of p16 and Arf in the control of senescence and possibly pigmentation in melanocytes. To eliminate coat color variations, which affect melanocyte pigmentation and sometimes growth, we previously back-crossed the Ink4a-Arf deletion to inbred C57BL/6J black mice over several generations. We report three aspects of p16/Arf biology and function in melanocytes that are not observed in mouse embryo fibroblasts.


    MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice

All animal work was performed according to protocols approved by the Dana-Farber Animal Care and Use Committee. Construction of Ink4a-Arf nullizygous mice with an exon 2/3 deletion was described previously (11).

Melanocyte and Other Cell Cultures

Melanocyte cultures were prepared as described (20) from 3-day-old Ink4a-Arf nullizygous (Ink4a-Arf–/–), hemizygous (Ink4a-Arf+/–), and homozygous (Ink4a-Arf+/+) littermate mice that were 97.5% or more C57BL/6J (the % non-C57 is halved per generation). Several litters were compared. Briefly, trunk skin was split with concentrated trypsin (5 mg/mL) and then dissociated with trypsin (250 µg/mL) and EDTA (200 µg/mL) before initial plating on a feeder layer of mitomycin-treated immortal murine XB2 keratinocytes (20). Feeder cells were omitted after the third passage. Cells were grown in RPMI-1640 medium with 10% fetal calf serum (FCS), 200 nM 12-O-tetradecanoyl phorbol 13-acetate (TPA), and 200 pM cholera toxin (melanocyte medium). A separate culture was made from each individual mouse. Immortal melanocyte and melanoblast lines from Ink4a-Arf+/+ mice were grown according to (20) and were derived by us, with the exception of mel-18 and mel-29 (21), which were provided by J.-J. Panthier (Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France). NC-m4 neural crest-like cells were derived by us from Ink4a-Arf+/+ mice and were grown in RPMI-1640 with 10% newborn calf serum and 2 nM TPA on gelatin-coated plates. Swiss 3T3 fibroblasts, originally obtained from E. Rozengurt (Imperial Cancer Research Fund, London, U.K.) were grown in Dulbecco's modification of Eagle's medium (DMEM) with 10% FCS. All cultures were grown in the presence of 10% v/v CO2 and 90% air.

Cell Counts and Melanin Assays

Explant cultures from different donor mice were kept separate. Melanocytes in each culture were counted at each passage, starting 10 days after explantation (when feeder cells were dead or very large and easily excluded). All subcultures were plated at 3 x 104 cells/mL. At each passage, the cells were detached with trypsin and EDTA, counted, and subcultured separately to triplicate dishes. The mean number of cells from triplicate dishes was determined by hemocytometer counts of at least 150 cells per sample. The ratio of the final to the initial cell number was calculated at each subculture to obtain the number of cumulative population doublings. Melanin assays were based on the optical density at 475 nm (OD475) of cell lysates, as described (20). Melanin content was normalized to cell number or to protein content, which was determined using a bicinchoninic acid assay (Pierce, Chester, U.K.) (20).

Immunoblot Analysis

For each cell sample, confluent cultures from 10-mL dishes were lysed in 0.5 mL cold RIPA buffer (10 mM Tris [pH 7.4], 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% [w/v] sodium deoxycholate, and 1% [w/v] sodium dodecyl sulfate) containing the protease and phosphatase inhibitors phenylmethylsulfonyl fluoride (5 mM), aprotinin (2 µg/mL), EDTA (2 mM), and sodium vanadate (10 mM). The suspension was passed through a needle several times to shear DNA and stored at –70 °C. After thawing the samples, protein concentrations in the lysates were determined by the bicinchoninic acid assay.

