VII^th Annual Meeting of the PASPCR

June 15th - 18th, 1997 The Westin Hotel Providence, RI, USA

Overview / Summary of Meeting - Walter Quevedo

On behalf of Hal Swartz and myself, I would like to congratulate the 129 scientists that participated in the 7th Meeting of the PanAmerican Society for Pigment Cell Research and the fine Providence weather for making it a good one. The clambake organized by Tom Holstein and the Reception and Banquet arranged by Fae Best Carletti were standout features of the social program. The scientific sessions were well attended throughout and discussions following presentations were of high quality. One had the feeling that pigment cell research is at the cutting edge of science not only in pursuing leads provided by workers in other fields but also in compensating such workers with new investigative directions that they might follow. Hal's concept of the sunrise sessions was vindicated by the consistently high attendance and unabating enthusiastic exchanges between "teachers" and "students" during classes that ended all too soon. There were moments of pride and sentimentality such as those accompanying Vince Hearing's Gelb Award, Joe Bagnara's Career Investigator Award, and Aaron Lerner's and Tom Fitzpatrick's acceptance of the Meeting Chairmen's Award for Outstanding Contributions to Pigment Cell Research and its Pursuit in New England. It was an honor for Providence to host the 7th Meeting.

This was an exciting symposium that elicited many questions and comments. Two major types of papers were presented: strategies related to the underlying basis for immunotherapy in human melanoma and characterization of molecular changes in certain genes related to pigmentation.

The symposium opened with a useful historical overview of the field of biologic and immunologic approaches to the management of melanoma (H Wanebo). To date, almost all approaches to melanoma management have produced no or modest benefit in the clinical situation, although the studies presented by D Shrayer and colleagues suggested that in an animal model of human disease that both primary melanoma tumor growth and lung metastases could be prevented. The emphasis of subsequent speakers was on the necessity of characterizing the specific features of immunologic alterations. Precise identification of human melanoma antigens by tumor infiltrating T lymphocytes (Y Kawakami), the immune response to melanosomal differentiation antigen induced by altered antigen (S Bartido). The new human melanocyte specific gene factor MSG1 was presented in two papers; T Shioda showed that the factor was a nuclear protein, and was induced by phorbol ester and was transcriptionally regulated. M Fenner provided further information that MSG1 promoter activity was correlated with pigmentation.

In two studies from Yale, it was shown that the regulation of FGF-2, a requirement for melanocyte proliferation, was regulated by c-Myb proto-oncogene (M Miglarese). In a second paper, R Halaban and her colleagues showed that when tyrosinase was inadvertently retained in the endoplasmic reticulum, its degradation was accelerated, contributing to the amelanotic phenotype, and suggesting perhaps that this property is phenotypical rather than from direct genetic alteration. Finally, a paper fitting neither of the two major areas, but dealing with an important, relatively neglected, aspect was presented (F Meyskens). NKFB and AP-1 were shown to be regulated differently in melanocytes and melanoma cells and the response in terms of redox suppression between the two cell types was markedly different. Overall, this symposium offered considerable anticipation that new approaches to the management of melanoma may evolved from these and other basic advances in understanding described throughout the conference.

