Cytoplasmic localization of the oncogenic protein Ski in human cutaneous melanomas in vivo: functional implications for transforming growth factor beta signaling.

The oncogenic protein Ski associates with Smad proteins and counteracts their activation of gene expression and growth inhibition in response to transforming growth factor beta (TGF-beta). Here we show that Ski protein levels are increased in all 44 human melanoma tumor tissues analyzed in vivo. In addition, Ski subcellular localization changes from nuclear, in preinvasive melanomas (melanomas in situ), to nuclear and cytoplasmic in primary invasive and metastatic melanomas. Furthermore, Ski/Smad association in the cytoplasm seems to prevent Smad3 nuclear translocation in response to TGF-beta. The biological significance of Ski overexpression in melanomas was established by showing that down-regulation of Ski levels, by antisense Ski vectors, restored TGF-beta-mediated growth inhibition. Such inhibition is apparently mediated by up-regulation of the cyclin-dependent kinase-I p21(Waf-1) and inhibition of cyclin-dependent kinase 2 activity. Our results suggest that high levels of Ski in human melanomas produce a disruption of TGF-beta signaling phenotypically similar to that in cells harboring mutations in TGF-beta receptors or Smad proteins, and this may represent a significant event in the progression of melanomas in vivo.

The human melanocyte: a model system to study the complexity of cellular aging and transformation in non-fibroblastic cells.

The melanocyte is a neural crest-derived cell that localizes in humans to several organs including the epidermis, eye, inner ear and leptomeninges. In the skin, melanocytes synthesize and transfer melanin pigments to surrounding keratinocytes, leading to skin pigmentation and protection against solar exposure. We have investigated the process of replicative senescence and accompanying irreversible cell cycle arrest, in melanocytes in culture. As was found in other cell types, progressive telomere shortening appears to trigger replicative senescence in normal melanocytes. In addition, senescence is associated with increased binding of the cyclin-dependent kinase inhibitor (CDK-I) p16(INK4a) to CDK4, down-regulation of cyclin E protein levels (and consequent loss of cyclin E/CDK2 activity), underphosphorylation of the retinoblastoma protein RB and subsequent increased levels of E2F4-RB repressive complexes. In contrast to fibroblasts, however, the CDK-Is p21(Waf-1) and p27(Kip-1) are also down-regulated. These changes appear to be important for replicative senescence because they do not occur in melanocytes that overexpress the catalytic subunit of the enzyme telomerase (hTERT), or in melanomas, which are tumors that originate from melanocytes or melanoblasts. In contrast to unmodified melanocytes, hTERT overexpressing (telomerized) melanocytes displayed telomerase activity, stable telomere lengths and an extended replicative life span. However, telomerized melanocytes show changes in cell cycle regulatory proteins, including increased levels of cyclin E, p21(Waf-1) and p27(Kip-1). Cyclin E, p21(Waf-1) and p27(Kip-1) are also elevated in many primary melanomas, whereas p16(INK4a) is mutated or deleted in many invasive and metastatic melanomas. Thus, the molecular mechanisms leading to melanocyte senescence and transformation differ significantly from fibroblasts. This suggests that different cell types may use different strategies to halt the cell cycle in response to telomere attrition and thus prevent replicative immortality.

Gaf-1, a gamma -SNAP-binding protein associated with the mitochondria.