Unfractionated cell lysates were electrophoresed through 12% polyacrylamide gels containing sodium dodecyl sulfate and transferred to ImmobilonTM-P membranes (Millipore Corp., Bedford, MA) by a semidry transfer at 25 V for 45 min in Towbin buffer (192 mM glycine, 25 mM Tris, 20% methanol). The membranes were blocked in 5% skimmed milk in phosphate-buffered saline (PBS) overnight at 4 °C. After four 5-min washes in PBS with 0.1% Tween 20, the membranes were incubated either with rabbit anti-mouse p16 (M156; Santa Cruz Biotechnology, Santa Cruz, CA) at 1 µg/mL or with rabbit antimouse Arf (p19) (R562; Abcam, Cambridge, U.K.) at 0.5 µg/mL in 5% skimmed milk in PBS for 2 h at room temperature. After four 5-min washes in PBS with 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (1 : 3000) (Amersham Pharmacia Biotech, Little Chalfont, U.K.) in 5% skimmed milk in PBS for 1 h at room temperature. After six 5-min washes in PBS with 0.1% Tween 20, the antibody-labeled proteins were detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) according to the manufacturer's instructions. To confirm that equivalent protein concentrations were loaded, membranes were stripped with 0.2 M NaOH and reprobed with mouse anti-actin antibody (1 : 2000) (Chemicon International, Inc., Temecula, CA) and an anti-mouse immunoglobulin G (1 : 3000) (Amersham Pharmacia Biotech).

Retroviral Gene Transfer Into Melanocytes

The full-length open reading frames for normal murine p16 and Arf were subcloned into pBABE-puro vectors (22), which express the insert from the Moloney murine leukemia virus (MoMuLV) long terminal repeat (LTR) promoter. Phoenix-ECO packaging cells (23) were transfected when nearly confluent with 7.5 µg of pBABE-puro-p16, pBABE-puro-Arf, or pBABE-puro as a control per 5-cm culture dish by the calcium phosphate method in 3.5 mL of DMEM containing 10% FCS. After 24 h, this medium was replaced with RPMI-1640 containing 10% FCS. This medium containing retroviruses was harvested after at least 6 h and passed through a 0.22-µm filter, and the process was repeated to give a second batch of medium. The medium was either used immediately or stored at –70 °C and thawed for use. The medium was supplemented with TPA (20 nM) and polybrene (8 µg/mL) and added to cultures of Ink4a-Arf–/– melanocytes for a total infection time of 24 h. The conditioned growth medium removed from the melanocyte cultures was kept at 4 °C during the infection and replaced on the cells from 24 to 72 h after infection, for recovery. This medium was then replaced by fresh melanocyte medium containing puromycin (1.5 µg/mL) to select for infected cells, which were subsequently used for cell counts or staining.

Acidic {beta}-Galactosidase Staining

We used a modification of the method of Dimri et al. (24) to detect acidic {beta}-galactosidase, a marker for senescence, in melanocytes. Melanocyte cultures were washed once in Dulbecco's PBS (with CaCl2 and MgCl2), fixed in glutaraldehyde (2.5 mg/mL) in the same PBS for 3–5 min at room temperature, and washed four times in this PBS supplemented with extra MgCl2 (1 mM). The cells were stained overnight at 37 °C in 1 mg/mL X-gal (Boehringer Mannheim, Mannheim, Germany), 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN) in PBS (pH 6.0) supplemented with extra MgCl2 (1 mM). Cytoplasmic acidic {beta}-galactosidase stains blue and can be seen by light microscopy.


    RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Removal of Linked Coat Color Variation

It was initially preliminarily reported that Ink4a-Arf–/– mice appeared paler than littermates (11). However, normal mice in the original mixed-strain background varied in pigmentation, so any effects of the deletion were ill defined. To remove irrelevant genetic variations, we back-crossed the deletion to the inbred C57BL/6J strain for more than four generations. C57BL/6J mice are genetically black (nonagouti or a/a, and +/+ at all other coat color loci). Initially, C57BL/6J mice homozygous for the Ink4a-Arf deletion had a brown rather than black coat color. However, with continued back-crossing, recombination was observed between the brown color and the Ink4a-Arf deletion. We therefore suspected the presence of a Tyrp1b (brown) mutation at the brown or Tyrp1 (tyrosinase-related protein 1) locus, which is tightly linked to the Ink4a-Arf locus. Tyrp1 is a melanocyte-specific protein required for the production of black melanin. Tyrp1b/Tyrp1b mice have nonfunctional Tyrp1, giving brown hair (25). Back-crossed brown but not black Ink4a-Arf nullizygous mice were shown by polymerase chain reaction and restriction fragment length polymorphism analysis for a diagnostic Taq1 site in Tyrp1 to be homozygous for the Tyrp1b allele (Matheu A, Serrano M: personal communication). Moreover, the brown color of cultured melanocytes from the brown Ink4a-Arf–/– mice was corrected to black after infection with the retrovirus pHS-TRP1 (25) carrying a wild-type Tyrp1 sequence (data not shown), as previously observed with other Tyrp1b/Tyrp1b melanocytes (25). Thus the brown color was entirely the result of a linked Tyrp1b allele in the Ink4a-Arf–/– mice, presumably derived from the WW6 ES cell line used for the targeted deletion of the Ink4a-Arf locus (11). By further back-crossing, we eliminated the Tyrp1b allele and established a true-breeding black C57BL/6J mouse stock carrying the Ink4a-Arf deletion. This stock has now been maintained for over 3 years. Mice homozygous or heterozygous for the Ink4a-Arf deletion now showed no obvious difference in color from normal C57BL/6J mice.

