MEETING REPORT

10th MEETING of the ESPCR

26-29 Sept 2001, Rome, Italy

INTRODUCTION

Contributed by Mauro Picardo

With great pleasure, this year, we organized and hosted the 10th Meeting of the European Society for Pigment Cell Research that, after thirteen years, has been held again in Italy. The inaugural Meeting indeed took place in Sorrento under the organization of one of the founders of the Society. We have tried to renew the same warm and friendly atmosphere and, must be said, Rome contributed to this wish with a extraordinary sunny September. Unfortunately, on 11th September, a date that everybody will never forget, the world was shocked by terrorist attacks in U.S.A., whose cruelty was of an exceptional extent. In spite of the discouraging circumstances, the overall attendance to the congress was only slightly affected and we did appreciate the participation of those who traveled to Rome, especially from overseas countries. We also understood fully some cancellations. For this reason, and for many others, on behalf of the organizing and scientific committees, I would love to thank the 210 scientists who participated in the 10th ESPCR Meeting, of whom 40 were non-ESPCR members.

The satellite symposium on hyper- and hypo- pigmentary disorders that preceded the opening of the meeting gave me the opportunity to introduce in a new dress the Dermatological Institute S. Gallicano that participated to the organization of the event. The goal of the clinically-oriented satellite symposium was to gather expertise of excellent clinicians-scientists in the treatment of benign pigmentary disorders to be transmitted to dermatologists. The satellite symposium, for its great deal of valuable contributions, represented a good premise to the following ESPCR meeting that was opened the day after in the Aula Minor at Angelicum University. In keeping with the interdisciplinary tradition of the Society, the scientific program foresaw numerous - 42 - invited contributions from excellent European and non-European researchers, in an attempt to offer overviews on very different areas of pigmentation to participants. The program was divided in eight sessions and two round tables that were all well attended, and discussions stemmed at the end of each presentation, always lively and rich of interesting cues. Organizers and scientific committee agreed in the introduction of innovative issues related to the regulation of melanogenesis, such as oxidative stress. In line with the latter, we judged of great interest an overview on the involvement of mitochondrial aberrant redox mechanisms in the pathogenesis of several diseases, held by Giuseppe Rotilio. Celebrative and of high scientific significance was the Fritz Anders Memorial Lecture on the genetic link between skin colour and susceptibility to skin cancer delivered by Richard Sturm. Very interesting submitted papers were also numerous and were selected for 46 oral presentations and 54 (plus 3 late) poster presentations. We tried to mirror the wider view on advances in the pigmentary cell research given during the meeting with an "overview" offered by the terrace on Via dei Fori Imperiali on the historical traces left by the ancients that, hopefully, was enjoyed by attendants.

We hope that the displayed continuous progress and improvement of our Society will further contribute to the success of the forthcoming meetings.

Mauro Picardo and the Organizing Committee


Session I. EUMELANINS, PHEOMELANINS AND SUSCEPTIBILITY TO UVR

Chaired by Marco d’Ischia, Stan Pavel and Tadeusz Sarna.

Contributed by Marco d’Ischia

The opening session of the 10th ESPCR meeting in Rome addressed at a multidisciplinary level the role of the different types of melanins in the individual susceptibility to UV radiation. The theme was brought to focus by three inspiring invited lectures and was further discussed in three proffered platform presentations. The opening lecture by G. Prota (Naples) was basically a keynote address in which the emerging complexities in the structures of melanin pigments from human hair were briefly overviewed. By means of improved microanalytical methodologies, based on hydrogen peroxide degradation, it was shown that the variety of human hair colour is due to at least four types of melanins. Such a diversity depends both on the well known branching point in the biosynthetic pathway, due to the intervention of cysteine leading to pheomelanins, and the occurrence of post-biosynthetic modifications due chiefly to hydrogen peroxide. This can cause even dramatic alterations of the basic polydihydroxyindole and polybenzothiazine structures of eumelanins and pheomelanins, whereby pyrrolic and (benzo)thiazolic moieties become prevailing.

The discussion that followed touched on several aspects of the presentation from the sensitivity of analytical methods to the possible consequences of the structural variety in relation to UV susceptibility. The emerging view was that melanins are not directly responsible for skin susceptibility but are rather markers of the genetic and biochemical features of the melanocytes from which they are produced.

