(with the Bulletin's Editor apologies for the delay)

The PASPCR meeting this year was held in Snowmass, CO under the chairmanship of Dr. David Norris. The Editor would like to thank the following authors (as noted) for contributing commentaries on each of the sessions held.

The Gelb lecture for the eighth meeting for the PASPCR was given by John Pawelek, Ph.D., and was entitled "Melanoma/macrophage hybrids and the development of metastases in melanoma". In his presentation, Dr. Pawelek provided a historical overview of reports by various investigators stating that such hybrids indeed exist and are associated with metastatic disease. Dr. Pawelek presented results from his laboratory, using mouse Cloudman melanoma cells that are known to have a poor metastatic potential. He reported that Cloudman melanoma X macrophage hybrids greatly enhanced the metastatic ability of tumor cells. It was observed that these hybrids became significantly more melanotic than the original melanoma cells. The proposed mechanism for the enhancement of metastasis and melanization is increased N-glycosylation of proteins, that included several melanogenic proteins. The melanoma/macrophage hybrids acquired a macrophage-like glycosylation system that resulted in enhancement of N-glycosylation. This mechanism offers one explanation for increased aggressiveness of melanoma tumors during disease progression.

The afternoon Plenary Session was on the Biochemical Control of Pigmentation.

1) Keynote Lecture: Perspectives on biochemical control of pigmentation and pigment cell survival, K. Schallreuter. Dr. Schallreuter discussed the hypothesis, proposed by Dr. John Wood and herself, that (6R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH4) through regulation of phenylalanine hydroxylase (PAH) and tyrosinase activities is a key, rate-limiting factor in the control of melanogenesis, wherein the availability of L-tyr is regulated by PAH and 6BH4. The proposal is based in part on their observations that L-Phe is actively transported by human melanocytes, whereas L-tyr enters cells much more slowly, through passive diffusion. 6BH4 regulates melanogenesis through specific binding sites on both tyrosinase and MSH. MSH activates tyrosinase by removing 6BH4 from a tyrosinase: 6BH4 inhibitory complex. UVB, through photooxidation of 6BH4, activates both PAH and tyrosinase.

2) Macrophage migration inhibitory factor (MIF) has enzyme activity towards oxidized catecholamines and recues cells from dopaminechrome induced death. J. Matsunaga et al. Evidence was presented that MIF is able to catalyze the conversion of DOPaminechrome and norepinephrinecrome, toxic qinone neurotransmitter by-products, to indole-quinone derivatives that may serve as precursors of neuromelanin. Cytotoxicity experiments showed that MIF can rescue cells from DOPaminechrome induced death in culture. Since MIF is highly expressed in brain, the possibility is raised that MIF detoxifies catecholamine products and therefore could have a protective role for neural tissues.

3) The function of the pink-eyed dilution protein. N. Puri and M.H. Brilliant. Using immunohistochemistry and confocal microscopy, the authors showed that at least one function of the pink-eyed dilution protein (p protein) is to regulate melanosomal pH. Acidic compartments of melanocytes were detected by DAMP incorporation and indirectly visualized using fluorescein-conjugated antibodies. In wild type melanocytes, virtually all melanosomes were acidic and virtually all acidic compartments were melanosomes. In contrast, melanosomes of p-deficient cell lines were almost never acidic. As tyrosinase activity in melanosomes is dependent on a low pH, the authors postulated that the minimal melanin synthesis observed in p-deficient mutants is due to insufficiently low pH.

4) Cell density-mediated induction of tyrosinase-related protein gene expression and differential regulation of TRP-2 glycoforms. T.J. Hornyak et al. The effects of cell density on the expression of TRP-1 and TRP-2 in cultured melan-a mouse melanocytes were investigated. The relative levels of mRNA for both proteins increased with increasing cell density and decreased when confluent cells were replated at low density. Compared to TRP-2, TRP-1 both decreased more rapidly with low density and increased more rapidly as density increased. Western blotting showed that TRP-2 existed in two distinct glycoforms under separate regulation with cell density changes. The results showed the potential importance of cell density or cell contact in determining the extent melanogenesis.