Effect of Ink4a-Arf Deletion on Senescence and Pigmentation in Melanocyte Cultures

Primary epidermal melanocyte cultures were prepared after back-crossing the Ink4a-Arf null allele to C57BL/6J mice for four generations or more, both before and after removal of the linked Tyrp1b mutation by additional back-crossing. Melanocytes from Tyrp1b/Tyrp1b, Ink4a-Arf nullizygous mice were visibly brown in culture, as expected (25), whereas melanocytes isolated after recombination (Tyrp1+/+) were black. Both Tyrp1b/Tyrp1b and Tyrp1+/+ melanocytes had similar rates of proliferation and are, therefore, not distinguished in Fig. 1Go. Fig. 1Go shows the rates of proliferation of melanocytes from mice with zero, one, and two copies of the Ink4a-Arf null allele. The kinetics of proliferation were reproducible among several independent cell cultures from individual mice with each of these genotypes. The averaged doubling times of presenescent cultures, which were derived from mean exponential growth rate constants between 10 and 28 days, were 4.2 days for Ink4a-Arf+/+ melanocytes, 3.5 days for Ink4a-Arf+/– melanocytes, and 2.4 days for Ink4a-Arf–/– melanocytes. Wild-type (Ink4a-Arf+/+) melanocytes reproducibly completed senescence after 4–5 weeks in culture, with cell numbers declining after 7 weeks. We have shown previously by [3H]thymidine labeling that the mouse melanocyte proliferation rate is very low by 4–5 weeks of culture (26). By contrast, Ink4a-Arf–/– melanocytes grew rapidly throughout the 3-month period shown in Fig. 1Go and have continued to grow exponentially for over a year, with no sign of entering senescence. It is interesting that Ink4a-Arf+/– melanocytes showed intermediate kinetics with some deceleration of growth but no arrest (Fig. 1Go). All Ink4a-Arf+/– cultures maintained for this long then accelerated in growth to resemble the Ink4a-Arf–/– cells, with doubling times as low as 2.3 days. This occurred at between 80 and 100 days in culture in two Ink4a-Arf+/– cultures followed throughout the first few months of culture. One of the cultures was then tested for p16 and Arf protein expression from the remaining Ink4a-Arf allele and showed loss of both proteins (see below).



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Fig. 1. Effect of Ink4a-Arf–/– and Ink4a-Arf+/– on melanocyte senescence. Melanocytes were isolated from neonatal Ink4a-Arf–/–, Ink4a-Arf+/–, and Ink4a-Arf+/+ mice. Each curve shows the cumulative growth of an independent melanocyte culture from one neonatal mouse. The total population doublings are relative to the first cell count at day 10 after explantation. Each point represents one subculture and is derived from the mean hemocytometer count of cells harvested with trypsin and EDTA from three replicate dishes. Doublings at each passage were calculated from the ratio between the numbers of cells plated and those harvested. Filled symbols = wild-type Ink4a-Arf+/+ cultures; crossed symbols = Ink4a-Arf+/– cultures; open symbols = Ink4a-Arf–/– cultures.