The subsequent presentation by S. Rosso (Turin) provided an overview of epidemiological ongoing approaches to determine relationships between skin type, melanins and melanoma risk. The author stressed that the relationship between genes polymorphism, molecular markers and risk of skin neoplasm in human population must be investigated with an appropriate epidemiological study design. To this aim, the GEM and HELIOS2 studies in molecular epidemiology maximise their efficiency in studying low prevalence markers, comparing sporadic versus multiple melanoma (GEM), and melanoma, basal-cell, squamous-cell carcinoma versus health controls (HELIOS2). As the author pointed out, molecular epidemiology of skin cancer is necessarily a work in progress, as from laboratory research new suggestions can be obtained on genes and molecular markers involved in carcinogenesis, and their biological mechanism. The presentation was well balanced and offered interesting opportunities for discussion. The third invited lecture was delivered by E. Kvam (Oslo), who studied the role of melanin or melanin precursors in UVA-induction of oxidative damage in human and mouse melanocytes and melanoma cells. He reported that some types of cells, in which melanin synthesis was induced prior to UVA-irradiation, were sensitized to UVA-induction of the premutagenic oxidative DNA base damage, 8-oxo-guanine. Since only cells producing pheomelanin were sensitized, pheomelanin synthesis is apparently associated with increased susceptibility to UVA-induction of premutagenic DNA damage.

In the first proffered paper, J.-F. Doré (Lyon) presented results from a collaborative study involving four groups in Brussels, Lyon, Tokyo and Milan, showing that individual response to UV light, as measured by apoptosis induced in peripheral blood lymphocytes by low dose UV radiation, constitutes a novel risk factor for melanoma at an early age, independently of other known risk factors. The discussion raised the possible relation between the functional assay and a polymorphism in DNA repair pathways. Considerable interest was also raised by the last two papers. In a joint contribution from Bradford University and Procter-Gamble, the effect of pro-opiomelanocortin (POMC) peptides was investigated for the first time in cultured human scalp hair follicle melanocytes. The results were reported by S. Kauser (Bradford) and indicated potent mitogenic, melanogenic and dendrogenic effects suggesting involvement in the regulation of differentiation and melanogenesis. An attractive, though still speculative possibility, is that a chronic alteration of POMC peptide homeostasis may be involved in hair greying, thus providing a new focus for dermocosmetic research in this area. Finally F. Rouzaud from Dr. Hearing’s lab at NIH (Bethesda) presented recent data on the effects of alpha-MSH on the extension gene transcripts. The remarkable finding was that alpha-MSH induces two types of transcripts, the second of which, designated T2, is shorter that the first (T1). This may help define the role of specific nucleotide sequences in each transcript and the significance of post transcriptional processes.

The central relevance of the theme to the whole field of pigment cell research, the multidisciplinary approaches surveyed, and the high quality of the presentations were undoubtedly strong incentives for the lively discussions during the session. Dissecting the complex interplay of etiological and contributory factors that emerged from the various presentations is certainly a primary goal in studies of UV susceptibility and is likely to open new perspectives for future research in this area.


Session II. REGULATORY MECHANISMS OF MELANOGENESIS

Chaired by José Carlos García-Borrón, Sheila Mac Neil and Anthony Thody

Contributed by José Carlos García-Borrón

The Session started with three consecutive invited lectures. The first one was presented by Robert Ballotti (Nice), who reviewed cAMP signalling in melanocytes. This signalling is complex and involves an interplay of several intracellular cascades. The best known is the one leading to protein kinase A-mediated activation of CREB and transcriptional up-regulation of Microphthalmia (Mitf). Mitf then binds to and activates the promoters of several melanogenic genes, notably TYR. But cAMP does much more than this. It activates the MAP kinase pathway, whose sustained activation inhibits melanogenesis by inducing a phosphorylation-dependent Mitf degradation. Therefore, the same signal mediates both stimulatory and inhibitory responses, that may allow for a limitation of the melanogenic response to a MSH. In addition ot this effect of cAMP on MAP kinases, cAMP also interacts with the phosphatidyl inositol 3-kinase pathway and with Rho protein, which accounts for its influence on melanocyte dendricity. The overall picture that emerges is that of a complex network of interactions and cross-talk between signalling cascades governing virtually all aspects of melanocyte biology.