5) Tyrosinase-related proteins (TRPs) modulate tyrosine hybroxylase activity of tyrosinase in genetically defined mouse melanocytes. R. Sarangarajan et al. Tyrosine hydroxylase (TH) activity of cultured mouse melanocytes from wild type B/B mice was compared to mice with mutations in TRP-1 and TRP-2. Using ³H-L-tyrosine in the Pomerantz assay, it was determined that TH activity was higher in wild type over mutant cells, even though tyrosinase itself was wild type in all cases except for albino (c/c) negative controls. Conclusion: TRPs are positive regulators of TH activity.

6) The regulation of pigmentation by serine proteases and their inhibitors. M. Seiberg and S.S. Shapiro. Multilayered epidermal equivalents expressing UVB-inducible melanogenesis were used to study the effect of protease inhibitors on melanogenesis. Several serine protease inhibitors were also effective melanogenesis inhibitors. The pigmented Yucatan swine treated with one of the protease inhibitors showed a visible lightening effect. Data suggested that the protease inhibitors interfered with melanosomal transfer from melanocytes to keratinocytes.

7) Supermelanotic and metastatic melanoma x macrophage fusion hybrids: Altered N-glycosylation as an underlying mechanism. S. Sodi et al. A number of fusion hybrids between weakly metastatic Cloudman S91 cells and peritoneal macrophages were shown to have increased metastatic potential and this correlated with a dramatic increase in both basal and MSH-inducible pigmentation. Using Western Blotting of LAMP-1 and TRPs, incorporation of ³H-glucoseamine, and glycosylation inhibitors, it was determined that the increased pigmentation was likely to be a result of a macrophage-like N-glycosylation system expressed in the metastatic hybrids, and that this glycosylation system might be an underlying cause for increased metastasis as well. Further, the data revealed for the first time that N-glycosylation may be an important pathway for MSH-induced melanogenesis.

8) Chemical characterization of dopamine-melanin: Application to identification of melanins in Cryptococcus neoformans. S. Ito, et al. Melanin has long been associated with virulence in C. neoformans and is believed to be produced by laccase. Several isolates of C. neoformans were incubated with dopamine or DOPA and subjected to improved melanin analyses using alkaline H₂O₂ oxidation and HI hybrolysis. The results indicated that laccase from C. neoformans indeed oxidizes catecholamines to produce eumelanic pigments.

10) An analysis of pigment patterns in leopard and golden mutant zebrafish and related taxa of danios. R. Morrison and K. Nagashima. The zebrafish, Danio rerio, was studied as a model organism for pigment pattern formation. There are at least four types of chromatophores in zebrafish: xanthophores, iridophores, leucophores, and melanophores. There is a distinct embryonic pigment pattern composed of four melanophore stripes that transform into the adult pattern of alternating blue-black and silver yellow stripes. The golden mutant, which lacks melanophores and iridophores as adults, and the leopard mutant, which shows a spotted phenotype were studied, along with the pearl danios, which lacks alternating striping elements as an adult but contains a fifth type of chromatophore, the erythophore, and the giant danios that has a different arrangement of chromatophores than that seen in wild-type zebrafish. It was proposed that analyses of these fish should further clarify the critical events and time-points in zebrafish pigment pattern formation.

11) LiCl is involved in the pigmentation of the embryonic zebrafish (Brachydanio rerio). E-J Jin and G. Thibaudeau. Zebrafish embryos were treated with various signalling-related molecules. LiCl and LiCl/forskolin treatments each increased pigmentation. The LiCl/forskolin-induced pigmentation was not accompanied by an increase in melanophore number, but did result in increased tyrosinase activity and increased expression of MSH-1, a pigment specific protein, and TRP-2 as assessed by immunoblotting.