 
Under our growth conditions, senescent normal mouse melanocytes typically become large, flat, and highly pigmented (27). Ink4a-Arf+/– melanocytes also showed this morphologic change to some extent (Fig. 2, AGo), although they continued to grow. However, Ink4a-Arf–/– melanocytes did not, remaining small, bipolar, and pale (Fig. 2, BGo) and resembling either presenescent or immortal mouse melanocytes (27). Pigmentation was quantified by determining the melanin content, using black (Tyrp1+/+) cultures throughout. Compared with wild-type Ink4a-Arf+/+ melanocytes at the time of senescence, Ink4a-Arf–/– melanocytes had a markedly lower melanin content (Fig. 3Go), whereas Ink4a-Arf+/– melanocytes had an intermediate melanin content. The differences were greater when expressed as melanin per cell than per unit protein, confirming that senescent Ink4a-Arf+/+ cells were substantially larger and contained more protein than Ink4a-Arf+/– cells, which in turn were larger than Ink4a-Arf–/– cells. However, normalizing to total protein content probably underestimates the visible difference in pigmentation because electron microscopy of highly pigmented melanocytes shows that much of the cytoplasm is occupied by melanosomes that would, therefore, account for much of the cellular protein. Of note, the melanin content of black Ink4a-Arf–/– cells appeared comparable to that of immortal black mouse melanocyte lines (Fig. 3, AGo).



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Fig. 2. Effect of genotype on appearance of melanocytes at passage 3. Melanocytes from Ink4a-Arf–/– and Ink4a-Arf+/– mice (from the stock of mice back-crossed to the C57BL/6J strain for at least four generations) were isolated, cultured for 5 weeks (passage 3), and photographed by bright-field optics 5 days after plating at the same density. Cultures shown are genetically black (Tyrp1+/+). A) Ink4a-Arf+/– melanocytes are relatively large, flat or irregular in shape, and dark. Pale spots indicate the nuclei. Wild-type cultures appeared similar but with fewer cells (not shown). B) Ink4a-Arf–/– melanocytes are pale, small, and more numerous than the Ink4a-Arf+/– melanocytes. Both micrographs are shown at the same magnification. Bar = 150 µm.

 


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Fig. 3. Pigmentation in Ink4a-Arf–/– and Ink4a-Arf+/– melanocytes. Melanocytes were isolated from normal C57BL/6J mice and from Ink4a-Arf–/– and Ink4a-Arf+/– mice (from the stock of mice back-crossed to C57BL/6J mice for at least four generations) and were genetically black (Tyrp1+/+). Melanin content was measured in diploid cultures at passages 3–6, the time normal Ink4a-Arf+/+ melanocytes entered senescence. Bars show the means expressed as nanograms of melanin per cell number (A) or as micrograms of melanin per milligram of protein (B) and 95% confidence intervals of at least three cultures from at least two independent mice. Shading indicates Ink4a-Arf genotype: open = –/–; single-hatched = ±; filled = +/+. The mean melanin content from three cultures of three spontaneously immortal, black melanocyte lines [melan-a (27), mel-18, and mel-29 (21)] is also shown (cross-hatching). These cell lines were harvested at higher passage levels.

 
No additional biologic alterations, such as depigmentation or morphologic transformation (i.e., accelerated growth, multilayering, and reduced density dependence) occurred in Ink4a-Arf–/– cell cultures over approximately 25 passages (about 1 year's growth), suggesting that the Ink4a-Arf deletion affected melanocyte growth primarily by affecting cell senescence. Likewise, no biologic differences were previously detected between Ink4a-Arf–/– and wild-type Ink4a-Arf+/+ mouse melanocytes in long-term cultures, in which the wild-type cells would have been immortal (28).

