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Z A Abdel-Malek

Publications and source records attributed to Z A Abdel-Malek.

At least 19 recordsLinked to original sources
PubMed2006

Cellular senescence in naevi and immortalisation in melanoma: a role for p16?

Cellular senescence, the irreversible proliferative arrest seen in somatic cells after a limited number of divisions, is considered a crucial barrier to cancer, but direct evidence for this in vivo was lacking until recently. The best-known form of human cell senescence is attributed to telomere shortening and a DNA-damage response through p53 and p21. There is also a more rapid form of senescence, dependent on the p16-retinoblastoma pathway. p16 (CDKN2A) is a known melanoma susceptibility gene. Here, we use retrovirally mediated gene transfer to confirm that the normal form of senescence in cultured human melanocytes involves p16, since disruption of the p16/retinoblastoma pathway is required as well as telomerase activation for immortalisation. Expression (immunostaining) patterns of senescence mediators and markers in melanocytic lesions provide strong evidence that cell senescence occurs in benign melanocytic naevi (moles) in vivo and does not involve p53 or p21 upregulation, although p16 is widely expressed. In comparison, dysplastic naevi and early (radial growth-phase, RGP) melanomas show less p16 and some p53 and p21 immunostaining. All RGP melanomas expressed p21, suggesting areas of p53-mediated senescence, while most areas of advanced (vertical growth-phase) melanomas lacked both p16 and p21, implying escape from both forms of senescence (immortalisation). Moreover, nuclear p16 but not p21 expression can be induced in human melanocytes by oncogenic BRAF, as found in around 80% of naevi. We conclude that cell senescence can form a barrier to melanoma development. This also provides a potential explanation of why p16 is a melanoma suppressor gene.

Cell Survival↗
PubMed2001

Melanocortin receptors: their functions and regulation by physiological agonists and antagonists.

The melanocortins are a family of bioactive peptides derived from proopiomelanocortin, and share significant structural similarity. Those peptides are best known for their stimulatory effects on pigmentation and steroidogenesis. Melanocortins are synthesized in various sites in the central nervous system and in peripheral tissues, and participate in regulating multiple physiological functions. Research during the past decade has provided evidence that melanocortins elicit their diverse biological effects by binding to a distinct family of G protein-coupled receptors with seven transmembrane domains. To date, five melanocortin receptor genes have been cloned and characterized. Those receptors differ in their tissue distribution and in their ability to recognize the various melanocortins and the physiological antagonists, agouti signaling protein and agouti-related protein. These advances have opened new horizons for exploring the significance of melanocortins, their antagonists, and their receptors in a variety of important physiological functions.

Animals↗
PubMed2001

Production of melanocyte-specific antibodies to human melanosomal proteins: expression patterns in normal human skin and in cutaneous pigmented lesions.

Multiple factors affect skin pigmentation, including those that regulate melanocyte and/or keratinocyte function. Such factors, particularly those that operate at the level of the melanosome, are relatively well characterized in mice, but the expression and function of structural and enzymatic proteins in melanocytes in human skin are not as well known. Some years ago, we generated peptide-specific antibodies to murine melanosomal proteins that proved to be instrumental in elucidating melanocyte development and differentiation in mice, but cross-reactivity of those antibodies with the corresponding human proteins often was weak or absent. In an effort to characterize the roles of melanosomal proteins in human skin pigmentation, and to understand the underlying mechanism(s) of abnormal skin pigmentation, we have now generated polyclonal antibodies against the human melanocyte-specific markers, tyrosinase, tyrosinase-related protein (TYRP1), Dopachrome tautomerase (DCT) and Pmel17 (SILV, also known as GP100). We used these antibodies to determine the distribution and function of melanosomal proteins in normal human skin (adult and newborn) and in various cutaneous pigmented lesions, such as intradermal nevi, lentigo simplex, solar lentigines and malignant melanomas. We also examined cytokeratin expression in these same samples to assess keratinocyte distribution and function. Immunohistochemical staining reveals distinct patterns of melanocyte distribution and function in normal skin and in various types of cutaneous pigmented lesions. Those differences in the expression patterns of melanocyte markers provide important clues to the roles of melanocytes in normal and in disrupted skin pigmentation.

Adult↗
PubMed2001

The melanocortin 1 receptor is the principal mediator of the effects of agouti signaling protein on mammalian melanocytes.

The agouti gene codes for agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length agouti signaling protein has shown that it competes with (&agr;)-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E(+)/E(+), e/e, or E(so)/E(so) mice to (alpha)-MSH and/or ASP. E(+)/E(+) melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and E(so)/E(so) is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to (alpha)-melanocyte stimulating hormone. Mouse E(+)/E(+) melanocytes, but not e/e or E(so)/E(so) melanocytes, respond to agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E(+)/E(+) melanocytes does agouti signaling protein abrogate the stimulatory effects of (alpha)-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to agouti signaling protein.