Responses of Skin to Damaging Agents - Raymond Boissy

This symposium began with the keynote address presented by James H Wyche entitled "A model system for studying cell death regulation". Dr Wyche presented a review of molecular events occurring in the initial stages of apoptosis prior to DNA damage by describing the molecules modulating susceptibility to apoptosis (i.e., bax, bcl2, etc.) and the various caspases involved in proteolysis during initiation of apoptosis. He highlighted this review by describing his research on staurosporine treated promyelocytic leukemia cells which exhibited activation of caspase-3 causing the proteolysis of the DNA-dependent protein kinase which then correlated with the initiation of apoptotic chromosomal DNA degradation. Zalfa Abdel-Malek then presented "Elucidation o f the signaling pathway which mediates the responses of human melanocytes to UVB light" in which she demonstrated that cultured human melanocytes exposed to UVB undergo proliferation arrest and can progress to apoptosis after overexpression of p53. In addition, cAMP inducers like MSH can accentuate the increased melanization response of cultured melanocytes to UVB and promote movement of cells to the S phase of proliferation. Fan Yang next presented "The effects of tyrosinase activity and commonly used mitogens on the cytotoxicity of 4-tertiary butyl phenol (TBP) to human melanocytes" in which she demonstrated that the development of apoptosis in cultured human melanocytes after exposure to 4TBP was not influenced by the level of tyrosinase activity or melanin content. In contrast, the presence of either MSH or bFGF could accentuate cytotoxicity of 4TBP exposed melanocytes. William Pavan then presented "Met-HGF signaling is critical for melanocyte development: Implications for Waardenburg Syndrome type II" in which he demonstrated, using genetically engineered mice as either knockouts or overexpressers for the Met tyrosine kinase receptor or its ligand HGF/SF respectively, that this signaling pathway is crucial for continued development of ckit/steel positive neural crest cells towards melanocytes. E. Michelle Southard-Smith next presented "Physical mapping and embryological analysis of Dominant Megacolon, a mouse model of Hirschsprung's disease" in which she described the mapping of the Dom locus to a 0.01cM region proximal to D15Mit2 by linkage disequilibrium and physical mapping. In addition, expression *** tructure". S Orlow (NY) began the session with a discussion of the pink-eyed dilution gene product and its role in melanogenesis and melanosome formation. Melanocytes cultured from p-null mice possess smaller, more numerous melanosomes, and this is corrected in part by culture in high concentrations of exogenous tyrosine, as are alterations in levels of tyrosinase and some other melanosomal proteins. V Setaluri (Winston-Salem) discussed the sequences responsible for exit from the ER and trafficking to melanosomes of gp75 (aka TRP-1). By the use of site-directed mutagenesis, the information necessary has been shown to be present in the short cytosolic tail of gp75, and the presence of a dileucine motif is an important criterion for proper trafficking, though it may not be the only one. VJ Hearing (Bethesda) gave the Gelb Keynote Address in the context of this symposium. He summarized a large body of work from his own lab and others, focusing especially on the differences between pheomelanosomes and eumelanosomes. Levels of tyrosinase are decreased and levels of TRP-1, TRP-2, p and silver proteins are almost undetected in pheomelanic hair follicle extracts. Cultured melanocytes can now be treated with recombinant agouti signal protein, enabling the process of pheomelanosome formation in response to ASP to begin to be dissected in vitro. J Hammer (Bethesda) presented work from his laboratory on myosin V^a, the dilute locus gene product. This unconventional myosin can be shown to be present at the melanosomal surface by immunofluorescence, immuno-EM and by biochemical techniques. In cells from mice with dilute mutations, melanocytes extend dendrites, but melanosomes fail to traffic down these dendrites and remain in a perinuclear distribution, thus implicating myosin V^a in the transport of melanosomes as a prelude to their transfer. J Bhawan (Boston) discussed various theories regarding the transfer of melanosomes from melanocyte to keratinocyte. Careful EM studies provide evidence in support of not one, but rather two or three modes of transfer that may be operative in sun-exposed skin.

P Samaraweera (Orlow lab) described a 65 kDa protein which localizes to the cytosolic face of the melanosome, and unlike previously described melanosomal proteins is peripheral rather than integral to the membrane. It shows altered detergent solubility in melanocytes cultured from buff mice, which may have a melanosomal transport defect. K Sato (Orlow lab) used site directed mutagenesis and the construction of chimeric molecules to show that both TRP-1 and TRP-2 are able to stabilize tyrosinase protein in cotransfection experiments. The amino terminal portion of TRP-1 is critical to its ability to effect this stabilization. B Potterf (Hearing/Gahl labs) examined the transport of sulfhydryl compounds by melanosomes. Although glutathione transport seems not to be an issue, melanosomes show robust transport of cysteine. The role of such transport in the control of melanogenesis was discussed. Finally M K Cullen (St. Louis) examined tyrosine uptake by isolated melanosomes. Cytosolic factors appear to regulate this accumulation, and interestingly, extracts from melanocytes cultured from mice with "melanolysosomal" defects such as ruby exhibit an altered ability to enhance this tyrosine transport.