The morning began with a sunrise session entitled "New Perspectives on the Treatment of Human Vitiligo". David Norris presented a review of the processes of programmed cell death (i.e., apoptosis) and necrosis and discussed the balance between these two mechanisms as they pertain to cell survival. Molecular regulators of apoptosis (i.e., the bcl family of proteins, FAS and the caspases) were reviewed. It was proposed that melanocyte destruction in vitiligo occurs via apoptosis and intervention of this may provide potential therapeutic opportunities. Raymond Boissy discussed occupational/contact vitiligo resulting from exposure to phenolic/catecholic agents. The phenolic agent, 4-tertiary butyl phenol, is cytotoxic to melanocytes via an apoptotic process. The antioxidant catalase could provide some protection against this form of melanocyte destruction. Pranab Das reviewed the immunological components of vitiligo. The role of T-cell autoreactive clones, conducting a hit and run affect on melanocytes in vitiligo was discussed. Immunomodulary therapy was proposed as a perspective for treatment for vitiligo. Finally, Karin Schallreuter discussed the biochemical aberration in skin of patients with vitiligo. The role of biopterin in the regulation of tyrosinase activity was reviewed. Successful results of the daily topical application of pseudocatalase on repigmentation in vitiligo was presented and discussed. It was concluded in this sunrise session that the etiology of vitiligo is both complex and diverse and that multiple theraputic regimes will have to be developed to successfully treat this disease.

A keynote lecture was then presented by Richard Spritz entitled "New Approaches in Genetics and Their Application to Pigment Cell Research" and subtitled "How to find a gene". Functional versus Positional Cloning methods were presented and the difference between discussed. Mapping a gene directly to a chromosome was first discussed and techniques using autoradiography, FISH, and chromosome abnormality was presented and the c-kit associated Waardenberg Syndrome and OCA2 were provided as examples. Genomic analysis (i.e., linkage) was discussed next. Examples of using RFLP assessment, Simple Tandem Repeats, and the utilization of chromosome site markers were presented. Finally, current methods for gene mapping were described. This included the need for large family trees, Yeast Artificial Chromosomes, Sequence-tagged-site, homozygosity mapping, linkage disequilibrium mapping, and the generation of a physical map.

A session focussing on the Hermansky-Pudlak Syndrome followed. Richard Spritz presented genetic and functional studies of Hermansky-Pudlak syndrome. The characteristics of this syndrome consist of oculocutaneous albinism, a platelet aggregation dysfunction, and the development of a ceroid like material in the lungs resulting in pulmonary fibrosis. The cloning of the gene (and the murine counterpart) and the identification of various protein-null mutations was presented. The HPS gene product appears to be soluble and unglycosylated. It is predominantly unassociated with organelles or granules in fibroblasts and lymphoblastoid cells and loosely associated with large granules in melanoma cells. The HPS gene product demonstrated some co-localization with tyrosinase, LAMP-1, and Myo5A (the product of the dilute locus). Richard King next presented the identification of an alternative 1.5 kb transcript (in addition to the 3.6 kb full length transcript) also present in bone marrow and a melanoma cell line. Raymond Boissy next discussed cytological aberrations in melanocytes cultured from patients with mutations in the HPS gene resulting in the lack of transcript expression. These hypopigmented melanocytes demonstrated muted tyrosinase activity, large membranous complexes, and DOPA positive 50 nm vesicles distributed throughout the cell. Tyrosinase, TRP-1 and ME491 were not efficiently trafficked to melanosomes in the mutant cells. Localization studies suggested that the HPS gene product was associated with the endoplasmic reticulum and melanosomes in the normal melanocytes.

The final session of the morning was entitled "Vitiligo: mechanisms of depigmentation and repigmentation". Pranab Das presented data demonstrating the appearance of immunocytes at the border of a vitiligo lesion and the associated development of apoptosis in some melanocytes. In addition, studies demonstrating that immune cells can lead to de novo generation of nitric oxide in melanocytes resulting in apoptosis. Caroline LePoole presented the immortalization of a line of vitiligo derived melanocytes using the E6 and E7 gene of HPV. These cells were passaged over 60 generations, demonstrated dilated RER, and exhibited alterations in proteins identified in fractionated RER. Marna Ericson presented data in which biopsies both pre and post treatment with topical steroids were immunocytochemically processed for the identification of melanocytes, Langerhans cells and nerve cells, and viewed with confocal microscopy and computer reconstruction. Demonstration of a decrease in the appearance of nerve fibers in the epidermis after successful treatment was provided. Fan Yang presented data demonstrating the 4-tertiary butylphenol (4-TBP) acts as a specific competitive inhibitor of tyrosinase at concentrations well below the threshold that generated a cytotoxic response in normal melanocytes. Finally, Caroline LePoole demonstrated that shortly after exposure of melanocytes to 4-TBP several transcripts are differentially expressed. One of these upregulated proteins was an adenosine receptor that has been implicated in the activation of apoptosis.