Agouti Signaling Protein↗
PubMed1998

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.

Cyclic AMP↗
PubMed1998

Endothelin-1 is a paracrine growth factor that modulates melanogenesis of human melanocytes and participates in their responses to ultraviolet radiation.

Endothelin (ET)-1, alpha-melanocyte stimulating hormone (alpha-melanotropin; alpha-MSH), and basic fibroblast growth factor (bFGF) are keratinocyte-derived factors that interact synergistically to stimulate human melanocyte proliferation. ET-1 has a dose-dependent mitogenic effect on human melanocytes and a biphasic effect on melanogenesis: a stimulatory effect at subnanomolar concentrations, and an inhibitory effect at concentrations equal to or higher than 1 nM. Human melanocytes express ET B receptors. Brief treatment of melanocytes with ET-1 caused up-regulation of alpha-MSH receptor mRNA but did not alter ET B receptor mRNA level. ET-1 modulates the response of human melanocytes to UV rays (UVRs). Treatment of melanocytes with 10 nM ET-1 immediately after exposure to UVRs enabled them to overcome the G1 growth arrest. However, ET-1 did not inhibit p53 accumulation or p21(Waf-1/SDI-1/Cip-1) overexpression, nor did it reverse the hypophosphorylated state of pRb or the reduction in Bcl2 level in irradiated melanocytes. These results substantiate the role of ET-1 as a paracrine regulator that modulates the response of human melanocytes to UVRs.

Cell Division↗
PubMed1997

Agouti signaling protein inhibits melanogenesis and the response of human melanocytes to alpha-melanotropin.

In mouse follicular melanocytes, the switch between eumelanin and pheomelanin synthesis is regulated by the extension locus, which encodes the melanocortin-1 receptor (MC1R) and the agouti locus, which encodes a novel paracrine-signaling molecule that inhibits binding of melanocortins to the MC1R. Human melanocytes express the MC1R and respond to melanotropins with increased proliferation and eumelanogenesis, but a potential role for the human homolog of agouti-signaling protein, ASIP, in human pigmentation has not been investigated. Here we report that ASIP blocked the binding of alpha-melanocyte-stimulating hormone (alpha-MSH) to the MC1R and inhibited the effects of alpha-MSH on human melanocytes. Treatment of human melanocytes with 1 nM-10 nM recombinant mouse or human ASIP blocked the stimulatory effects of alpha-MSH on cAMP accumulation, tyrosinase activity, and cell proliferation. In the absence of exogenous alpha-MSH, ASIP inhibited basal levels of tyrosinase activity and cell proliferation and reduced the level of immunoreactive tyrosinase-related protein-1 (TRP-1) without significantly altering the level of immunoreactive tyrosinase. In addition, ASIP blocked the stimulatory effects of forskolin or dibutyryl cAMP, agents that act downstream from the MC1R, on tyrosinase activity and cell proliferation. These results demonstrate that the functional relationship between the agouti and MC1R gene products is similar in mice and humans and suggest a potential physiologic role for ASIP in regulation of human pigmentation.

Agouti Signaling Protein↗
PubMed1996

Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis.

alpha-Melanocyte stimulating hormone (alpha-MSH) and ACTH increase the proliferation and melanogenesis of cultured human melanocytes. To further analyze how melanotropins produce these biological effects, we investigated the regulation of the melanocortin receptor MC1R expression by alpha-MSH and ACTH using Northern blot analysis and determine the relative affinity of the receptor for the structurally similar peptides alpha-MSH, ACTH, beta-MSH, and gamma-MSH. We also determined the relative potencies of these hormones to stimulate cAMP formation, tyrosinase activity, and melanocyte proliferation. The order of affinity and potency of the noted melanotropins in these assays were alpha-MSH = ACTH > beta-MSH > gamma-MSH. Because the binding affinity of each of these melanotropins for the MC1R correlated with its ability to stimulate human melanocyte proliferation and melanogenesis, we conclude that these effects are mediated specifically by binding to and activation of the MC1R. gamma-MSH stimulated cAMP formation without affecting proliferation or melanogenesis. However, we found that relative to alpha-MSH, the effect of gamma-MSH on cAMP formation was transient. Our results suggest that alpha-MSH, ACTH, and possibly beta-MSH, but not gamma-MSH, are capable of a physiological role in regulating human pigmentation, and that melanocytes in human skin are a specific target for these hormones.

Adrenocorticotropic Hormone↗
PubMed1995

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.

Cell Cycle↗
PubMed1995

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.

Cells, Cultured↗
PubMed1995

Long-term proliferation of human melanocytes is supported by the physiologic mitogens alpha-melanotropin, endothelin-1, and basic fibroblast growth factor.