The role of alpha/beta-SNAP (Soluble NSF Attachment Protein) in vesicular trafficking is well established; however, the function of the ubiquitously expressed gamma-SNAP remains unclear. To further characterize the cellular role of this enigmatic protein, a two-hybrid screen was used to identify new, gamma-SNAP-binding proteins and to uncover potentially novel functions for gamma-SNAP. One such SNAP-binding protein, termed Gaf-1 (gamma-SNAP associate factor-1) specifically binds gamma- but not alpha-SNAP. The full-length Gaf-1 (75 kDa) is ubiquitously expressed and is found stoichiometrically associated with gamma-SNAP in cellular extracts. This binding is distinct from other SNAP interactions since no alpha-SNAP or NSF coprecipitated with Gaf-1. Subcellular fractionation and immunofluorescence analysis show that Gaf-1 is peripherally associated with the outer mitochondrial membrane. Only a fraction of gamma-SNAP was mitochondrial with the balance being either cytosolic or associated with other membrane fractions. GFP-gamma-SNAP and the C-terminal domain of Gaf-1 both show a reticular distribution in HEK-293 cells. This reticular structure colocalizes with Gaf-1 and mitochondria as well as with microtubules but not with other cytoskeletal elements. These data identify a class of gamma-SNAP interactions that is distinct from other members of the SNAP family and point to a potential role for gamma-SNAP in mitochondrial dynamics.

Tyrosine levels regulate the melanogenic response to alpha-melanocyte-stimulating hormone in human melanocytes: implications for pigmentation and proliferation.

Melanocyte-stimulating hormone (alpha-MSH) increases cytosolic levels of cAMP as well as tyrosinase activity in murine melanocytes. These activities depend upon the presence of melanin precursors and may differ in human melanocytes. In this study, we demonstrate that high levels of tyrosine (3.7 mM), the chief melanin precursor, reduced the proliferative effect of alpha-MSH and altered human melanocyte morphology as compared to treatment with low (25-30 microM, half-physiological) levels of tyrosine. The anti-proliferative effect of high levels of tyrosine was not restricted to alpha-MSH; tyrosine also reduced proliferation induced by forskolin, a direct activator of the cAMP pathway. Exposure to low tyrosine levels and alpha-MSH induced a dendritic morphology; in the presence of high tyrosine and alpha-MSH, melanocytes displayed large, pigmented cell bodies and less dendricity. Exposure to alpha-MSH in the presence of low tyrosine for up to 9 days did not appreciably increase melanin levels, but culturing the human melanocytes in high levels of tyrosine with alpha-MSH increased melanin levels 10-50-fold, depending on the pigmentation background of the donor. A greater induction of melanin accumulation was observed in melanocytes derived from light-skinned donors than was observed in cells obtained from dark-skinned donors. The poor ability of alpha-MSH to stimulate melanin synthesis was not caused by a lack of induction of melanogenic proteins, as alpha-MSH increased the expression of microphthalmia (MITF), tyrosinase, dopachrome tautomerase (DCT), and Pmel-17, compared to untreated cells or cells stimulated by phorbol ester alone, regardless of tyrosine levels. DCT levels were greatly induced by low tyrosine with alpha-MSH, but were dramatically decreased by high tyrosine with alpha-MSH. Interestingly, in this same medium (high tyrosine), MITF levels also decreased after 2 weeks and were barely detectable by the third week. Despite the absence of MITF at 3 weeks of treatment in high tyrosine medium, tyrosinase levels remained high, thereby suggesting that additional factors must be responsible for tyrosinase transcription in human melanocytes. Our results indicate that tyrosine levels can regulate the proliferative activity induced by alpha-MSH, as well as the extent of melanogenesis in normal human melanocytes. The significance of this work is that tyrosine levels may be part of the mechanism that switches melanocytes out of a proliferative status and into a melanin-synthesizing, terminally differentiated phenotype.

Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes.