Role of Transcription Factors in Pigment Cell Biology - Seth J Orlow

A symposium on Ocular Melanocytes and RPE/ Transcription Factors in Pigment Cell Biology was chaired by Seth Orlow. Richard Sidman (Southborough) delivered the keynote address. He and his colleagues have examined the effects of two white-spotting mutations, namely mi^vit and W^sh, on RPE and choroidal melanocytes. Careful analysis of the RPE of mi^vit mutant mice reveals that this gene controls not only pigmentation but proliferation as well, with more numerous taller RPE cells, many of them hypopigmented, noted early on in the mutants. An unusual tigroid pattern can be discerned in ocular tissue from W^sh mice, with the additional unexpected observation that giant clumped melanosomes are present in pigment cells from this mutant, reminiscent of those in beige mice. Since W^sh mi^vit double mutant mice are indistinguishable from the mi^vit single mutants, Dr Sidman deduced that Kit may act downstream of MITF. Ongoing studies are geared towards determining how nuclear localization may play a role in controlling MITF function.

Sylvia Smith (Augusta) presented work on the expression of the TRP family in RPE/choroid of mi^vit mice. Levels of TRP-1 are dramatically reduced even at the earliest time points examined, and levels of tyrosinase diminished to a lesser extent, whereas levels of TRP-2 were unaffected, suggesting that MITF in vivo plays its most critical role in TRP-1 gene expression regulation. A Zervos (Charlestown) and his group have identified an MITF-interacting protein using the yeast 2-hybrid system. This protein is the human homolog of RKR2, which contains both multiple zinc finger repeats and basic regions. The protein interacts with and inhibits the transcriptional regulatory function of MITF.

Rivka Rachel (NY) discussed her efforts to determine how the ability of RPE cells to make melanin affects the retinofugal projections during embryonic development. She has looked at both albino mice which lack all tyrosinase activity as well as those from mice homozygous and heterozygous for dark-eyed albinism (c^44H) which have low but demonstrable levels of tyrosinase activity to ask whether the pigment-producing effects are graded or there is a "gate" level needed below which normal development will not occur. Her results suggest that the reality may be a mixed-situation. Dan-Ning Hu (NY) and Ray Boissy (Cincinnati) each discussed their laboratories efforts to study melanogenesis and growth of cultured human ocular melanocytes. These cells can now be cultured from the uveal tract and will synthesize melanin and melanosomes in culture. Agents which upregulate cyclic AMP stimulate growth and melanogenesis by these cells, including via the bet adrenergic receptor. By contrast muscarinic agonists inhibit their growth. MSH receptors appear not to be expressed by ocular

melanocytes in vitro. Finally Brian Potterf (Hearing/Gahl labs) expanded on his previous work to show that melanosomal tyrosine transport is upregulated by pretreatment of cells with MSH or dbcAMP. Whether this regulation is at the translational or transcriptional levels is under investigation. Of a wide range of small molecules, those that compete with tyrosine best are L-phenylalanine, L-DOPA and L-leucine. The potential role of tyrosine transport in the regulation of melanogenesis and in the switch from eumelanin to pheomelanin was discussed.