The next invited lecture was delivered by Colin Goding (Oxted) who presented data on the differential regulation of Mitf in melanocytes and melanoma cells. Since Mitf is a key regulator of melanocyte development and differentiation, the control of its expression is a major aspect of melanocyte biology. The transcription factors positively controlling the Mitf promoter are relatively well characterized. However, much less is known about repressors of the Mitf promoter activity. Melanoma cells display aberrant patterns of tyrosine kinase activity and a constitutive activation of the Wnt signalling pathway. Dr. Goding presented strong evidence showing that Brn-2, a member of the POU domain family of transcription factors binds to and represses Mitf promoter activity, both in vitro and in vivo, as shown by chromatin immunoprecipitation experiments. Interestingly, the levels of expression of Brn-2 are very low in normal melanocytes, but high in melanoma cells. This can be related with the positive control of Brn-2 expression by Ras, Rho and b -catenin signalling, three pathways deregulated in melanoma.

The last invited lecture of the Session was presented by Jose Carlos García-Borrón (Murcia) and dealt with structure-function relationships in the tyrosinase family proteins. The main question that was addressed was the identification of structural elements accounting for the very different enzymatic activities of tyrosinase, Tyrp2/Dct and Tyrp1. Concerning Tyrp2, its dopachrome tautomerase activity is explained by the binding of zinc, rather than copper, as the metal cofactor. However, the structure of the metal ion binding sites of tyrosinase and Tyrp2 is very similar, and mutation of selected residues of Tyr copper binding sites to mimic the Tyrp2 sites fails to switch the metal cofactor specificity. Therefore, the determinants of a differential metal ion specificity may lay outside the active site and be related to the interaction with different chaperones. Evidence was also obtained for a differential docking of monophenolic and diphenolic substrates to the active site or Tyr, since the effects on the hydroxylase and oxidase activities of some site-directed mutants were markedly different. Finally, Tyrp1 does not display detectable enzymatic activities when expressed in heterologous cells, but the protein is not adequately processed to the final glycosylation isoform. This is in contrast to Tyr, whose glycosylation pattern is normal after transient expression in the same host cells. Therefore, the requirements for efficient processing of Tyr and Tyrp1 are different. Overall, the data presented suggest that important determinants of full enzymatic activity are located outside the highly conserved metal ion binding sites, but do have a strong effect on the glycosylation status of these sites and on their metal binding specificity.

The next presentation, by J. Vachtenheim (Prague) highlighted the complexity of the regulation of the chromatin-integrated tyrosinase gene. In human melanoma cells, Tyr mRNA levels correlate very poorly with MITF protein levels. Moreover, evidence for still uncharacterised mechanisms of transcriptional activation of the Tyr gene was obtained from studies of E1a mutants.

The next two papers came from A. Thody’s laboratory (Bradford). In the first one, J. Ancans discussed the possible implications of the genetic background in P-locus product abundance. The P protein appears to activate melanogenesis by controlling the melanosomal pH. Melanocytes from skin types I to IV show little or no difference in the levels of P mRNA, and the same is true for melanocytes from Caucasian or Asian donors. Conversely, P mRNA abundance increases upon treatment with a MSH or sun exposure. The data suggest that the presence of P gene polymorphisms, rather than differences in the levels of expression, might act as genetic determinants of skin colour. The second paper, presented by M. Hoogduijn discussed the regulation of calcium fluxes in human melanocytes. The striking feature in this presentation was the demonstration of spontaneous fluctuations in intracellular calcium concentrations within individual melanocytes, whose significance remains to be elucidated.

The last talk of the Session was delivered by Messod Benathan (Lausanne), and dealt with the effects of glutamine on thiol availability and tyrosinase activity in human melanocytes and melanoma cells. Cells grown in media containing a low glutamine concentration contained high levels of free cysteine and 5-S-cysteinyldopa (5-S-CD), but low tyrosinase activity. In the presence of higher concentrations of glutamine, cysteine and 5-S-CD decreased in a concentration-dependent manner, whereas tyrosinase activity increased. These effects of glutamine might be dependent on the conversion into glutamate.


Session III. GENETICS

Chaired by Miguel Seabra and Eugene Healy

Contributed by Eugene Healy

The first talk of this session was by Eugene Healy (Southampton) who discussed the causal association between melanocortin 1 receptor (MC1R) gene variants and red hair and fair skin type. Following on from the original association studies, more recent work involving transfected cell lines demonstrated that MC1R variants compromise signalling via cAMP in melanoma cells. The generation of recessive yellow mice transgenic for human MC1R confirmed that MC1R variants alter the pigmentation phenotype in vivo, and preferentially result in the synthesis of phaeomelanin.