Replicative senescence of human diploid fibroblasts (HDFs) or melanocytes is caused by the exhaustion of their proliferative potential. Stress-induced premature senescence (SIPS) occurs after many different sublethal stresses including H(2)O(2), hyperoxia, or tert-butylhydroperoxide. Cells in replicative senescence share common features with cells in SIPS: morphology, senescence-associated beta-galactosidase activity, cell cycle regulation, gene expression and telomere shortening. Telomere shortening is attributed to the accumulation of DNA single-strand breaks induced by oxidative damage. SIPS could be a mechanism of accumulation of senescent-like cells in vivo. Melanocytes exposed to sublethal doses of UVB undergo SIPS. Melanocytes from dark- and light- skinned populations display differences in their cell cycle regulation. Delayed SIPS occurs in melanocytes from light-skinned populations since a reduced association of p16(Ink-4a) with CDK4 and reduced phosphorylation of the retinoblastoma protein are observed. The role of reactive oxygen species in melanocyte SIPS is unclear. Both replicative senescence and SIPS are dependent on two major pathways. One is triggered by DNA damage, telomere damage and/or shortening and involves the activation of the p53 and p21(waf-1) proteins. The second pathway results in the accumulation of p16(Ink-4a) with the MAP kinase signalling pathway as possible intermediate. These data corroborate the thermodynamical theory of ageing, according to which the exposure of cells to sublethal stresses of various natures can trigger SIPS, with possible modulations of this process by bioenergetics.

Melanin accumulation accelerates melanocyte senescence by a mechanism involving p16INK4a/CDK4/pRB and E2F1.

Cellular and molecular evidence suggests that senescence is a powerful tumor-suppressor mechanism that prevents most higher eukaryotic cells from dividing indefinitely in vivo. Recent work has demonstrated that alpha-melanocyte stimulating hormone (alpha-MSH) or cholera toxin (CT) can activate a cAMP pathway that elicits proliferative arrest and senescence in normal human pigmented melanocytes. In these cells, senescence is associated with increased binding of p16INK4a to CDK4 and loss of E2F-binding activity. Because senescence may provide defense against malignant transformation of melanocytes, and because pigmentation is a strong defense against melanoma, we examined the ability of melanocytes derived from light and dark skin to respond to CT. Here we demonstrate that in melanocytes derived from dark-skinned individuals, CT-induced melanogenesis is associated with accumulation of the tumor suppressor p16INK4a, underphosphorylated retinoblastoma protein (pRb), downregulation of cyclin E, decreased expression of E2F1, and loss of E2F-regulated S-phase gene expression. In contrast to other senescent cell types, melanocytes have reduced or absent levels of the cyclin-dependent kinase inhibitors p27Kip1 and p21Waf-1. Importantly, melanocytes derived from light-skinned individuals accumulated smaller amounts of melanin than did those from dark-skinned individuals under the same conditions, and they continued to proliferate for several more division cycles. This delayed senescence may result from reduced association of p16 with CDK4, reduced levels of underphosphorylated pRb, and steady levels of cyclin E and E2F1. Because cyclin E-CDK2 inhibition is required for p16-mediated growth suppression, upregulation of p16 and downregulation of cyclin E appear essential for maintenance of terminal growth and senescence. Given the rising incidence of melanoma, identification of major growth regulatory proteins involved in senescence should shed light on the biology of this genetically mysterious tumor.

Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor.

The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type beta transforming growth factor (TGF-beta) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-beta. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-beta-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-beta-mediated growth inhibition in a prostate-derived epithelial cell line.

Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9.

The basic helix-loop-helix/leucine zipper (bHLH/ZIP) microphthalmia-associated transcription factor (MITF) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells. To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with MITF. The majority of clones that showed positive interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168-325) encoded the ubiquitin conjugating enzyme hUBC9. The association of MITF with hUBC9 was further confirmed by an in vitro GST pull-down assay. Although hUBC9 is known to interact preferentially with SENTRIN/SUMO1, in vitro transcription/translation analysis demonstrated greater association of MITF with ubiquitin than with SENTRIN. Importantly, cotransfection of MITF and hUBC9 expression vectors resulted in MITF protein degradation. MITF protein was stabilized by the proteasome inhibitor MG132, indicating the role of the ubiquitin-proteasome system in MITF degradation. Serine 73, which is located in a region rich in proline, glutamic acid, serine, and threonine (PEST), regulates MITF protein stability, since a serine to alanine mutation prevented hUBC9-mediated MITF (S73A) degradation. Furthermore, we identified lysine 201 as a potential ubiquitination site. A lysine to arginine mutation abolished MITF (K201R) degradation by hUBC9 in vivo. Our experiments indicate that by targeting MITF for proteasome degradation, hUBC9 is a critical regulator of melanocyte differentiation.

