Dissection of RAS downstream pathways in melanomagenesis: a role for Ral in transformation.

Cutaneous malignant melanoma is considered one of the most deadly human cancers, based on both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wild-type NRAS. NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here, we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine exchange factor (RalGEF), using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we discovered that constitutive RalGEF activation had a major impact on several malignant phenotypes, particularly anchorage-independent growth, indicating that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target.

The gene mutated in cocoa mice, carrying a defect of organelle biogenesis, is a homologue of the human Hermansky-Pudlak syndrome-3 gene.

Hermansky-Pudlak syndrome (HPS) is a group of human disorders of organelle biogenesis characterized by defective synthesis of melanosomes, lysosomes, and platelet dense granules. In the mouse, at least 15 loci are associated with mutant phenotypes similar to human HPS. We have identified the gene mutated in cocoa (coa) mice, which is associated with an HPS-like mutant phenotype and thus represents a strong candidate for human HPS. Analysis of coa-mutant mice and cultured coa-mutant mouse melanocytes indicates that the normal coa gene product is involved in early stages of melanosome biogenesis and maturation.

Agouti signaling protein and other factors modulating differentiation and proliferation of immortal melanoblasts.

The melanocyte lineage potentially forms an attractive model system for studies in cell differentiation, developmental genetics, cell signaling, and melanoma, because differentiated cells produce the visible pigment melanin. Immortal lines of murine melanoblasts (melanocyte precursors) have been described previously, but induction of differentiation involved a complex culture system with keratinocyte feeder cells. Here we describe conditions for both growth and induced differentiation of the melanoblast line melb-a, without feeder cells, and analyze factors that directly control proliferation and differentiation of these pure melanoblasts. Several active factors are products of developmental and other coat color genes, including stem cell factor (SCF), melanocyte-stimulating hormone (alphaMSH), and agouti signaling protein (ASP), a natural antagonist at the MSH receptor (melanocortin 1 receptor, MC1R) encoded by the agouti gene. A stable analog of alphaMSH (NDP-MSH) stimulated differentiation and inhibited growth. ASP in excess inhibited both effects of NDP-MSH, that is, ASP could inhibit pigmentation and stimulate growth. These effects provide an explanation for the interactions in mice of melanocyte developmental mutations with yellow agouti and Mc1r alleles, and a role for embryonic expression patterns of ASP.

Cell volume measurement using scanning ion conductance microscopy.

We report a novel scanning ion conductance microscopy (SICM) technique for assessing the volume of living cells, which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The technique can measure a wide range of volumes from 10(-19) to 10(-9) liter. The cell volume, as well as the volume of small cellular structures such as lamelopodia, dendrites, processes, or microvilli, can be measured with the 2.5 x 10(-20) liter resolution. The sample does not require any preliminary preparation before cell volume measurement. Both cell volume and surface characteristics can be simultaneously and continuously assessed during relatively long experiments. The SICM method can also be used for rapid estimation of the changes in cell volume. These are important when monitoring the cell responses to different physiological stimuli.

Impaired growth and differentiation of diploid but not immortal melanoblasts from endothelin receptor B mutant (piebald) mice.

Endothelin 3 (Edn3) and its preferred receptor, endothelin receptor B (Ednrb), are implicated in development, especially that of two neural-crest-derived cell lineages: melanocytes and enteric ganglion cells. Mice and humans with a null mutation at either locus can show major deficiencies in both cell types: congenital white spotting and aganglionic megacolon (Hirschsprung disease in human). Numbers of early (migrating) embryonic melanoblasts are low in Ednrb(ls) mutant mice, while added Edn3 appears to promote the growth of melanocyte precursors in neural crest cultures. However, it is hard to assess cell differentiation in these mixed cultures, and it is not known whether Ednrb has any role in the postnatal melanocytic lineage. We have therefore studied primary cultures of neonatal melanoblasts homozygous for the piebald (Ednrb(s)) mutation. These mutant melanoblasts showed severe impairment of both net cell growth and differentiation compared to wild-type melanoblasts. They were also unresponsive to stimulation of growth by cholera toxin. We have established three immortal lines of melanoblasts and one of melanocytes homozygous for Ednrb(s). These immortal lines, however, had no detectable deficiency of growth or differentiation as judged by cell counts, induced pigmentation and immunocytochemistry for melanocytic markers. Consistent with this, neither Ednrb nor Edn3 mRNA was detected in 3/3 tested immortal lines of mouse melanoblasts and 5/5 lines of melanocytes, of various genotypes. We also report for the first time a method to grow immortal melanoblasts in pure culture, without feeder cells.