Valeria Marigo (Naples) reported on Oa1-deficient mice generated by gene targeting as a model for ocular albinism type 1, an X-linked form of albinism affecting the eye. Although the Oa1-deficient mice are phenotypically indistinguishable from wild type mice, abnormally large melanosomes are detected by microscopic examination of the retinal pigment epithelium of the affected mouse. Similar to that seen in human OA1 patients, a reduction in ipsilateral optic nerve fibres is seen in the Oa1 mouse, and the model may therefore help in the elucidation of the mechanism whereby this abnormality arises.

Celia Jiménez-Cervantes (Murcia) discussed two new MC1R variants which had recently been identified in the Spanish population. Cell transfection experiments suggested that the Ile40Thr and Val122Met, which were seen more frequently in subjects with skin types I-II, are likely to affect the ability of alpha-MSH to signal via MC1R. Both variant receptors had slightly lower binding affinities than the wild type receptor, and resulted in attenuated cAMP responses, consistent with these variants being partial loss of function mutations.

Jochen Utikal (Ulm) showed evidence, using fluorescence in situ hybridization (FISH), for additional copies of Cyclin D1 being present in short term cultures of primary and metastatic melanomas. In addition, high quality pictures of interphase FISH on paraffin-embedded sections were presented, which demonstrated that primary uncultured melanomas contained extra copies of Cyclin D1 (as compared with a centromere 11 probe). The results suggested that aberrations of Cyclin D1 may be important in the pathogenesis of cutaneous melanoma.

The next presentation by Tomonori Motokawa (Nagoya) reported on the expression of several pigment-related genes in lentigo senilis. Increased expression of Tyr, TRP-1, TRP-2, Pmel-17, P, and MITF was detected in cells matching the distribution of melanocytes, whereas POMC was over-expressed in epidermal keratinocytes. The possibility of increased POMC expression resulting in the transcription of the other pigment-related genes in the melanocytes was discussed. However, it was noted that the frequent observation of lentigo senilis in red haired subjects, many of whom have two variant MC1R alleles, might suggest that the higher POMC expression was not a causal event.

Paola Grammatico (Rome) spoke about CDNK2 mutations in patients with melanoma, and reported that three novel mutations had been identified, as well as five previously described mutations, in Italian subjects with this condition. It was noted that subjects with CDNK2 mutations more frequently had multiple melanoma, and that CDNK2 mutations were sometimes present in families where only one or two members had developed melanoma.

In the final talk, Annerose Anders (Giessen) presented interesting data on melanomagenesis in a Xiphophorus fish line. In this model, more melanomas developed in successive generations of fish who were descendants of those exposed to UVB and X-ray irradiation (although the offspring themselves had not been exposed to these noxious stimuli) than the numbers of melanomas arising in descendents of fish not exposed to radiation. Paragenetic elements, possibly involving a retrotransposon, are likely to be responsible, and result in anticipation with earlier onset of melanoma in later generations of fish.