The cytotoxicity and apoptosis induced by 4-tertiary butylphenol in human melanocytes are independent of tyrosinase activity.

It has been known for several decades that cutaneous depigmentation, i.e., contact/occupational vitiligo, can be caused by some phenolic derivatives that have a similar structure to tyrosine. Among these phenolic depigmenting agents, 4-tertiary butylphenol is the most potent. The cutaneous depigmentation induced by phenolic derivatives results from the loss of functional melanocytes. Tyrosinase is a melanocyte specific copper-containing enzyme that catalyzes the conversion of the amino acid tyrosine, through a complex series of intermediates, to melanin. In this study we tested the hypothesis that the cytotoxicity induced by 4-tertiary butylphenol is mediated by tyrosinase and occurs via an apoptotic process. Melanocyte cultures derived from African-American and Caucasian donors exhibiting a 3-fold difference in tyrosinase activity and 14-fold difference in melanin content demonstrate comparable concentration-dependent sensitivity to 4-tertiary butylphenol. In addition, cultures of dermal fibroblasts and epidermal keratinocytes exhibited similar and reduced sensitivity, respectively, to 4-tertiary butylphenol compared with autologous melanocytes. Two melanoma cell lines, one melanotic and one amelanotic lacking the expression of both tyrosinase protein and activity, when transfected with the tyrosinase cDNA, exhibited no alteration in its sensitivity to 4-tertiary butylphenol. These data suggest that 4-tertiary butylphenol cytotoxicity is not mediated via tyrosinase. Melanocytes treated with 4-tertiary butylphenol, however, did exhibit plasma membrane blebbing, DNA fragmentation, and phosphatidylserine relocalization indicating that 4-tertiary butylphenol induced melanocyte destruction occurs by an apoptotic process.

High levels of expression of p27KIP1 and cyclin E in invasive primary malignant melanomas.

Cancer cells have abnormal cell cycle regulation which favors accelerated proliferation, chromosomal instability, and resistance to the senescence response. Although the p16INK4a locus is the most prominent susceptibility locus for familial melanomas, the low frequency of p16 mutations in sporadic melanomas suggests additional alterations in other cell cycle regulatory genes. Here we used primary melanoma tumors to reveal early cell cycle alterations that could be masked in advanced metastatic lesions due to their inherently high genetic instability. Unexpectedly, the cyclin-dependent kinase inhibitors p27KIP1 and/or p21Waf-1/SDI-1 were found to be expressed in 13 of 18 (72%) of the primary melanomas with a Breslow thickness greater than 0.076 mm. In general, p27 and/or p21 staining in the primary tumors correlated with low Ki-67 index. Importantly, most of the p21- and p27-positive tumors expressed high levels of cyclin D1 and cyclin E. In proliferating cells p27 is predominantly associated with cyclin D-CDK4 complexes, but does not inhibit the kinase activity, whereas in quiescent cells p27 is found associated with inactive CDK2 complexes. p27 was also expressed at high levels in proliferating primary melanomas in culture, and found to be associated with active cyclin E-CDK2 complexes containing high levels of cyclin E. It is thus likely that accumulation of cyclin E overcomes the potent inhibitory activity of p27 and p21 in CDK2 complexes. Of the primary melanomas with no indication of invasiveness, only three of 15 (20%) were positive for p27 and/or p21. We propose that high levels of p27 and p21 may confer upon melanoma tumors their characteristic resistance to conventional therapies. In turn, high levels of cyclins E and D1 may contribute to unlimited proliferation in primary melanomas that express the tumor suppressor p16INK4. J Invest Dermatol 113:1039-1046 1999

Activation of a cAMP pathway and induction of melanogenesis correlate with association of p16(INK4) and p27(KIP1) to CDKs, loss of E2F-binding activity, and premature senescence of human melanocytes.