Next, to investigate the similarity between Ink4a-Arf–/– melanocytes and immortal melanocytes, we assessed the expression of p16 and Arf proteins in six independent lines of spontaneously immortalized melanocytes and their precursors (melanoblasts and neural crestlike cells) (20,27,29) (Fig. 4Go). These cell lines were derived from nonmelanoma normal mouse skin and were nontumorigenic where tested (27). It is interesting that although most melanocyte and melanoblast lines expressed Arf, none expressed p16 protein (Fig. 4Go). The NC-m4 line, a line of neural crestlike precursors of melanoblasts (Sviderskaya E, Hill S, Easty D, Bennett DC: unpublished data), expressed neither p16 nor Arf. At passage 7 (a higher passage than those in the growth curves of Fig. 1Go), one strain of Ink4a-Arf+/– melanocytes did not express p16 or Arf proteins. This suggested that a loss of heterozygosity or coordinate silencing of both products of the normal allele (Ink4a-Arf+) was associated with immortalization. Results of an RNAse protection assay (30) (data not shown) identified RNAs corresponding to two transcripts each for p16 and Arf in these cells, indicating transcription from both the normal and disrupted alleles (this is possible because the first exon of both p16 and Arf was not deleted). Thus, the absence of the p16 and Arf proteins was not because of a loss of heterozygosity but because of a post-transcriptional effect and is under further investigation.




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Fig. 4. Expression of p16 and Arf protein in immortal melanocytic lines. Immunoblot analysis of cell lysates (30 µg of protein) from fibroblasts (3T3 fibro), immortal mouse melanocytes [melan-a, -b, and -c (27)], melanoblasts [melb-a and melb-s1 (29)], and a melanoblast-precursor or neural crestlike line (NC-m4; Sviderskaya E, Hill S, Easty D, Bennett DC: unpublished data), all from Ink4a-Arf+/+ mice, and cultures of Ink4a-Arf–/– and Ink4a-Arf+/– melanocytes. Unfractionated cell lysates were electrophoresed through 12% polyacrylamide gels and transferred to an Immobilon-P membrane. Membranes were incubated with rabbit polyclonal antibodies to mouse Arf (A) and p16 (B) and with peroxide-conjugated anti-rabbit immunoglobulin G. The bound antibodies were detected by enhanced chemiluminescence. An unidentified protein of approximately 39 kDa was detected by the Arf antibody in Ink4a-Arf–/– and other melanocytes, melanoblasts, and neural crestlike cells, but not in fibroblasts. Membranes were stripped and reprobed with a mouse anti-actin antibody to confirm that equivalent amounts of protein were loaded.

 
Effect of Retroviral Expression of p16 or Arf in Nullizygous Ink4a-Arf–/– Melanocytes

To investigate the individual roles of p16 and Arf in melanocyte senescence, p16 or Arf cDNAs were inserted into pBABE-puro retroviral vectors containing the MoMuLV LTR promoter and a selectable marker (puromycin resistance) (22). Black (Tyrp1+/+) Ink4a-Arf–/– mouse melanocytes (melan-Ink4a-1) at passage levels 11–12 were infected with pBABE-puro, pBABE-puro-p16, or pBABE-puro-Arf. After 3 days following initiation of infection, the cells were selected for resistance to puromycin. The proliferation of surviving cells was measured in a growth-curve assay starting 5 days after drug selection began. Melan-Ink4a-1 cells infected with the pBABE-puro empty retroviral vector grew exponentially, whereas those infected with pBABE-puro-p16 grew very little during the period of the growth curve (Fig. 5Go). Melan-Ink4a-1 cells infected with pBABE-puro-Arf declined in number, indicating cell death, as supported by microscopy.



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Fig. 5. Effect of retroviral infection of wild-type p16 and Arf complementary DNAs (cDNAs) on the growth of Ink4a-Arf–/– melanocytes. Retroviruses containing the full-length p16 and Arf cDNAs were constructed in the pBABE-puro vector. Ink4a-Arf–/– melanocytes were infected with the retroviruses pBABE-puro ({diamondsuit}), pBABE-puro-p16 ({blacksquare}), or pBABE-puro-Arf ({blacktriangleup}) and were selected with puromycin; the number of cells was counted at the indicated times after infection. Points represent the means, and lines represent 95% confidence intervals. The cell number in cultures infected with pBABE-puro-Arf declined by day 14 to about 25% of that at day 8, whereas the number remained approximately stable over this time in cultures infected with pBABE-puro-p16.

 
After the period of growth measurement, most melan-Ink4a-1 cells infected with pBABE-puro-p16 appeared large, flat, and highly pigmented and resembled senescent melanocytes. Because these cells were in groups of about one to eight cells, some divisions may have occurred before growth curves were started, but growth was apparently arrested after zero to three cell divisions.