Session IV. PIGMENT CELL DEVELOPMENT

Chaired by Elisabeth Dupin, Colin Goding and Lionel Larue

Contributed by Lionel Larue

This session regrouped two aspects of development. The first one was associated to the embryological development and the second one to the technological development. The first presentation of this session associated both aspects. Unfortunately, William Pavan (IL23) cancelled his trip to Rome after the events of September 11th, 2001. Bill was supposed to present his work on a functional genomic approach to discover genes involved in the development and the transformation of melanocytes. They produced cDNA micro array containing thousands of melanocyte expressed EST and known genes associated with melanocytes. On these micro arrays we can find Endothelin-3 (ET-3) and Steel (Sl) ligands as well as their corresponding receptors (Ednr-B and Kit). After hybridization, they could define different "blocks" of cDNA. Importantly, they developed an efficient method to study the function of these EST-cDNAs in melanoblast-derived cells. Two papers were given on the role of one of the major ligands/receptors of the melanocyte development. These papers may lead to the idea that during evolution, the signaling and the role associated to ET-3 and Steel evolved. In birds, using the well established neural crest cell in vitro culture, Dupin et al. (IL24) showed the importance of ET-3 during proliferation and survival of the glia-melanocyte bipotent cells and during the melanocyte differentiation. A precise study on this aspect of the development of melanocytes was presented. In mammals, only one EdnrB was cloned, the chance that a second receptor exists is very low. In birds, these authors isolated a second receptor (Ednr-B2); the effect of ET-3 is mediated by Ednr-B and Ednr–B2, which are temporally and spatially regulated differently. Pla and Larue (SP20) showed the importance of Ednr-Bs during the migration of neural crest cells. They used a novel migration assay in which they graft various ES cell mutants in the chicken embryo. They could show that Ednr-B2 can direct the migration towards the dorso-lateral pathway. Another aspect of the migration was presented by Wehrle-Haller et al. (SP36). The role of the cell adhesion molecule, a vb 3 integrin, was studied using a time-lapse approach. This approach allowed the authors to show the importance of the local density of this protein. The take home message is that a slow turnover of a vb 3 is associated to low-density contacts and to a moderate migration, and a high turnover of a vb 3 is associated to a high-density contact and to a high migration. Originally, beta-catenin was found to be associated to the cell adhesion molecule and tumor-suppressor, E-cadherin. An oncogenic form of b -catenin was detected in carcinomas. Martinozzi et al. (SP18) expressed oncogenic forms of b -catenin in melanocytes in vivo. At the time of this presentation, no melanoma was observed. An overall down-pigmentation and white-belly spots were observed. The molecular mechanisms remain unclear. Szabad et al. (SP21) isolated and characterized an immortalized human epidermal melanocyte cell line. This cell line possesses effectively a large number of melanocyte characteristics. However and interestingly, it has to be noticed that Cyclin D1 is overexpressed, tyrosinase is not well expressed and that a defect at the level of chromosome 15 was discovered. This cell line will certainly be useful for many laboratories in the future. Finally, Beck et al. (SP19) focused their interest in the proper coculture (melanocyte/keratinocyte) cell growth conditions. It is known for a long time that the proper heterophilic cell-cell interactions are crucial to study the normal and pathological behavior of melanocytes. They discover that Green’s medium with collagen I or Defined keratinocyte medium with the plasma polymer facilitate effective co-culture of keratinocytes and melanocytes.


Session V. OXIDATIVE STRESS AND MELANOGENESIS

Chaired by Robert Ballotti, Mauro Picardo, Nico Smit

Contributed by Nico Smit

In this session several factors involved in the induction of oxidative stress in melanocytes were discussed.

Mac Neil and Haycock (Sheffield) presented their work on the different physiological responses of a -MSH especially with regard to its function mediating the defence mechanism of melanocytes against inflammation and oxidative stress. The effect of a -MSH on cAMP levels was measured in melanocyte cultures and melanoma cell lines. When the cAMP pathway is inhibited using an adenosine agonist a calcium response can be found when the cells are treated with a -MSH. This is in accordance with results presented by Hoogduijn in another session (II) who also showed influences on calcium levels in individual cells by a -MSH and ACTH. Other responses of a -MSH may depend on the ratio of the cAMP and calcium signalling. One of these responses next to effects on pigmentation (and tyrosinase activation) is the inhibition of cytokine (TNF-a ) induced NFk B. This induction of NFk B was nicely demonstrated by Dr Haycock using digital imaging of the NFk B/p65 subunit by immunofluorescence microscopy. a -MSH and MSH 11-13 tripeptide were shown to inhibit the induction of NFk B. In HBL melanoma cells this inhibition was strong whereas in C8161 melanoma cells no inhibition was found. The HBL cells show a good cAMP response and a limited calcium response. For the C8161 cells the opposite was found suggesting that the signalling via cAMP or calcium may influence the further biological responses to MSH such as the defence against inflammation and oxidative stress.

A relevance for induction of NFk B in melanocytes was obvious from the paper presented by McNulty (Irvine). In this case the observed increased expression of NFk B in nevi and melanoma could have also been induced by cytokines or directly, as a response to (oxidative) stress. Melanoma cells exhibit constitutive NFk B binding activity by RelA. Nevi showed activated RelA staining in the cytoplasm while metastatic melanomas showed both nuclear and cytoplasmic staining. In comparison to nevi the melanomas also showed reduced staining of NFk B inhibitory proteins Ik B-alpha and –epsilon. This loss of the inhibitory proteins may be responsible for accumulation of RelA in the nucleus.