There is strong evidence that the senescent phenotype, whether induced by telomere shortening, oxidative damage, or oncogenic stimuli, is an important tumor suppressive mechanism. The melanocyte is a cell of neural crest origin that produces the pigment melanin and can develop into malignant melanomas. To understand how malignant cells escape senescence, it is first crucial to define what genes control senescence in the normal cell. Prolonged exposure to high levels of cAMP results in accumulation of melanin and terminal differentiation of human melanocytes. Here we present evidence that activation of a cAMP pathway correlates with multiple cellular changes in these cells: (1) increased expression of the transcription factor microphthalmia; (2) increased melanogenesis; (3) increased association of the cyclin-dependent kinase inhibitors (CDK-Is) p27(KIP1) and p16(INK4) with CDK2 and CDK4, respectively; (4) failure to phosphorylate the retinoblastoma protein (pRB); (5) decreased expression of E2F1, E2F2, and E2F4 proteins; (6) loss of E2F DNA-binding activity; and (7) phenotypic changes characteristic of senescent cells. Senescent melanocytes have potent E2F inhibitory activity, because extracts from these cells completely abolished E2F DNA-binding activity that was present in extracts from the early proliferative phase. We propose that increased activity of the CDK-Is p27 and p16 and loss of E2F activity in human melanocytes characterize a senescence program activated by the cAMP pathway. Disruption of cAMP-mediated and melanogenesis-induced senescence may cause immortalization of human melanocytes, an early step in the development of melanomas.

Multiple primary melanoma.

BACKGROUND: Incidence rates of cutaneous malignant melanoma (CMM) have been increasing for decades among Caucasian populations worldwide. Multiple factors identify persons at increased risk of CMM, including those with a family history of melanoma and those with atypical moles. Approximately 6-12% of melanomas are familial and approximately 12% of patients with familial melanoma have multiple primary melanomas. OBJECTIVE: To report a case of a patient with atypical moles and with 17 multiple primary melanomas. To review the literature on multiple primary melanomas as well as to review the genetics and treatment of melanoma. CONCLUSION: Patients with numerous atypical moles and a family or personal history of melanoma are at greatest risk for developing CMM. Patients from this population tend to develop CMM approximately 10 years earlier than the general population and have an increased risk for developing multiple primary melanomas. Since genetic tests capable of detecting individuals with an inherited susceptibility to CMM are not available, it is important to identify those patients with an increased risk and monitor them closely with regular total-body examinations.

Aging in epidermal melanocytes: cell cycle genes and melanins.

With aging, melanocytes become unevenly distributed in the epidermis. In light skin individuals, hypopigmentation is found in association with focal hyperpigmentation (lentigo senilis). Apparently this results from progressive loss of active melanocytes and focal increase in melanocyte proliferation and/or aggregation. This paper summarizes the present knowledge on aging of melanocytes in vivo and in vitro, with a focus on the role of melanin as a determinant for proliferation and terminal differentiation. We describe that excessive melanin deposition by cyclic AMP-inducing agents results in increased expression of the cyclin-dependent kinase inhibitors p27Kp-1 and p21SDI-1/Waf-1, increased binding of p16 to CDK4, and terminal differentiation. Importantly, the efficiency with which the melanocytes exit the cell cycle depends on the melanin background of the donor's cells. Melanocytes from skin types IV-VI that accumulate large amounts of brown black melanin (eumelanin), lose expression of the transcription factors E2F1 and E2F2, two key regulatory proteins, and withdraw from the cell cycle more rapidly than melanocytes from skin types I and II that accumulate red/yellow melanin (pheomelanin). Thus, we propose that terminal differentiation is a tumor suppressor mechanism that becomes less efficient under imperfect eumelanization.