Synopsis pending

Synopsis pending

In his introduction to the session, Joe Bagnara pointed out that in most life science disciplines, the word "comparative" is inferred immediately to mean cold-blooded vertebrates and, as such, are looked at as model systems by those who investigate mammals. He emphasized that this is a misconception and that lower and higher vertebrate pigmentation systems are related primarily by homology rather than by analogy. He enumerated some of these homologies and several of these facets were discussed in more detail by subsequent speakers. Thus, Ken Mason next discussed molecular, genetic, and evolutionary aspects of the pigmentation of the Mexican axolotl, a salamander species for which many mutants are available. Comparative analysis of these mutants was discussed in relation to relevant mammalian mutants. Mason indicated that "the cladistic analysis of the TRP-1 cDNA from the axolotl along with members of the tyrosinase gene family from a number of species, clarified the evolutionary relationships of these molecules." The molecular biology of other axolotl pigmentary enzymes was discussed. Several shorter presentations related to the development of pigmentation were presented later. Thus, Mark Reedy disclosed that, in the chick embryo, later chromatoblasts emigrating from the neural crest follow a dorsolateral route and differentiate into melanocytes. He suggested that specification of these neural crest cells as melanoblasts occurred in situ and dictated their later migration and their route. Mark Moody then discussed his work on developmentally regulated expression of TRP-1 in the axolotl. The axolotl was also used by Susan Holder who discussed developmental regulation of xanthophore differentiation by region-specific influences. Perhaps, one such region-specific factor is melanization inhibiting factor (MIF) which appears to function much as does the product of the agouti gene implicated in several aspects of mammalian pigmentation. Thus, J Newton presented his work on the cloning and molecular characterization of two genes, the agouti and extension, that are involved in the regulation of pheomelanogenesis in breeds of domestic dogs. The Smyth line chicken (SL), long considered a model for autoimmune human vitiligo, was discussed by G.P. Sreekumur who presented a penetrating analysis of the genetic and molecular linkage of the Smyth line chicken system. For his presentation, he was later honored with the award for the most outstanding presentation by a graduate student. The functional aspects of chromatophores were reviewed by Mac Hadley who emphasized the role of MSH and its receptor MC1R. The physiology of chromatophores was further discussed by Ana Maria Castrucci who considered the roles of a variety of neural and hormonal agents in the normal regulation of chromatophore function in fishes. A remarkable means of chromatophore control was considered by Mark Rollag who discussed photo-transduction in Xenopus melanophores. He reported that such light-sensitive melanophores contain a unique opsin that represents a class of opsins that became distinct at about the time that vertebrate and invertebrate opsins diverged during evolution.

This symposium was held on Tuesday, June 17th and was cochaired by Drs. Harold M Swartz and Helene Z Hill. Three of the papers dealt with effects of iron in one form or another on melanin. Jeffrey Tosk noted that the brain stem has one of the highest concentrations of ferritin in the body and this iron-rich substance may interact with catecholaminergic precursors to form melanin. This could account for the presence of melanin in this area of the brain. Dr. Eisner is also interested in neuromelanins and the structure around the Fe-site. Natural and synthetic neuromelanins were compared and showed small but significant differences. Dr. Jacobson's talk focused on the role of extracellular melanin of pathogenic yeast as a redox buffer the activity of which is enhanced by Fe++. Iron may provide the reducing equivalents needed in order for melanin to neutralize pro-oxidants generated by the immune system, thereby enhancing the pathogenesis of the yeast.

Determining the routes taken by various precursors to synthesize the various melanins is key to our understanding of the balance of pigments in the final products and ultimately to the understanding of pigment function. Dr. Ito showed in a most elegant fashion that phaeomelanin synthesis is favored in the presence of high cysteine and low melanogenic activity. Eumelanin will dominate when the opposite conditions exist. In the presence of TRP1, DHICA is oxidized to its corresponding quinone and this in turn will oxidize DHI resulting in a mixed polymer.

Understanding the nature of the radical spectra generated by melanins of various types and sources is key to understanding the myriad effects attributed to melanins. Mark Nilges, in his studies of the free radicals generated by melanins, cast doubt on the hypothesis that dehydrohydroxybenzothiozine is the source of the radical spectrum of phaeomelanin.

'Is melanin photoprotective, photosensitizing or neutral?' remains a question that has yet to be answered. The literature is rife with conflicting results which have only served to muddy the waters. Endpoints studied are survival, pyrimidine dimers, growth arrest, to mention a few. Yet it is to skin cancer and especially melanoma that we should direct our attention. In tissue culture studies, this boils down to studying cellular transformation and mutation. These endpoints are technically difficult to investigate. Ms. Kaur, a graduate student in Dr. Hill's lab, has developed an artificial melanocyte so-to-speak by transfecting a plasmid containing the tyrosinase gene into Chinese hamster ovary cells which are exquisitely sensitive to mutation. Her preliminary studies show that cells which express pigment are less sensitive to killing by ionizing radiation and UVA and more sensitive to UVC than plasmid controls lacking the gene. The responses of the pigmented cells were no different from the controls after exposure to UVB and FS20 radiation. The pigment in the cells was eumelanin by ESR analysis. These cells will be useful in future mutational analyses.