Next to the effects of oxidative stress at the transcriptional level that influence cell cycle regulation, mutations in certain genes may be responsible for the development of melanoma from the normal melanocyte in skin and atypical nevi. Events that could lead to hypermutability in atypical nevus cells may be related to increased oxidative stress in these cells. This was the topic of the two papers from our laboratory by Smit and Van Nieuwpoort (Leiden). Different fluorescent probes were used for demonstration of hydroperoxides in the cell. The non fluorescent dihydrodichlofluorescein diacetate and dihydrorhodamine 123 can react with intracellular hydrogen peroxide (http://www.probes.com) to form the fluorescent dichlorofluorescein or rhodamine 123. Results indicate that basic levels of ROS detected with these probes are high in cultured melanocytes of skin type VI as compared to skin type I and II melanocytes. Furthermore the melanocytes from an atypical nevus showed higher fluorescence intensity than the corresponding normal melanocytes from the same individual. Hydrogen peroxide may cause high levels of oxidative DNA damage (8-hydroxy-2’-deoxyguanosine) especially when iron is present at the site of the nucleus. Using methylene blue as a photosensitizer very strong induction of 8OHdG was found in lightly pheomelanic M14 melanoma cells after UVA irradiation and much lower levels in the eumelanic An melanoma cells. This could be a result of the photosensitising properties of pheomelanin. The paper by Maresca (Rome) indicates such properties of pheomelanin, since this caused changes in the electrophoretic behaviour of catalase as shown by zymography, whereas eumelanin did not induce any modification.

Session VI. EXTRACUTANEOUS MELANINS

Chaired by Dan-Ning Hu, Giuseppe Prota and Francisco Solano

Contributed by Francisco Solano

The session was opened by Prof. Prota, who emphasized the importance of the advances about the structure and functions of extracutaneous melanins in the context of the new markers identified from oxidative degradation studies (further details in his presentation IL15, Session 1). He then introduced the first speaker, Dr. Sarna, who presented new data concerning the properties and function of ocular melanin. Dr. Sarna (Krakow) and collaborators have characterized melanosomes from RPE donors of very different age by atomic force microscopy. The presentation clearly showed that the surface of melanosomes is coated with lipid material, mainly lipofuscin. This coating material increases a lot with aging, decreasing the accessibility of melanin to putative cytotoxic intracellular compounds. Thus, RPE melanosomes of aged donors loose part of their antioxidant and photoprotective efficiency. Next, Dr. Hu, from the New York Medical College, presented some data also concerning ocular melanins, although the talk was initiated with an emotive mention to the Twin Towers catastrophic event of past September 11th, less than 3 miles away from the Eye and Ear Infirmary Hospital where Dr. Hu is working. He established differences between the two different pigment cells in the eye concerning melanin content, variability with the iris color and expression of the melanogenic enzymes. Using cultures of uveal melanocytes, Dr. Hu has studied the effect of a plethora of agents including growth factors, cytokines, hormones, neurotransmitters and prostaglandins. Most of them affect pigmentation, so that the pathogenesis of changes in the iris color can be explained by alteration of these factors. The last invited lecture of the Session was delivered by Dr. Rosei from Univ. Roma, "La Sapienza", who presented an interesting study of synthetic melanins studied by Micro-Raman spectroscopy in comparison with other models, such as amorphous carbon films. Surprisingly, all synthetic polymers presented two main characteristic bands independently of the preparation method or the presence of peptidic bonds. However, Raman spectra were dependent on the excitation wavelength. In general, the phonon modes of melanins closely resemble those observed in carbon films. Thus, this technique may be used to estimate the size of carbon clusters in this untreatable material.

After the 3 invited lectures, the session accumulated a considerable delay, and the invited presentations were initiated by Dr. Miranda (Rome) presenting the purification and kinetic characterization of a tyrosinase from truffles (Tuber melanosporum). This tyrosinase is one of the scarce plant catechol oxidases presenting tyrosine hydroxylase and dopa oxidase activities. The enzyme is reversibly inhibited by dimethyl-sulfide and methyldithiometane. Both chemicals are present in the endogenous flavour of truffles. So, Dr. Miranda and his Italian team demonstrated that the tyrosinase activity decreases as the fruit matures and becomes flavoured, and the correlation between tyrosinase activity and the color of the truffle, from black to whitish. These studies might have a great economic interest for the truffle's market.

The excursion to plant melanin was terminated to come back to animal melanins with the new presentation, performed by Dr. S. Svensson (Linköping), who presented a method for screening the melanin binding ability of a number of antineoplastic agents. In fact, they used commercial melanin from Sepia officinalis to test the capacity of this polymer to bind the chemotherapeutic agent cisplatin and the antibiotics doxorubicin and daunorubicin. According to the presented results, the first agent did not bind to melanin, but the latter antibiotics do.

Due to the delay accumulated, the session was interrupted for lunchtime. The other two scheduled presentations, by Dr I. Canton (collaboration between Univ. Sheffield, UK and Univ. Basque Country, Spain) and Dr. D.J. Tobin (Bradford Univ, UK) were postponed to the afternoon session. Those presentations dealt with the inhibitory effect of a-MSH on the invasion of ocular melanoma cells and the consideration of the human hair follicle as a suitable target for pro-POMC peptides.


