Faculty Bibliography

We recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential. With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content and increased responsiveness to MSH compared to parental cells. Here we investigated the hybrid melanotic phenotype in more detail, comparing the pigmentary systems of hybrids and parental Cloudman S91 cells by several techniques. Cells were studied by electron microscopy, cell lysates were analyzed for tyrosinase (E.C.1.14.18.1) activity, and melanosomal proteins were analyzed by gel electrophoresis and immunoblotting. Melanosomes in parental Cloudman melanoma cells were few in number and relatively amorphous, whereas those in the hybrids were numerous and heavily pigmented, containing highly organized lattice structures. Both basal and MSH-inducible tyrosinase activities were elevated several fold in hybrids compared to parental cells. Tyrosinase, TRP-2, and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages, which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. By using 3H-glucosamine as a marker of N-glycosylation, its incorporation into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors, and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids

BACKGROUND: The pathogenesis of infantile hemangiomas is not yet understood. Growth factors and hormonal and mechanical influences have been thought to affect the focal abnormal growth of endothelial cells in these lesions. However, these influences may represent secondary responses to an underlying primary molecular event leading to the development of hemangiomas. OBSERVATIONS: We report the rare familial occurrence of hemangiomas and/or vascular malformations in 6 kindreds, suggesting autosomal dominant inheritance. In these families, multiple generations (2-4) were affected by hemangiomas or vascular malformations. In contrast to the generally accepted female-male ratio of 3:1 to 4:1 associated with sporadic hemangiomas, the families with hemangiomas in our study demonstrated a 2:1 ratio. Additionally, vascular malformations and hemangiomas were present in different members of the same family. The vascular lesions appeared to be transmitted in an autosomal dominant fashion with moderate to high penetrance. CONCLUSIONS: We have identified 6 families demonstrating autosomal dominant segregation of childhood hemangiomas. Additionally, family members with vascular malformations were identified in these kindreds. Physicians caring for children with hemangiomas and vascular malformations should include in their medical histories inquiries about vascular lesions in other family members, even when obvious lesions are not present in the parents. The identification of the mutation(s) underlying vascular lesions will provide insight into the pathogenesis of these familial hemangiomas and, potentially, common sporadic hemangiomas. In addition, such research would shed light on the regulation of angiogenic processes during development

Malignant cutaneous melanomas and metastases were taken directly from in situ lesions of genetically identical (C57BL/6 strain) Tyr-SV40E transgenic mice, and samples were analyzed by Western immunoblotting with antisera specific for the COOH terminus of each of four melanocytic proteins. These were tyrosinase, TRP-1, TRP-2, and Pmel 17/silver. Of the 13 melanomas examined, there were 5 melanotic primary tumors, 5 amelanotic primary tumors, and 3 amelanotic metastases. The melanotic tumors expressed all of the markers to some extent. In contrast, the amelanotic tumors lacked detectable levels of one, two, or three of the proteins, except for an apparently amelanotic tumor sample in which all were expressed, but in which some melanotic cells were likely to have been present. Thus, despite some variability, there is clearly a downward trend in the presence of these proteins as the tumors become amelanotic, a pigmentary change associated with ongoing malignant progression. In the amelanotic tumors, tyrosinase was most often deficient, whereas TRP-2 was most often persistently expressed. These results, obtained from melanomas of syngeneic origin, indicate that tumors in the relatively early stages of malignancy might be more responsive than later-stage tumors to immunotherapy involving an ensemble of antigenic peptides of the tested gene products. Moreover, TRP-2 peptides may be especially useful for therapeutic intervention at the later stages

Mitf (Microphthalmia transcription factor), a basic-helix-loop-helix zipper protein, encoded at the microphthalmia (Mitf) locus, regulates the transcription of the gene encoding tyrosinase, the rate-limiting enzyme in melanin biosynthesis, by binding the DNA sequence CATGTG. This binding site is present also in the genes encoding two tyrosinase related proteins, TRP-1 and TRP-2. To gain insight into the function of Mitf in vivo, we determined whether there was a difference in the levels of these proteins in the RPE/choroid of the vitiligo (Mitfvit) mouse, in which there is a mutation of the Mitf gene. This mouse has alteration of RPE pigmentation and function that presumably leads to slow progressive loss of photoreceptor cells. The RPE/choroid was dissected from eyes of vitiligo and C57BL/6 wild-type mice at postnatal ages 2, 4, 7, 10, 14, 21 and 42 days. Extracts of pooled tissues were subjected to electrophoresis and immunoblotting. The levels of tyrosinase, TRP-1 and TRP-2 were determined densitometrically following immunodetection with rabbit antipeptide antisera. In addition, the tyrosine hydroxylase activity of tyrosinase as assayed radiometrically. Levels of TRP-1 were 3-7 fold greater in control RPE/choroid compared with mutants. This marked difference in protein level was observed at the earliest age examined (P2) and persisted throughout the first two weeks. Tyrosinase levels in mutants were similar to controls at P2 and P4, but were reduced at P10 and beyond. Tyrosinase activity was diminished also in mutants by P10. Levels of TRP-2 were similar between mutants and controls, although the typical decrease seen in controls after P14 was attenuated in the mutant mice. There is a significant reduction in the level of TRP-1 in the RPE/choroid of the Mitfvit mouse. The data suggests that transcription of the gene encoding TRP-1 is extremely dependent upon functional Mitf. It provides in vivo evidence that Mitf regulates the transcription of the gene encoding TRP-1 as well as tyrosinase

X-linked ocular albinism (OA1), Nettleship-Falls type, is characterized by decreased ocular pigmentation, foveal hypoplasia, nystagmus, photodysphoria, and reduced visual acuity. Affected males usually demonstrate melanin macroglobules on skin biopsy. We now report results of deletion and mutation screening of the full-length OA1 gene in 29 unrelated North American and Australian X-linked ocular albinism (OA) probands, including five with additional, nonocular phenotypic abnormalities (Schnur et al. 1994). We detected 13 intragenic gene deletions, including 3 of exon 1, 2 of exon 2, 2 of exon 4, and 6 others, which span exons 2-8. Eight new missense mutations were identified, which cluster within exons 1, 2, 3, and 6 in conserved and/or putative transmembrane domains of the protein. There was also a splice acceptor-site mutation, a nonsense mutation, a single base deletion, and a previously reported 17-bp exon 1 deletion. All patients with nonocular phenotypic abnormalities had detectable mutations. In summary, 26 (approximately 90%) of 29 probands had detectable alterations of OA1, thus confirming that OA1 is the major locus for X-linked OA

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

We evaluated the frequency of an association of cutaneous cervicofacial hemangiomas in a 'beard' distribution (including the preauricular areas, chin, anterior neck, and lower lip) with symptomatic hemangiomas of the upper airway or subglottic areas. Of 529 patients seen, 187 were pediatric patients with hemangiomas of the head and neck. Sixteen of the 187 patients (8.5%) had cutaneous lesions with a beard distribution, with a score of 4 or greater. Ten of these 16 (63%) patients had some degree of symptomatic airway involvement, and four of the 10 (40%) required tracheotomy. The presence of cutaneous hemangiomas in a beard distribution should alert the evaluating physician to the potential association of upper airway or subglottic involvement