The session on malignant melanoma covered a diversity of topics. Meenhard Herlyn started the session with an eloquent keynote presentation of their skin reconstruction model, which is used to study progression. Using the model, Hsu has shown that adherence of keratinocytes by E-cadherin to melanocytes controls their growth and phenotype and when this adhesion molecule is shut off a cell cluster, a nevus, forms. The overall important point was made that the model more closely resembles the biology in intact skin than that exhibited by cells under typical culture conditions. There were several papers on the role of various proteins in controlling melanocyte growth including its positive regulation by retino-blastoma tumor suppressor protein (R. Halaban, Yale), and negative regulation by p15(INK4B) (T. Pacheco, University of Colorado). In a similar vein Rearden at the University of Colorado used T-cell receptor knock-out mice to show that transduction of B16 F10 melanoma cells with M-CSF increased survival of the inoculated animals suggesting that cells other than T lymphocytes (eg NK or macrophages) are involved in anti-melanoma immunity, at least in this model.

In a fasciniating presentation, Bill Robinson of Australia presented the zebrafish as a powerful developmental model for understading the role of p16 and other regulators in melanocyte proliferation. This model impresses this reviewer as possibly one of the most important new systems to come along in quite some time.

The keynote lecture by Karl H. Pfenninger presented basic insights into cell motility through the use of nerve growth cones as an example. Thus, he set the stage for the subsequent three presentations which were specifically directed toward an understanding of the migration of melanocytes and melanoma cells. Dr. Pfenninger illustrated the importance of pseudopod attachment, release, and reattachment as the basis for cell migration and he considered the means by which these steps are accomplished. The subsequent presentation by Hiroaki Yagi demonstrated through the use of Boyden chamber assays that insulin-like growth factor-1 (IGF-1) is a potent chemoattractant for both human melanocyes and melanoma cells. Endothelin-1 (ET-1) and basic fibroblast growth factor (bFGF) enhanced migration of normal human melanocytes and enhanced the invasion activities of WM35 cells (a human melanoma with low invasiveness). IGF-1 induced maximal movement in both these cell types and this action could be blocked by CDC, a selective 12-lipoxygenase inhibitor. cPLA2, a critical enzyme in pseudopod activation in both cell types is stimulated by ET-1, bFGF and IGF-1. The implications of these findings toward the progression of melanoma were discussed. This presentation was followed by that of Tatsuya Horikawa, et al. who considered the motility and proliferative responses of an H-ras-transfected murine melanocyte cell line, melan-A. In a Boyden chamber assay it was found that H-ras-transfected melanocytes show a higher incidence of migration than do wild type melanocytes. Cell motility was induced by ET-1 and bFGF in wild type melan-A, but not in H-ras-transfectants. TPA was required to grow parental melan-A cells, but the ras-transfectant was TPA-independent and in fact was inhibited by TPA. It was suggested that H-ras plays a key role in the induction of melanocyte locomotion and proliferation. The last talk on acquired MSH-sensitive chemotaxis by highly metastatic melanoma/macrophage fusion hybrids by Rachkovsky et al. was presented by John Pawelek. It was a most fitting follow up to the very first presentation of the conference, John Pawelek's Gelb Lectureship on melanoma/macrophage hybrids and melanoma metastases. In this symposium talk, it was pointed out that of the various fusion hybrids between normal macrophages and Cloudman S91 melanoma cells, the most metastatic also showed dramatically increased motility. Metastatic hybrids demonstrated increased migration in response to 3T3-conditioned medium, lung fibroblast-conditioned media, and lung explants. Treatment of the hybrid cells with MSH/BMX markedly increased migration through enhanced chemotaxis rather than chemokinesis. Evidence was presented suggesting that different glycosylation pathways were expressed in hybrid and parental cells and that motility was probably regulated by means of N-glycosylation. It was suggested "that the enhanced metastatic potential of macrophage x melanoma hybrids may have its basis in a new, MSH-inducible chemotactic phenotype, with altered N-glycosylation as one of the underlying regulatory mechanisms."