Activation of the cyclic AMP pathway by alpha-melanotropin mediates the response of human melanocytes to ultraviolet B radiation.

A hallmark of sun exposure is increased melanin synthesis by cutaneous melanocytes which protects against photodamage and photocarcinogenesis. Irradiation of human keratinocytes or melanocytes with ultraviolet (UV) rays stimulates the synthesis and release of alpha-melanotropin (alpha-MSH) and adrenocorticotropic hormone (ACTH), which induce cyclic AMP (cAMP) formation and increase the proliferation and melanogenesis of human melanocytes. We report that stimulation of cAMP formation is obligatory for the melanogenic response of cultured normal human melanocytes to UVB radiation. In the absence of cAMP inducers, UVB radiation inhibited, rather than stimulated, melanogenesis. UVB radiation (28 mJ/cm2) arrested melanocytes in the G1 phase of the cell cycle, and concomitant treatment with 0.1 microM alpha-MSH enhanced their proliferation but did not increase the surviving fraction. Irradiation with UVB, with or without alpha-MSH, caused prolonged expression of p53 and p21(waf-1, cip-1), maintained pRB in a hypophosphorylated state, and reduced the expression of Bcl2. However, alpha-MSH allowed UVB-irradiated melanocytes to enter S phase, suggesting that alpha-MSH acts as a mitogen rather than a survival factor, and that overexpression of p53 is mainly a signal for cell death. Our results underscore the importance of the cAMP pathway and its physiological inducers in mediating the response of human melanocytes to UV radiation.

Retroviral infection with human tyrosinase-related protein-1 (TRP-1) cDNA upregulates tyrosinase activity and melanin synthesis in a TRP-1-deficient melanoma cell line.

A human tyrosinase-related protein-1 (TRP-1) cDNA was inserted into the retroviral vector, pBAbe-puro. Sense and anti-sense constructs were identified and transfected, as well as vector-alone, into a retrovirus packaging cell line by a liposome-mediated technique and used in turn to infect a human melanoma line deficient in TRP-1 protein/transcript. Polymerase chain reaction (PCR) amplification of genomic DNA from these infectants, using TRP-1 cDNA-specific primers, demonstrate that PCR products were only identified from the sense- and anti-sense-infected clones, not from the parental cells or vector-alone infectants. Northern analysis demonstrated that TRP-1 sense and antisense infectants produced TRP-1 cDNA-related transcripts. Immunoblotting analysis with TA99 (a monoclonal antibody for TRP-1) demonstrated a single band of normal molecular weight from melanoma cells infected with sense cDNA, not from cells infected with sense cDNA, not from cells infected with anti-sense or vector-alone, or from the uninfected-parental melanoma cells. The quantitative and qualitative analysis of melanin in the sense and anti-sense infectant cells demonstrated an increase and decrease in pigmentation, respectively, compared with vector alone. Tyrosine hydroxylase and DOPA oxidase activities of tyrosinase hydroxylase and DOPA oxidase activities of tyrosinase were both increased in sense cDNA infected cells plus unaltered or slightly decreased, respectively, in anti-sense cDNA-infected cells compared with control cells. Immunoblotting analysis with anti-tyrosinase antibody (alpha Ty-SP) demonstrated the amount of tyrosinase was slightly increased in TRP-1 overexpressing cells but slightly decreased in anti-sense infectant cells. We have demonstrated that the expression of exogenous TRP-1 cDNA melanoma cells stimulated the activity of tyrosinase and promoted melanogenesis, indicating that TRP-1 plays a role in regulating tyrosinase activity.

Thy-1+ dendritic cells express truncated form of POMC mRNA.