Session VII. MELANOSOMES AND MULTIORGANELLAR DISEASE

Chaired by Ghanem Ghanem and Shosuke Ito

Contributed by Ghanem Ghanem

- First, some most interesting work on melanosome biology and transport has been presented by M. Seabra. The importance of Rab27a (ashen) and Myo5a (dilute) genes in melanosome transport and clustering has been studied in mouse mutant models of Griscelli syndrome. It has been found that Rab27a is associated with melanosomes as well as myosin V as demonstrated by fluorescent antibody studies. However, myosin V (product of Myo5a) did not colocalize with melanosomes in ashen or leaden models. Rab27a is mutated in ashen mice. It has been also found that melanophilin is the product of the leaden gene. On the other hand, Rab27a seems necessary for the secretion of CTL lytic granules leading to a lack in CTL killing activity in Griscelli and ashen models. Likewise itself, myosin Va and Rab27 appear to play a role in melanosome capture at the cell periphery. Rab27a is suggested as a marker for lysosome-related organelles like melanosomes.

- The second contribution, presented by S. Höning, discussed the function of AP3 adaptor complex in melanosome biogenesis. Lysosomal and melanosomal membrane proteins like tyrosinase and TYRPs may interact with cytosolic adaptor complexes AP1® 4. After the binding, coated vesicles may be formed and and transport the proteins from one cellular compartment to another, possibly via endosomes. In melanosomes, AP complexes bind to tyrosinase tail and AP1 and AP2 bind to TYRP-1. AP3 complexes colocalize with tyrosinase and show more than 80% labelling in melanosomes. The authors propose that sorting of tyrosinase to endosomes and melanosomes is a variation of the intracellular membrane proteins sorting to lysosomes.

- K. Lefort presented data supporting a GADD45 activation by UVB specific to melanocytes as compared to skin fibroblasts and keratinocytes. The authors focused on GADD45 functions by also on the analysis of the promoter activity. They were able to localize a minimal promoter region of 50 bp responsible for GADD45 activation by UVB, and that binds to the POU family gene products oct-1 and N-oct3. The latter is poorly expressed in melanocytes leading the authors to conclude to an original alternative mechanism for UVB response in these particular cells.

- A. Calcabrini investigated the resistance mechanisms to melphalan in a series of M14 melanoma cell lines resistant to doxorubicin known to be due to P-gp expression. The authors found by clonogenic assays that the same is true with melphalan and that this resistance can be reversed by the use of cyclosporin A. They suggest that melphalan is actively transported out of the cell by P-gp thus explaining the observed resistance to the drug.

Two additional contributions have been moved from the previous session due to a lack of time and were presented:

- I. Canton showed data suggesting that MSH is able to significantly inhibit the invasion of ocular melanoma cells at concentrations as low as 10-11 M. The authors stressed that MSH is present in the aqueous humour and that its immunosuppressive properties may explain why primary melanoma of the iris is less aggressive than the one arising in the posterior compartment of the eye.

- D.J. Tobin focused on the human hair follicle pigmentary unit and studied the presence to different POMC derived peptides by immunohistochemistry. ACTH, a -MSH and b -endorphin expression was evaluated in gp100 positive melanocytes. The authors concluded that all three immunoreactive peptides are differentially expressed in the fully functioning anagen hair follicle pigmentary unit. ACTH and a -MSH may function during the critical stages of hair follicle regeneration during the hair growth cycle. They finally highlighted the expression of b -endorphin in hair bulb melanocytes explaining the maintenance of high melanogenic activity during the anagen phase.


Session VIII. NEW TRENDS IN MELANOMA-BASIC ASPECTS

Chaired by Jean Marie Naeyaert and Pier Giorgio Natali

Contributed by Jean Marie Naeyaert

Dorothy Bennett (London) discussed cellular senescence in the melanocytic system. Understanding cellular senescence is of major importance since it constitutes a barrier to tumour development. It is controlled by telomere shortening and by the tumour suppressor gene CDKN2A that encodes two distinct cell cycle inhibitory proteins, p16 (INK4a) and p14 (ARF). P16 activates the RB1 pathway while ARF is an activator of the p53 pathway. In mouse melanocytes, p16 can induce senescence without ARF, while immortalized cells expressed ARF but not p16. Two independent strains of p16-null, ARF-positive human melanocytes showed severe abnormalities of senescence. Immortalization was independent of the p53 pathway. This could help explain the rarity of p53 mutations in sporadic melanoma. These studies are in favour of a predominance of the p16/RB1 pathway in effecting melanocyte senescence.