Normal tyrosine transport and abnormal tyrosinase routing in pink-eyed dilution melanocytes.

The pink-eyed dilution phenotype in mice arises from mutations in the p gene; in humans, analogous mutations in the P gene result in oculocutaneous albinism type 2. Although the molecular mechanisms which underlie this phenotype remain obscure, it has been postulated that mutations in p result in defective tyrosine transport into murine melanosomes, resulting in hypopigmentation and diminished coat color. However, we previously reported no difference in melanosomal tyrosine transport in unpigmented, melanoblast-like pink-eyed dilution (pcp/pcp), and in pigmented (melan-a) murine melanocytes. In this study, we utilized melan-p1 cells, more differentiated pink-eyed dilution (pcp/p25H) melanocytes which can be induced to produce melanin, to characterize the melanogenic lesion(s) more definitively. Uptake of [3H]tyrosine into melan-a melanosomes did not differ significantly from uptake into melanosomes derived from melan-p1 melanocytes, further arguing against its critical role as a tyrosine transporter. Pink-eyed dilution melanocytes incubated in high tyrosine concentrations became extremely pigmented as they became confluent and secreted large amounts of black material into the medium. Total cellular tyrosinase activity in melan-p1 melanocytes was significantly higher than that in melan-a melanocytes (which are wild-type at the p locus), but the localization of tyrosinase to melanosomes was impaired in melan-p1 melanocytes compared to melan-a melanocytes. These results indicate that mechanisms other than deficient tyrosine transport are involved in the pink-eyed dilution phenotype and that this protein may serve a chaperone-like or stabilizing function in melanocytes.

Involvement of microphthalmia in the inhibition of melanocyte lineage differentiation and of melanogenesis by agouti signal protein.

In mouse follicular melanocytes, production of eumelanins (brown-black pigments) and pheomelanins (yellow-brownish pigments) is under the control of two intercellular signaling molecules that exert opposite actions, alpha-melanocyte-stimulating hormone (alphaMSH) which preferentially increases the synthesis of eumelanins, and agouti signal protein (ASP) whose expression favors the production of hair containing pheomelanins. In this study, we report that ASP does not only affect mature melanocytes but can also inhibit the differentiation of melanoblasts. We show that both alphaMSH and forskolin promote the differentiation of murine melanoblasts into mature melanocytes and that ASP inhibits this process. We present evidence that the expression of a specific melanogenic transcription factor, microphthalmia, and its binding to an M box regulatory element, is inhibited by ASP. We also show that, in B16 murine melanoma cells, ASP inhibits alphaMSH-stimulated expression of tyrosinase, tyrosine-related proteins 1 and 2 through an inhibition of the transcription activity of their respective promoters. Further, ASP inhibits alphaMSH-induced expression of the microphthalmia gene and reduces the level of microphthalmia in the cells. Our data demonstrate that ASP can regulate both melanoblast differentiation and melanogenesis, pointing out the key role of microphthalmia in the control of these processes.

Melanosomal defects in melanocytes from mice lacking expression of the pink-eyed dilution gene: correction by culture in the presence of excess tyrosine.

Mutations in the murine pink-eyed dilution (p) gene, or its human homologue P, result in oculocutaneous albinism. Melanocytes cultured from mice lacking p gene expression exhibit defective melanogenesis, but following culture in the presence of high concentrations of L-tyrosine, increased melanin deposition is observed. Electron microscopy and image analysis demonstrated that untreated p mutant melanocytes exhibited small melanosomes, largely of stages I-II. Following tyrosine treatment, increased proportions of stage III-IV melanosomes, almost normal in size, were observed. Levels of tyrosinase protein and to a lesser extent of tyrosinase-related protein-1 (TRP-1) were subnormal but rose dramatically following stimulation by tyrosine. Levels of TRP-2 and Pmel17/silver gene product were not altered, nor were the levels of mRNA for tyrosinase, TRP-1, TRP-2, or the Pmel17/silver gene product. As expected, the 110-kDa product of the p gene was absent from both stimulated and unstimulated p mutant cells. In a melanoblast line derived from the same mice, excess tyrosine failed to stimulate visible melanogenesis or increase the low levels of tyrosinase. The melanosomes in these cells were smaller still than those in the mutant melanocytes even when cultured in the presence of excess tyrosine. Thus, absence of the p gene product affects melanosomal structure and protein composition at the posttranscriptional level. These defects are correctable at least in part by supplementation with L-tyrosine.

