VIIITH ANNUAL MEETING OF THE PASPCR
Aug 15-18, 1998 - Snowmass, CO, USA
(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.
Sunday morning Sessions by Zalfa Abdel-Malek
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 first plenary session was entitled "Control of Melanocyte Development and Differentiation". The abstract by Southard-Smith et al. was presented by Bill Pavan. He stated that premature termination of SOX 10, one of the SRY-like HMG box transcription factors resulted in the absence of neural crest derivatives in Dom mice, a model of Waardenburg-Hirschprung disease. SOX 10 was found to function intrinsic to melanocytes and to be expressed in early melanoblasts. The potential target genes for SOX 10 are to be identified using cDNA expression microarrays. The abstract by Donatien and Bennett was presented by Dorothy Bennett who described the procedure for establishing long-term culture of human fetal melanoblasts. For this, murine keratinocyte feeder cells were used, and the growth medium consisted of RPMI 1640 supplemented with 10% fetal calf serum, 1.5 µM hydrocortisone, 20 pM thyroxine, 40 pM basic fibroblast growth factor, 20 ng/ml stem cell factor, 20 pM cholera toxin, 100nM endothelin-3, and 20 nM TPA. In this medium, the cells doubled every 3-5 days, and expressed TRP-2 and Pmel-17, but not P protein, and were DOPA-negative. Growth could be further enhanced by the addition of NDP-MSH, and melanin content could be increased by the addition of oleoyl acetyl glycerol or cAMP inducers. The abstract by Xu et al., was presented by Estela Medrano. The authors used the yeast two-hybrid system to identify human proteins that interact with the transcription factor MITF. By screening the melanoma cell line IIB-Mel-J cDNA library, they found that most clones with a positive interaction with MITF encoded the human ubiquitin-conjugation enzyme hUBC9 (UBE21). This was confirmed by in vitro GST "pull down" assay, using 6 x His-tagged MITF, by co-localization of MITF and hUBC9 in the nucleus, and by increased degradation of MITF upon co-transfection of MITF and hUBC9 into Cos 7 cells. Dong and Vijayasaradhi investigated the role of the transcription factor MITF in the coordinated regulation of tyrosinase and TRP-1 in human melanocytes and melanoma cells. Upon treatment of these cells with the differentiation inducer hexamethylene bis-acetamide (HMBA), the level of TRP-1 mRNA was drastically reduced, an effect that was not due to increased rate of mRNA degradation, and could be abolished by inhibition of de novo protein synthesis. The protein level of TRP-1 correlated with the level of its mRNA. On the other hand, tyrosinase and MITF mRNA levels were up regulated, with no change in the protein level of MITF. It was concluded that while expression of tyrosinase and other pigmentary genes correlated with MITF expression, TRP-1 expression was regulated independently of MITF and required other transcriptional factors.
The second Plenary Session was entitled "Control of Pigmentation by MSH, The Melanocortin Receptor and The Agouti Signaling Protein". Suzuki et al. reported on the regulation of the human MC1R expressed on human epidermal melanocytes. Brief treatment of melanocytes with a-MSH, ACTH or endothelin-1 resulted in increased MC1R mRNA level. This might account for lack of desensitization of the receptor following prolonged treatment with its ligands. Treatment of melanocytes with agouti signaling protein (ASP) or with UV radiation down regulated MC1R mRNA level. Unlike melanocytes, human keratinocytes did not express functional MC1R, as determined by Northern blot analysis, receptor binding assay, and cAMP radioimmunoassay. Matsunaga et al.used aPEP 16, a rabbit polyclonal antiserum generated against a synthetic peptide that corresponds to the carboxy teminus of mouse agouti protein, to characterize the expression of this protein in 3, 6, and 9 day old non-agouti black, agouti, and lethal yellow mouse skin specimens. Expression of agouti protein was very low in black mice, was noticeable and increased with age in hair matrix cells adjacent to melanocytes in lethal yellow mice, and was observed during the pheomelanogenic phase in agouti mice. Abdel-Malek et al. addressed the question whether ASP antagonizes the effects of a-MSH by exclusively binding to the MC1R, or by additionally binding to another unknown receptor. Melanocytes cultured from C57 BL6J E+ /E+ mice, congenic e/e or Eso /Eso mice were compared for their responsiveness to ASP. Only E+ /E+ responded to ASP with inhibition of basal and a-MSH stimulated tyrosinase activity, significant reduction of tyrosinase, TRP-1 and TRP-2 protein levels, and inhibition of a-MSH induced cAMP level. This demonstrates that expression of normal MC1R is pivotal for the responsiveness of mammalian melanocytes to ASP. Miltenberg et al. examined the biological significance of the highly conserved basic domain, adjacent to the cysteine-rich agouti C-terminal. Deletion of this entire region and expression of the mutant gene in transgenic mice resulted in mice with yellow coat color, but without hypoglycemia, insulinemia, or obesity. This mutation rendered the protein inactive in neural tissues, but functional in regulating pigmentation, with a reduced capacity to inhibit MC1R-induced stimulation of cAMP level. This suggests that the basic domain in the agouti gene is involved in the proteolytic processing of agouti signaling protein, affects the secretory pathway, and/or facilitates melanocortin receptor binding. Virador et al. attempted to identify bioactive domains of ASP by synthesizing overlapping 15 mer peptides that encompass its entire sequence. The biological effects of these