Marco Paggi (Rome) presented work on the in vitro and in vivo tumour growth inhibition by a p16-mimicking peptide in p16-defective, pRB-positive human melanoma cells (A375M). A2058 cells (p16-positive, pRB-defective) were refractory to the action of the p16-mimicking peptide. The authors conclude that p-16 mimicking peptide may be a promising tool for targeted cancer therapy in selected melanoma phenotypes.

Böhm and coworkers (Münster) presented data on the activation status of mitogen-activated protein kinases (MAPK) in melanoma cells in vitro and in situ. Using Western blotting with phospho-specific MAPK antibodies and kinase assays, they demonstrated constitutive activation of MAPK-1 and MAPK-2 in melanoma cells as compared to normal human melanocytes. This was not due to increased protein expression or to suppression of the MKP-1 phosphatase. In situ MAPK-1/2 tyrosine phosphorylation correlated with invasiveness of primary cutaneous melanoma. Melanoma metastases showed a heterogeneous staining pattern.

Malorni et al (Rome) discussed subcellular mechanisms responsible for interferon-induced sensitisation to cisplatin-induced apoptosis in melanoma cells. By CIFN gene transfer into melanoma cells they could demonstrate a direct effect on mitochondria, i.e. a transient hyperpolarisation, that sensitises these cells towards type II apoptotic stimuli (cisplatin, staurosporin, radiation), but not to type I triggers (FAS, TRAIL).

Dr McNulty (Irvine) presented data on the reactive oxygen species (ROS) regulation of NFkB and AP-1 in melanoma cells. It looks like the binding activity of both transcription factors is still responsive to ROS (NFkB to intracellular hydrogen peroxide and AP-1 to superoxide anion). However, there is no concomitant control of apoptosis, maybe providing an explanation for the fact that melanoma cells can escape the noxious injury of chemotherapeutic agents.

The final paper of the Session was presented by Baldi et al (Rome). They performed cDNA array analysis on two cell lines from one patient, one from a primary cutaneous melanoma and from a metastatic lymph node. 31 differentially expressed genes were identified, 27 down-regulated and 4 up-regulated in the metastatic cell phenotype. The authors chose a down-regulated gene, PRSS11, for further study. Its gene product is a human serine protease homologue of the E coli protease htrA. Metastatic clones stably transfected with PRSS11 cDNA showed a significant decrease in proliferation and chemotaxis. The studies suggest that decrease of PRSS11 expression could be an indicator of melanoma aggressiveness.


Session IX. DIAGNOSTIC, PROGNOSIS AND TREATMENT OF MELANOMA

Chaired by Jean François Doré, Silvia Moretti and Alain Taieb

Contributed by Alain Taieb

K. Teuchner et al (Berlin) presented a promising new diagnostic technique based on femtosecond pulse stepwise two photon excitation of the fluorescence of pigmented tissues. This mode of excitation yields a selective fluorescence for melanin in tissues in vivo, which has been applied to clinical samples including excised primary melanomas and melanocytic nevi. There is a modification of the fluorescence spectrum, with a pronounced shift in the red for primary melanomas. The distinction between nevus and melanoma remains to be studied on a larger scale.

The strategies used in chemotherapy to obtain a better efficacy/tolerance ratio could be influenced by the studies presented by Leonetti et al (Rome), which suggest that the concomitant administration of vitamin E (4,3 mg/kg) and cisplatin in mice injected with a human melanoma cell line reduces the overall toxicity of cisplatin and more specifically its peripheral nerve toxicity, without affecting its efficacy. If these findings can be generalized, they offer a simple and readily available therapeutic improvement in the management of metastatic melanoma.

A systematic comparison of cutaneous and extracutaneous melanoma is currently carried out at the Karolinska Institute of Stockholm and was presented by B. Ragnarsson-Olding et al. New data were presented at this meeting concerning the rare vulvar melanomas as compared to common skin melanomas for p53 gene mutations and protein expression studied by immunohistochemistry. The data were similar, with immunopositivity in 42% of cases and controls as wells as no differences for mutations. This type of approach, although here negative, may provide clues for the understanding of the molecular mechanisms involved in melanoma, especially to detect environmental influences such as UV irradiation.