The murine misty mutation: phenotypic effects on melanocytes, platelets and brown fat.

Although the recessive murine mutation misty (m) is well known, its phenotype has never been reported beyond brief descriptions of a dilution of coat color and white spotting of the belly and extremities, suggesting a developmental mutation. A report in abstract has also suggested effects on white fat and body weight. Here, we report effects of the homozygous misty mutation on an unusual combination of three cell types: melanocytes, platelets, and brown fat. Brown fat appeared to be completely absent from all expected locations in neonatal m/m mice. A prolonged bleeding time was observed; platelet count and platelet serotonin and ATP levels were normal, but the level of ADP in m/m platelets was low. Primary cultures and immortal lines of melanocytes from m/m mice showed several abnormalities. There was a marked deficiency in net proliferation, suggesting that the color dilution and spotting in vivo may result from reduced numbers of melanocytes and their precursors. m/m melanocytes were also hyperdendritic in morphology, overproduced melanin, and had deficient responses to the cAMP agonists cholera toxin and melanocyte-stimulating hormone, which normally promote melanin production. The misty gene product may be involved in adenine nucleotide metabolism or signaling.

Specialized scanning ion-conductance microscope for imaging of living cells.

A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch-clamp apparatus, which uses a glass micropipette as the sensitive probe. In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary. We have successfully imaged murine melanocytes in growth medium. The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three-dimensional relief of the cells. This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real-time microscopy, electrophysiology, micromanipulation and drug delivery.

Complementation of hypopigmentation in p-mutant (pink-eyed dilution) mouse melanocytes by normal human P cDNA, and defective complementation by OCA2 mutant sequences.

Mutations in the P gene of humans and the homologous p-locus of mice, respectively, result in the homologous disorders oculocutaneous albinism type 2 (OCA2) and pink-eyed dilution. Although clearly required for melanin biosynthesis, the specific function of the P gene product, a melanosomal transmembrane protein expressed in melanocytes of the skin, hair, and eyes, is not yet known. Here we describe lines of immortal melanocytes and melanoblasts from mice of the null genotype p(cp)/p(25H). These p-null melanocytes were severely hypopigmented, although they and the melanoblasts expressed mRNAs for a number of melanosomal proteins. Proliferation of the p-null melanoblasts was normal. Both diploid and immortal p-null melanocytes grew more slowly than wild-type melanocytes, however, and were unusually susceptible to the antibiotic G418; these abnormalities were corrected by culture in high concentrations of L-tyrosine. Transfection of the p-null melanocytes with full-length normal human P cDNA resulted in complementation of deficient melanin biosynthesis and hypopigmentation. In contrast, transfection with mutant human P cDNAs containing amino acid substitutions (A481T, V443I) found in patients with OCA2 resulted in minimal or partial correction, consistent with the corresponding pigmentation phenotypes in patients with these mutations. These results demonstrate the utility of this model system for distinguishing true OCA2 mutations from nonpathologic polymorphisms and for quantitating the effect of these mutations on P function.

Cloning of the murine homolog of the ocular albinism type 1 (OA1) gene: sequence, genomic structure, and expression analysis in pigment cells.