peptides on melan-a cells were investigated by determining total melanin synthesis, and the expression of tyrosinase and TRP-1 mRNA. Peptides in the regions 30-52 and 57-91 generated about 10% reduction in total melanin production, compared to 30% reduction generated by recombinant ASP. The functional agouti region was narrowed down to 5 amino acids, Lys82Pro86, which resulted in significant reduction in melanin formation, tyrosinase expression and function. Johansen et al. described the isolation and sequencing of the bovine and porcine agouti genes, and the expression of this gene in bovine tissues. Using murine agouti cDNA as a probe, they found that cattle and pigs possess an agouti gene with 161 bp products that hybridize to murine agouti exon 2. This bovine and porcine agouti region was 75 and 76% similar to murine, 82 and 79% similar to human, respectively, and 88% similar to each other. The predicted amino acid sequences showed about the same percent homology. Furumura et al. reported the results of differential display study in which they identified three genes that were up regulated during the switch from eumelanin to pheomelanin synthesis. One of these genes is ITF2, an E type basic helix loop helix transcription factor. Upon transfection of Melan-a cells with the murine ITF2 gene, ITF2 trans-activated the TRP-1 promoter to the same extent as MITF, was less efficient than MITF in trans-activating the tyrosinase promoter, and had no trans-activating effect on the TRP-2 promoter. ITF2 over expression up regulated TRP-1 level, but down regulated the levels of tyrosinase and TRP-2, suggesting that ITF2 regulates melanogenesis by acting as an E-box binding protein. Abdel-Malek et al reported on the differential responsiveness of cultured melanocytes established from different skin types to a-MSH. Five out of five cultures with high constitutive melanin contents, and four out of five cultures with very low melanin contents exhibited the typical dose-dependent responses to a-MSH, evidenced by stimulation of cAMP formation, proliferation, and tyrosinase activity, beginning at a dose of 0.1 nM. One of the five cultures with a very low melanin content demonstrated a significantly reduced response to a-MSH, evident as a shift to the right in the dose-response curves for cAMP formation and tyrosinase activity, with a minimal effect dose of a-MSH equal to 10 nM. RT-PCR and sequencing of the entire coding region of the MC1R gene revealed the presence of these point mutations in this culture: Phe147Leu, Ile155Leu, Arg160Trp,Thr177Arg, and Ile264Met. The significance of these mutations on the loss of function of the MC1R is being investigated.
Sunday afternoon Sessions by John Pawelek
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.
9) Molecular characterization of c-kit from the Mexican axolotl. K.A. Mason et al. Using alignments of known c-kit sequences, degenerate PCR primers were designed and used to amplify a small fragment of c-kit from axolotl RNA by RT-PCR. With this fragment, 3 positive clones were identified in an axolotl cDNA library. Two clones were identical and appeared to encode a complete cDNA sequence for axolotl c-kit. Genetic mapping excluded c-kit as a candidate for the axolotl white mutant, but showed that c-kit is tightly linked to PDGFa, seen in other vertebrates, and additionally confirming that this represented at least one form of axolotl c-kit.
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.
Monday morning Sessions by Raymond Boissy
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.
Monday afternoon Sessions by Frank Meyskens
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 other Keynote lecture was entertainingly provided by John Cohen of the University of Colorado and apoptosis was reviewed in detail. Two papers were presented dealing with apoptosis in melanoma. Shellman from Colorado convincingly showed that apoptosis was controlled by the dimension status of tumor growth; i.e.under monolayer growth conditions, ras-alterated cells underwent apoptosis in response to various stress conditions while under 3-dimensional spheroid growth no or little apoptosis occurred. Since most culture studies are done in monolayer these studies have obvious and important consequences for the interpretation of studies of melanoma growth and drug resistance in the in vivo setting. From my own laboratory, Spillane has found that human melanoma cells have high levels of endogenous reactive oxygen species that may induce high levels of constitutive NFKB and a protective stress response. Interestingly, the complex antioxidant PDTC induced apoptosis while classical antioxidants(e.g. a-tocopherol) did not; suprisingly heavy metal chelators could largely mimic the effect PDTC. The most intriguing and interesting study of the session (and perhaps of the conference) was presented by Stan Pavel of the Netherlands. They measured the sulfer and phaeomelanin content of atypical nevi; and found their concentrations to be elevated, as was the concentration of calcium. Normal melanocytes from these same subjects showed no such changes suggesting that an abnormality in melanosomal regulation exists. Since phaemelanin metabolites are considerably more toxic that those of eumelanin. metabolites. One wonders if the generation of atypical nevi results from a simple error of metabolism, e.g. in the Agouti signaling protein. All in all this session was quite stimulating and a lot of new ideas were heard and vigorous discussion ensued.
Tuesday morning Sessions by Joe Bagnara & Vince Hearing
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.
Poster Session by Roger Bowers
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.