We determined whether melan-Ink4a-1 cells infected with pBABE-puro-p16 expressed an acidic {beta}-galactosidase active at pH 6.0, a marker of cell senescence and sometimes referred to as "senescence-associated {beta}-galactosidase" (13,14,24). It should be noted that nonsenescent human melanocytes express a {beta}-galactosidase active at pH 6.0 (24). Our studies of cultured human melanocytes have confirmed this, but the activity is associated with subcellular granules, possibly melanosomes, and the pH range appears to be different from that of the senescence-associated enzyme (data not shown). Moreover, no clear acidic {beta}-galactosidase activity was detected in growing melan-Ink4a-1 mouse melanocytes infected with pBABE-puro (Fig. 6, AGo). By contrast, prominent blue staining indicative of acidic {beta}-galactosidase activity was observed in the growth-arrested melan-Ink4a-1 cells infected with pBABE-puro-p16 (Fig. 6, BGo). This indicated that these cells were senescent. No staining of melan-Ink4a-1 cells infected with pBABE-puro-Arf was observed. However, the activation of acidic {beta}-galactosidase in these cultures was not ruled out because at the time of staining the few remaining adherent cells all looked highly pigmented (Fig. 6, C and DGo), and it was unclear whether they were alive. Most cells infected with pBABE-puro-Arf were small and dendritic rather than large and flat (Fig. 6, C and DGo): they did not resemble senescent cells morphologically. After puromycin selection, too few cells infected with pBABE-puro-Arf could be obtained for any apoptosis assay. Melanocytes expressing exogenous Arf without p16 had a high rate of cell death without evidence of senescence.



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Fig. 6. Cell morphology and {beta}-galactosidase expression after infection of Ink4a-Arf–/– melanocytes with retroviral vectors containing p16 or Arf complementary DNAs. Melan-Ink4a-1 mouse melanocyte cultures, 12 days after infection, were stained overnight to show activity of acidic {beta}-galactosidase at pH 6.0 (24). A) Control cultures, infected with pBABE-puro only, typically formed quite large clonal colonies that did not stain blue for acidic {beta}-galactosidase. B) Cultures infected with pBABE-puro-p16 formed mostly small colonies or single cells that were generally large and flat. Most cells showed juxtanuclear blue stain for acidic {beta}-galactosidase. Bar = 100 µm. C and D) Cultures infected with pBABE-puro-Arf were generally composed of single, highly pigmented cells that were bipolar or dendritic rather than flat. However, few cells were viable. These cells lacked staining for acidic {beta}-galactosidase, although a faint stain might have been masked by the melanin. Bar = 200 µm in A, C, and D.

 

    DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have identified several aspects of the biology of Ink4a-Arf-deficient melanocytes that seem pertinent to why the human INK4A-ARF locus, and p16 in particular, constitutes a melanoma susceptibility locus. First, Ink4a-Arf+/– (hemizygous) as well as Ink4a-Arf–/– (nullizygous) melanocytes did not senesce 4–5 weeks after explantation, the time that Ink4a-Arf+/+ (wild-type) epidermal mouse melanocytes consistently did so. This behavior differs from that of Ink4a-Arf+/– fibroblasts, which showed growth kinetics indistinguishable from those of wild-type fibroblasts (11). Although hemizygous melanocytes showed partial morphologic features associated with senescence (i.e., became fairly large, flat, and dark), the whole culture continued to divide. Later, the growth of these cells accelerated and resembled that of nullizygous melanocytes, and in fact, one line was found to have lost expression of p16 and Arf from the remaining Ink4a-Arf allele.