After a short break, Barbara Gilchrest presented a Keynote Lecture on the "Effects of UV on melanocytes: speculative relationship to the epidemiology of melanoma." Melanocyte responses to UV were reviewed with special emphasis on their DNA repair mechanisms. The role these responses play in melanocytes and keratinocytes might explain the differences in malignant transformation in those cell types to produce melanomas and carcinomas, respectively. The importance of sunscreen use to protect from UV damage throughout life was emphasized.

N. Kobayashi then reported on studies examining the photoprotective effects of melanins. Immuno-histochemistry with antibodies specific for different types of DNA photoproducts allows assessment of DNA damage in UV irradiated skin at the cellular level. A direct correlation was found between melanin content in a cell and protection from UV light. Current studies are aimed at examining the efficiency of different types of melanins using mouse tail skins of different genotypes as models. Z. Abdel-Malek then reported on responses of melanocytes from different human skin phototypes to UV light. The sensitivities of melanocytes from different donors to UV light was measured for DNA damage, bcl2 expression and other parameters of cellular injury. There were significant differences in the responses measured depending on skin phototype in melanocytes, keratinocytes and fibroblasts, and the increased sensitivity of keratinocytes and fibroblasts to UV damage was proposed as the reason for the higher incidence of carcinomas in those cell types. Finally, F. Meyskens reported on the response of metastatic melanoma cells to UVB stimulation, particularly with respect to activation of the NFkB transcription factor. There were dramatic differences in activation of 2 NFkB family members (p50 and p75) in malignant melanoma cells and in normal human melanocytes, often by an order of magnitude, suggesting basic differences in response mechanisms of normal and transformed melanocytes. It was suggested that such changes are associated with the metastatic potential of those malignant cells, and that understanding the reason behind such differences may offer novel approaches to the prevention, diagnosis and therapy of melanoma.

The poster sessions at this PASPCR meeting were well attended. There were only 12 posters due to the numerous oral presentations but they were of the highest quality. Holder and Thibaudeau showed that increased melanization in the melanoid defect in axolotls may involve aspects in addition to cellular plasticity whereas cellular plasticity is implicated to be involved in enhanced xanthophores from albino axolotl embryos. Parker and Mason demonstrated that extracts from the white axolotl mutant caused a decrease of pigment cells and inhibited their movement in cultured melanoma cells. They are currently working to characterize this extract. Roberson and Thibaudeau showed a two stage time-dependent differentiation of in vivo melanophores in zebrafish. Similar results were found in in vitro zebrafish melanophores and LiCl increased pigmentation in these same cells. Nurcahyani and Thibaudeau demonstrated cellular parameters influencing ant./post. responses of neural crest-derived pigment cell linages in axolotls. For example, ant. neural crest cells gave rise to more pigment cells whereas post. neural crest cells yielded more melanophores. Gonzalez, Buckner, Ruiz and Bowers, with the use of the glutathione inhibitor BSO, other treatments and parameters, showed the importance of antioxidants in the viability of avian melanocytes. Maxwell, Walsh and Maxwell demonstrated the infection of human melanoma cells by parvoviruses and suggested therapeutic uses of these viruses and their vectors for melanoma. Murakami, Baba, Kawa and Mizoguchi showed that inflammation in atopic dermatitis plays a role in developing acquired dermal melanocytosis along with the hereditary disposition of having immature dermal melanocytes. Sarangarajan, LePoole and Boissy demonstrated that NHE-1 isoform of sodium hydrogen exchanger is expressed in M14 melanoma cells and in keratinocytes but not in human melanocytes. Ahn, Jang, Cho, Lee, Hong and Lee showed that a kojic acid derivative, kojyl caffeic acid, has a greater depigmenting effect than kojic acid. Forest, Nofsinger, Drake and Simon demonstrated that the photochemistry of melanin in the UV is wavelength dependent. Shi, Krauss and Woodward showed evidence for the presence of EP2- and IP- receptors coupled to melanin production via stimulation of tyrosinase activity in S91 Cloudman cells. Butts and Naughten demonstrated that melanocytes decreased with time in new wound closure epidermis in the corneal region of the frog.