The present study was designed to examine the expression of proopiomelanocortin (POMC) and its related derivative peptide adrenocorticotropic hormone (ACTH) in murine derived Thy-1+ dendritic cells. Immunostaining using a polyclonal antibody specific to ACTH and parent POMC molecule indicated the presence of POMC and its derivative peptide, ACTH, in cultures of Thy-1+ dendritic cells. To explore whether the POMC peptide is present as a reservoir or synthesized de novo in Thy-1+ dendritic cells. Northern blot analysis using 30-mer oligonucleotide probe for alpha-MSH/ACTH precursor POMC was carried out in total RNA from these cells. Northern blot analysis revealed the presence of POMC like mRNA transcript. However, the observed size of transcript was smaller (approximately 0.9 kb) than that expressed by murine AtT20 cells (approximately 1.2 kb), an anterior pituitary tumor cell line used as a positive control. These observations suggest that the epidermal Thy-1+ lymphocytes, like thymic lymphocytes, might serve the epidermis as one source for the synthesis of POMC. The synthesis and presence of POMC in the epidermis may be related to some of the pigmentary anomalies observed in many mucocutaneous disorders.

A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.

Comparison of the responses of human melanocytes with different melanin contents to ultraviolet B irradiation.

Melanin is thought to serve in photoprotection. To investigate this, we have compared the responses of cultured human melanocytes derived from different pigmentary phenotypes (skin types I-VI) to a single irradiation with different doses of UVB light, ranging between 11.7 and 70.1 mJ/cm2. After UVB irradiation, heavily pigmented melanocytes had the same percent survival but a greater capacity to resume proliferation than their lightly pigmented counterparts. A significant increase in melanin content was observed in heavily pigmented but not in lightly pigmented melanocytes. Irradiation with UVB light blocked melanocytes, regardless of their melanin content, in G1, and induced the expression of the tumor suppressor p53 protein within 4 h. This induction steadily increased up to 48 h in lightly pigmented melanocytes; however, in heavily pigmented melanocytes, p53 level peaked at 24 h after UVB treatment and declined thereafter. Additionally, DNA from lightly pigmented melanocytes contained significantly higher numbers of cyclobutane pyrimidine dimers than did DNA from heavily pigmented melanocytes after irradiation with increasing doses of UVB light. We speculate that the prolonged induction of p53 in lightly pigmented melanocytes arrests them in G1 for a long time period in order to repair extensive DNA damage. The above described differences might partially explain the increased susceptibility of individuals with lightly pigmented skin compared to individuals with dark skin to the photodamaging and photocarcinogenic effects of sun exposure.

Ultraviolet B light induces G1 arrest in human melanocytes by prolonged inhibition of retinoblastoma protein phosphorylation associated with long-term expression of the p21Waf-1/SDI-1/Cip-1 protein.

UVB irradiation inhibits melanocyte proliferation by causing arrest in G1 (D. Barker, K. Dixon, E. E. Medrano, D. Smalara, S. Im, D. Mitchell, G. Babcock, and Z. A. Abdel-Malek. Cancer Res., 55: 4041-4046, 1995). To determine how, after UVB irradiation, signal transduction pathways, DNA damage, and cell cycle arrest interact in the human melanocyte, we analyzed here the possible activation of tyrosine kinases, the serine-threonine kinases Baf-1 and ERK2, the status of the transcription factor c-fos, and the activation of cell cycle checkpoints induced by expression of p53 protein. We found that in contrast to the UVC response, exposure to UVB irradiation did not stimulate the above kinases. UVB light induced a prolonged c-fos expression, suggesting a mechanism of induction different from the transient expression elicited by growth factors. The tumor suppressor p53 and the p53-inducible cyclin-dependent kinase inhibitor protein p21Waf-1/SDI-1/Cip-1 were expressed at high levels for at least 2 days after UV-irradiation. In parallel, phosphorylation of Rb, the retinoblastoma tumor suppressor gene product, was halted in UVB-irradiated cells and correlated with the expression of the protein p21Waf-1/SDI-1/Cip-1. Our data define for the first time how UVB irradiation affects the expression of crucial regulatory events needed for cell cycle progression in the human melanocyte.