Second, there was a relationship between the number of Ink4a-Arf alleles and melanin content at the time of senescence of wild-type melanocytes. Ink4a-Arf+/+ senescent melanocytes had the highest melanin levels, Ink4a-Arf+/– melanocytes had an intermediate level, and Ink4a-Arf–/– melanocytes had the lowest level. Indeed, Ink4a-Arf–/– nullizygous melanocytes had a level comparable to those of immortal melanocytes. This relationship between Ink4a-Arf, pigmentation, and senescence is interesting in relation to the observation that the RB1 protein can bind the melanocytic transcription factor Mitf (31), which appears to be central in the control of melanocytic differentiation (32). Moreover, this interaction is specific for the hypophosphorylated form of RB1 (Goding CR: personal communication), which raises the possibility that hypophosphorylated RB1 is an activator of Mitf. Similarly, RB1 protein activates the related bHLH (basic helix–loop–helix) transcription factor MyoD1, which mediates differentiation in cells of the skeletal muscle lineage (33). The idea that RB1 is involved in melanocyte differentiation is consistent with our observation that pigmentation in Ink4a-Arf–/– melanocytes is abnormally low at the time normal Ink4a-Arf+/+ melanocytes senesce but is increased with restoration of p16 expression, concomitant with the apparent induction of senescence. Recalling that p16 activates RB1 by inhibiting its phosphorylation, this idea also provides one possible mechanism for the commonly deficient differentiation of cultured melanoma cell lines (34,35) because 22 of 22 melanoma lines were found to have some defect, often p16 loss (36), in the RB1 pathway.

Third, a particularly notable property of Ink4a-Arf–/– melanocytes was that apparent cell senescence—determined on the basis of growth arrest, appropriate morphologic changes, and the expression of acidic {beta}-galactosidase—could be induced after restoring wild-type p16 expression in the absence of Arf. This observation was different from that with fibroblasts: mouse embryo fibroblasts lacking Arf but with intact p16 do not senesce normally (6), whereas mouse fibroblasts lacking p16 but with intact Arf do senesce normally (37,38), although they show some increased incidence of immortalization (37). Thus, it appears that senescence in mouse melanocytes may be more dependent on p16 than on Arf, although it should be noted that here p16 was expressed from a viral promoter and thus was possibly overexpressed. Nonetheless, our results are supported by the observation that no spontaneously immortal murine melanocyte or melanoblast lines expressed p16 protein, whereas most expressed substantial levels of Arf protein.

The expression of Arf but not p16 in spontaneously immortal melanocytes raises several questions for future study, including: By what mechanism is p16 expression selectively lost? How do melanocytes and melanoblasts grow well while expressing Arf, a growth inhibitor and activator of p53? It is possible that there is some block in the p53 pathway in melanocytic cells; this would be consistent with the tendency of melanomas to express high levels of normal p53 (39,40) and for senescent human melanocytes to lack increased p21 expression (41). It is unclear why spontaneously immortal melanocytes expressed Arf and grew well but those that expressed exogenous Arf from a viral promoter died. One possibility is a difference in the level of expression, with the latter possibly overexpressing the protein. However, because of the high death rate, it was not possible to obtain enough cells to measure the level of protein expression.

Retrovirally mediated expression of wild-type Arf without p16 in Ink4a-Arf–/– melanocytes produced a different outcome from expression of p16 without Arf; namely, a decline in cell numbers from excessive cell death. This observation is reminiscent of the high rate of apoptosis in several other cell lineages, e.g., the lens, in Rb1-deficient mice with intact p53 and Arf (7,42), and it suggests interacting roles for the Rb1 and p53 pathways in the control of melanocyte survival, as with these other lineages. Binding of Rb1 to MDM2 has been implicated in the suppression of p53-mediated apoptosis by Rb1 (43).

These potentially distinct roles for p16 and Arf in controlling mouse melanocyte senescence, differentiation, and death provide a model for the role of INK4A-ARF mutations in human melanoma susceptibility. There is strong evidence that immortalization is a crucial step in tumorigenesis (17,44); in other words, senescence appears to be an important anticancer mechanism. In this study, we found impaired senescence in Ink4a-Arf+/– melanocytes; this is important because most familial melanoma patients with an INK4A-ARF defect are heterozygous, with only one defective copy (2,3). We found evidence that p16 is important in melanocyte senescence, which may explain why melanoma families often have mutations in the INK4A sequence but not in the ARF sequence. We also found reduced differentiation in Ink4a-Arf+/– melanocytes. This is a common property of cancer cells, including melanoma cells (34,35) and is likely to contribute to neoplastic growth, since more cellular resources are available for the support of growth rather than for the synthesis of specialized products. These dual effects, impairment of both senescence and differentiation, may select strongly for cells with deficiencies in the RB1 pathway during melanoma development. It is unknown, however, why p16 rather than RB1 deficiencies are associated with melanoma. One possibility is that the other two RB family members, p107 and p130, may share some roles of RB1 in melanocytes. A loss of p16 would also reduce the activity of these proteins, as all three are inhibited by CDK4 and CDK6, the kinases inhibited by p16 (3,5). Deficiencies in ARF, which are seen in a proportion of familial and sporadic melanomas, may be associated with a reduced death rate in melanocytes or melanocytic lesions. To summarize, mutations of the INK4A-ARF locus may favor tumorigenesis from melanocytes by impairing senescence, cell differentiation, and (where ARF is disrupted) cell death.