We report the isolation of the mouse homolog of OA1, the gene responsible for ocular albinism type 1. The mouse Oa1 gene encodes a putative protein of 405 amino acids displaying a high level of homology (78% identity, 87% similarity) to the human gene. All disease-associated missense mutations reported in patients with ocular albinism involve conserved amino acid residues in the mouse protein. Moreover, the murine homolog shows six putative transmembrane domains, as observed for the human gene, indicating that the overall structure of the two proteins is conserved. The genomic organization is also conserved between the two species across the entire coding region with splice sites located in the same positions. Like its human counterpart, the expression pattern of Oa1, apart from the eye, is restricted to the epidermal melanocyte lineage. A transcript of approximately 1.8 kb was readily detected by this probe in 5 out of 5 murine melanocyte lines, 4 out of 4 murine melanoblast lines, 1 out of 2 murine melanoma lines, and 1 out of 2 human melanoma lines tested, but it was not detected in 2 out of 2 lines of a developmentally earlier normal cell type, melanoblast precursor cells, suggesting that the gene is transcriptionally activated in epidermal melanocytes at the same stage as most other tested melanosomal proteins. Together, these data suggest that the function of the OA1 gene is conserved between human and mouse and point to the mouse as a model to facilitate the understanding of ocular albinism pathogenesis.

Cellular stress causes accumulation of the glucose transporter at the surface of cells independently of their insulin sensitivity.

The stimulation of glucose transport in response to various types of stress has been studied. There is no relationship between effects of stress-inducing agents on glucose transport and their effects on cellular protein synthesis. Although the effect of stress on glucose transport appears analogous to its stimulation by insulin, cells that are slightly insulin-sensitive in terms of glucose transport (BHK cells) show a similar degree of stimulation as highly insulin-sensitive cells (differentiated 3T3-L1 cells). External labeling of the transporter protein with a photoactivatable derivative of mannose, 2-N-4-(1-azi-2,2,2-trifluoroethyl) benzoyl-1, 3-bis-(D-mannos-4-yloxy)-propylamine, shows that most of the increased glucose transport activity correlates with an increase in the amount of the transporter on the cell surface. Cells subjected to K(+)-depletion, which inhibits endocytosis and results in an accumulation of receptors at the cell surface, show the same increase in glucose transport as cells exposed to stress; stressed cells show no further increase in glucose transport when subjected to K+ depletion. These results support the view (Widnell, C.C., Baldwin, S.A., Davies, A., Martin, S., Pasternak, C.A. 1990. FASEB J 4:1634-1637) that cellular stress increases glucose transport by promoting the accumulation of glucose transporter molecules at the cell surface.

A cloned, immortal line of murine melanoblasts inducible to differentiate to melanocytes.

Cultures of differentiated melanocytes can readily be grown from the dissociated epidermis of neonatal mice, and immortal cell lines often develop from these. However, the first cells that grow and transiently dominate the cultures, while similar to melanocytes, are unpigmented. These have been shown to be precursors of melanocytes and may be termed melanoblasts. Under our previous standard culture conditions, involving the use of keratinocyte feeder cells, foetal calf serum, the phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA) and cholera toxin, all the melanoblasts spontaneously differentiated to pigmented melanocytes within about 3 weeks. We now describe some factors affecting the proliferation and differentiation of diploid murine melanoblasts in the presence of serum. Murine stem cell factor/steel factor (SCF), basic fibroblast growth factor (bFGF) and murine leukaemia inhibitory factor/differentiation-inhibiting activity (LIF/DIA) all increased melanoblast numbers. SCF and LIF also slightly inhibited melanoblast differentiation, while cholera toxin and TPA promoted differentiation. Using some of these findings, and by regular replacement of keratinocyte or fibroblastoid feeder cells, we have established a clonal line of immortal murine melanoblasts, 'melb-a'. These cells express tyrosinase-related protein-2 but not, in general, tyrosinase. They can be induced to differentiate irreversibly to functional melanocytes (also proliferative and immortal) by plating in the absence of feeder cells. Thus a new immortal melanocyte line, 'melan-a2', has also been produced.

Staphylococcus aureus alpha-toxin-induced pores: channel-like behavior in lipid bilayers and patch clamped cells.

The conductance of pores induced by Staphylococcus aureus alpha-toxin in Lettre cells has been compared to that in bilayers composed of synthetic lipids or Lettre cell membrane constituents. Previously described characteristics of toxin-induced conductance changes in lipid bilayers, namely rectification, voltage-dependent closure, and closure at low pH or in the presence of divalent cations (Menestrina, 1986) are displayed also in bilayers prepared from Lettre cell membranes and in patch clamped Lettre cells. It is concluded that endogenous proteins do not affect the properties of alpha-toxin-induced channels significantly and that the relative lack of ion channels in Lettre cells makes them ideal for studies of pore-forming toxins by the patch clamp technique.