We have successfully established normal neonatal and adult human melanocyte cultures in a growth medium containing the physiologic mitogens basic fibroblast growth factor (bFGF; 0.6 ng/ml), endothelin-1 (endo-1; 10 nM), and alpha-melanocyte stimulating hormone (alpha-MSH; 10 nM). The latter two factors replaced the commonly used mitogens 12-O-tetradecanoylphorbol 13-acetate (TPA) and bovine pituitary extract (BPE), respectively. Basic FGF alone maintained the viability but did not induce the proliferation of melanocytes. The addition of endo-1 to the bFGF-containing medium resulted in reduction of tyrosinase activity without enhancement of proliferation. However, the addition of alpha-MSH to the bFGF-containing medium potentiated melanocyte proliferation and tyrosinase activity. The concomitant addition of endo-1, alpha-MSH, and bFGF significantly increased the entry of melanocytes into S phase and potentiated their proliferation. Melanocytes maintained under these conditions had the same tyrosinase activity as those maintained in a medium containing alpha-MSH and bFGF. The signal transduction pathway induced by either endo-1 or bFGF, but not alpha-MSH, includes the activation of the mitogen-activated (MAP) kinase pathway. The addition of both endo-1 and bFGF had more than an additive effect on the MAP kinase extracellular signal-regulated kinase 2 (ERK2). This effect was further increased by the addition of alpha-MSH to these two growth factors. In summary, we have devised a growth medium for human melanocytes based on the use of physiologic mitogens that substituted for routinely used artificial and undefined growth factors. The resulting cultures should be desirable for clinical uses and permissive for the expression of in vivo relevant responses to regulatory factors.

Adult↗
PubMed1994

Synthesis of interleukin-1 alpha and beta by normal human melanocytes.

Normal human melanocytes and melanoma cells have been reported to produce several cytokines. Previously we demonstrated that neonatal human melanocyte proliferation and tyrosinase activity are inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, and interleukin-6. We have now also shown that interleukin-1 beta induces an inhibiting effect on neonatal melanocyte tyrosinase activity with little effect on melanocyte proliferation. We investigated the ability of neonatal and adult human melanocytes to synthesize interleukin-1 alpha and beta. By immunocytochemistry, using monoclonal antibodies against interleukin-1 alpha and beta, we observed that neonatal and adult melanocytes stain positively for both cytokines. Flow-cytometric analysis revealed that the percentage of melanocytes positive for interleukin-1 alpha was always greater than that for interleukin-1 beta. The ability of neonatal and adult melanocytes to synthesize interleukin-1 alpha and beta was further confirmed using the polymerase chain reaction. These results clearly indicate that human melanocytes synthesize interleukin-1 alpha and beta, and that these cytokines may function as autocrine and/or paracrine regulators of cells in the epidermis.

Adult↗
PubMed1994

Proliferation and propagation of human melanocytes in vitro are affected by donor age and anatomical site.

We report the effects of two factors, donor age and anatomical site, on the proliferation and melanization of human melanocytes (MC) derived from (1) neonatal foreskins, (2) adult foreskins, or (3) adult breast or arm skin. Two different growth media have been used for this purpose. Medium I supports the long-term proliferation of neonatal MC, and medium II supports the growth of both neonatal and adult MC. We found that neonatal foreskin MC proliferated equally well in both media for more than 12 passages. However, adult MC behaved differently in the two growth media. In very early passages (passages 1-5), all three types of MC proliferated well and reached comparable final cell densities in medium I, but adult foreskin MC proliferated at a higher rate than neonatal or adult non-foreskin MC in medium II. The non-foreskin adult MC had the least proliferative rate in medium II. Unlike neonatal MC, both adult foreskin and non-foreskin MC underwent a drastic reduction in their proliferative rate after only a few (9-10) passages in culture. While the three types of MC differed in their proliferative rates, they responded similarly to melanogenic stimulation by cholera toxin (CT) and isobutyl methylxanthine (IBMX). These results demonstrate that the proliferation of human MC in standard culture media is affected by the donor age and the anatomical site from which these cells are derived. We conclude that human MC are heterogeneous, and caution must be used in extrapolating the results of studies on MC derived from different anatomical sites or age groups.

Adult↗
PubMed1991

Endothelin stimulates chloride secretion across canine tracheal epithelium.