Despite the biologic abnormalities in Ink4a-Arf–/– melanocytes, melanoma incidence is not increased in Ink4a-Arf–/– mice, even after prolonged, suberythrogenic doses of UVB irradiation in adult animals (11), in contrast to the predisposition of humans with INK4A-ARF mutations to develop melanoma after exposure to sunlight. There are, however, a number of possible explanations. One possibility is that Ink4a-Arf–/– mice died of other tumors induced by the Arf deletion before melanomas developed. This is supported by recent findings on mice with engineered alterations of Ink4a but not Arf. Mice either nullizygous for Ink4a (37) or with a point mutation in Ink4a and deletion of both Ink4a and Arf at the other allele (38) did develop melanomas among other tumors after carcinogen treatment (37,38). By implication, carcinogen-treated Ink4a-Arf–/– mice should also have developed melanomas had they survived long enough. It is possible that melanomas are rare in mice because there are fewer epidermal melanocytes in typical (hairy) skin of mature mice (around 50/mm2, with variation between strains) (45) than in typical (sparsely haired) human skin (1000–2000/mm2) (46). Moreover, in the hairy skin of mice, most melanocytes are in the hair follicles. In hairless, pigmented skin, such as that of the ear, there are more epidermal melanocytes (around 300/mm2, poorly pigmented), but most are in the dermis (47,48). Even after shaving, these deep sites are well protected from penetration by UVB, the more mutagenic waveband of UV light in sunlight that reaches ground level, although not by UVA. When Ink4a-Arf–/– mice were crossed with transgenic mice expressing an activated Ras gene from the tyrosinase promoter–enhancer, giving melanocyte-specific expression, the incidence of melanoma did increase (49).

In summary, on the basis of our study, we conclude that melanocytes nullizygous or hemizygous for Ink4a-Arf are defective in senescence, that senescence of mouse melanocytes appears to be controlled by the p16/Rb1 pathway more than the Arf/p53 pathway, and that melanocytes nullizygous or hemizygous for Ink4a-Arf show reduced pigmentation at the time of normal melanocyte senescence. These findings provide some potential reasons for specific melanoma susceptibility in humans with INK4A (and ARF) alterations.


    NOTES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Present address: D. J. Easty, Biotechnology Centre, University College Dublin, Ireland.

Supported in part by Wellcome Trust grant 046038/Z/95/Z (to D. C. Bennett), by Cancer Research Campaign grant SP1923/0503 (to D. C. Bennett), and by a British Council Commonwealth Scholarship (to S. C. Cheong). L. Chin is supported by Public Health Service grant U01CA84313 (National Cancer Institute), National Institutes of Health (NIH), Department of Health and Human Services (DHHS) and is a Culperer Medical Scholar. R. A. DePinho is supported by Public Health Service grant U01CA84313 (National Cancer Institute), NIH, DHHS, and by the American Cancer Society (ACS) and is an ACS Research Professor.

We are indebted to M. Serrano (Spanish National Center of Biotechnology, Madrid, Spain) for reagents, M. Serrano and C. Goding (Marie Curie Memorial Foundation Research Institute, Oxted, U.K.) for communication of unpublished information, H.-W. Lee (Albert Einstein College of Medicine, Bronx, NY) for help and advice, and T. Hirobe (National Institute of Radiological Sciences, Chiba-shi, Japan) for useful discussions.


    REFERENCES
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Manuscript received August 16, 2001; revised January 8, 2002; accepted January 22, 2002.


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