The endothelins (ET) are a group of isopeptides produced by a number of cells, including canine tracheal epithelial cells. Because these compounds are endogenous peptides that may activate eicosanoid metabolism, we investigated the effects of ET on Cl secretion in canine tracheal epithelium. Endothelin 1 (ET-1) was found to produce a dose-dependent change in short-circuit current (Isc) that increased slowly and reached a maximal value within 10-15 min. When isopeptides of ET were compared, 300 nM ET-1 and ET-2 produced comparable maximal increases in Isc, whereas ET-3 produced smaller changes in Isc (half-maximal concentrations of 2.2, 7.2, and 10.4 nM, respectively). Ionic substitution of Cl with nontransported anions, iodide and gluconate, reduced ET-1-induced changes in Isc. Furthermore, the response was inhibited by the NaCl cotransport inhibitor, furosemide. In paired tissues, ET-1 significantly increased mucosal net 36Cl flux without significant effect on 22Na flux. The increase in Isc induced by ET was diminished by pretreatment with indomethacin. The second messengers mediating the increase in Isc were investigated in cultured canine tracheal epithelial cells. ET-1 stimulated the release of [3H]arachidonate from membrane phospholipids, increased intracellular Ca2+ (occasionally producing oscillations), and increased adenosine 3',5'-cyclic monophosphate accumulation. The latter was diminished by indomethacin. Thus ET is a potent agonist of Cl secretion (with the isopeptides having the following potency: ET-1 greater than or equal to ET-2 greater than ET-3) and acts, in part, through a cyclooxygenase-dependent mechanism.

Animals↗
PubMed1989

A new role for epidermal cell-derived thymocyte activating factor/IL-1 as an antagonist for distinct epidermal cell function.

It is known that many immunologic responses to IL-1 are antagonized by the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH). This led us to investigate the possible reciprocal effects of IL-1 and the functionally related epidermal cytokines, epidermal cell-derived thymocyte activating factor (ETAF) and IL-6, on the melanogenic effect of alpha-MSH on murine Cloudman melanoma cells. When these cells were treated with ETAF in combination with alpha-MSH or its potent analog [Nle4,D-Phe7]-alpha-MSH, the melanotropin induced increase in tyrosinase activity, and thus melanin synthesis, was abrogated. This inhibitory effect of ETAF was not mediated by competitive binding to the melanotropin receptor, because ETAF also blocked the melanogenic response of melanoma cells to isobutyl methylxanthine (IBMX) and to PGE1 and PGE2. ETAF had no effect on cellular proliferation. Inhibition of the stimulated tyrosinase activity by ETAF was not due to diminished cAMP synthesis or increased cAMP degradation. Cells treated concomitantly with ETAF and alpha-MSH, IBMX, or PGE1 had the same cAMP levels as cells treated with alpha-MSH, IBMX, or PGE1 alone. In contrast to ETAF, human rIL-1 alpha or IL-1 beta alone or in combination did not have an inhibitory effect on melanogenesis. IL-6 significantly inhibited the basal level of tyrosinase and partially abrogated the alpha-MSH-induced tyrosinase activity. IL-6 also stimulated cellular proliferation when added alone or in combination with alpha-MSH. Granulocyte-macrophage colony stimulating factor (GM-CSF) did not alter either the tyrosinase activity or cellular replication at the concentrations tested. IL-1 alpha, GM-CSF, and IL-6 or IL-1 alpha and GM-CSF added together did not significantly affect the MSH-induced tyrosinase activity. These results ascribe a new potential function for ETAF and IL-6 as modulators of the melanogenic response of pigment cells.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Stimulation of Cloudman melanoma tyrosinase activity occurs predominantly in G2 phase of the cell cycle.

A widely accepted notion is that an increasing cellular cyclic AMP (cAMP) concentration is prerequisite for increasing tyrosinase activity and melanin synthesis and for regulating proliferation of pigment cells. alpha-Melanocyte stimulating hormone (alpha-MSH) increases cAMP and tyrosinase activity in Cloudman melanoma cells. Prostaglandins (PGs) E1 and E2 increase melanoma cell tyrosinase activity and inhibit proliferation. Both PGs, but not alpha-MSH, block the progression of Cloudman melanoma cells from G2 phase of the cell cycle into M or G1. Only PGE1 and not PGE2 causes an elevation of cellular cAMP concentrations. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) at 5 x 10(-4) M effectively blocks the increased cAMP synthesis by cells treated with 10 micrograms/ml PGE1. The addition of DDA, however, enhances the melanogenic response of melanoma cells to 10 micrograms/ml PGE1 or PGE2, 10(-7) M alpha-MSH, 10(-4) M isobutylmethylxanthine, 10(-4) M dibutyryl cyclic AMP. DDA also augments the effects of PGE1 or PGE2 on the melanoma cell cycle. Moreover, when DDA is added concomitantly with alpha-MSH, more cells are recruited into G2 than observed in untreated controls. Neither alpha-MSH nor DDA alone has any effect on the cell cycle. These findings undermine the role of cAMP in the melanogenic process and suggest that blocking melanoma cells in G2 may be required for the remarkable stimulation of tyrosinase activity observed with PGE1 or PGE2 alone or in combination with DDA. The observed block in G2 may be essential for the synthesis of sufficient mRNA, which is required for stimulation of tyrosinase activity.

1-Methyl-3-isobutylxanthine↗