Analysis of N- and K-Ras Mutations in the Distinctive Tumor Progression Phases of Melanoma

Mutations in the ras genes are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in cutaneous melanoma. To investigate whether these mutations occur in early or late tumor progression phases, we searched for point mutations in the N- and K-ras genes in 69 primary cutaneous melanoma, 35 metastases, and seven nevocellular nevi in association with cutaneous melanoma. Lesions were microdissected in order to procure pure tumor samples from the distinctive growth phases of the cutaneous melanoma; the very sensitive denaturing gradient gel electrophoresis technique was used to visualize the mutations, and was followed by sequencing. Point mutations in the N-ras gene but not in the K-ras gene were detected on denaturing gradient gel electrophoresis. Twenty-three primary (33%) and nine metastatic (26%) melanomas showed bandshifts for N-ras. In the majority of cases, mutations occurring in early growth phases (i.e., the “intraepidermal” radial growth phase), were preserved in later growth phases (i.e., the invasive radial growth phase, vertical growth phase, and metastatic phase), which proves the clonal relationship between the successive growth phases. In three cases, however, the mutations differed between the distinctive growth phases within the same cutaneous melanoma, due to the occurrence of an additional mutation (especially in codon 61) in a later tumor progression phase. Our approach also permitted us to analyze the mutational status of nevi, associated with cutaneous melanoma. Six out of seven associated nevi carried the same sequence (mutated or wild-type) as the primary cutaneous melanoma, whereas in one case the sequence for N-ras differed between the primary melanoma and the associated nevus. In conclusion, this approach allowed us to demonstrate the clonal relationship between subsequent growth phases of melanoma and associated nevi; our results suggest that N-ras exon 1 mutations preferentially occur during early stages of tumor progression and hence may be involved in melanoma initiation, whereas those in N-ras exon 2 are found preferentially during later stages and hence are more probably involved in metastatic spread of cutaneous melanoma

Treatment of onychomycosis with a solution of miconazole 2% in alcohol

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Contact sensitivity to rubber additives in Belgium

The frequency of positive patch tests to rubber additives in Belgium is recorded. Allergy due to rubber protective golves represents 41% of the overall sensitivity to rubber.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Why are some patients with onychomycosis still not responding to the newer antifungal agents ?the challenge in the near future

Poster. 7th Congress of the European Academy of Dermatology and Venereology (Nice, 7-11.9.1998FLWINinfo:eu-repo/semantics/publishe

1,25-Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis, Jun kinase activation, and interleukin-6 production in primary human keratinocytes

We investigated the capacity of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)-irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)(2)D(3) on UVB-irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)(2)D(3) suppressed UVB-induced apoptotic cell death. An ELISA, detecting DNA-fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM for 24 h reduced UVB-stimulated apoptosis by 55-70%. This suppression required pharmacological concentrations 1,25(OH)(2)D(3) and a preincubation period of several hours. In addition, 1,25(OH)(2)D(3) also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB-induced apoptosis. Furthermore, we demonstrated that 1,25(OH)(2)D(3) reduced two important mediators of the UV-response, namely, c-Jun-NH(2)-terminal kinase (JNK) activation and interleukin-6 (IL-6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM diminished UVB-stimulated JNK activation with more than 30%. 1,25(OH)(2)D(3) treatment (1 microM) reduced UVB-induced IL-6 mRNA expression and secretion with 75-90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D(3) and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB-induced skin damage and carcinogenesis.status: publishe

Loss of membranous expression of β-catenin is associated with tumor progression in cutaneous melanoma and rarely caused by Exon 3 mutations

beta-Catenin plays a fundamental role in the regulation of the E-cadherin-catenin cell adhesion complex. It also plays a role in the Wnt signaling pathway by activating T-cell factor- and lymphoid enhancer factor-regulated gene transcription. The level of beta-catenin in cells is tightly controlled in a multiprotein complex, and mutations in the glycogen synthase kinase 3beta (GSK-3beta) phosphorylation sites of the beta-catenin gene (CTNNB1) result in nuclear and/or cytoplasmic accumulation of beta-catenin and constitutive transactivation of T-cell factor and lymphoid enhancer factor target genes, a mechanism occurring in many cancers. Melanoma cell lines may harbor beta-catenin mutations; in vivo, however, cellular accumulation of beta-catenin is rarely caused by CTNNB1 mutations. In our study, 43 primary cutaneous melanoma and 30 metastases were screened for CTNNB1 exon 3 mutations by using a denaturing gradient gel electrophoresis technique and sequencing. beta-Catenin mutations were found in 2 primary melanomas and 1 metastatic melanoma and were not correlated with nuclear accumulation of beta-catenin in these cases. Cellular expression of beta-catenin was evaluated by immunohistochemistry and by reverse polymerase chain reaction (RT-PCR) in 80 and 70 cases, respectively. Immunohistochemistry revealed a significant loss of membranous P-catenin staining between the primary and metastatic melanomas as well as between radial and vertical growth phase. RT-PCR showed a significant inverse correlation between the amount of RNA and the proportion of cells with membranous expression of beta-catenin (P = .0015); no correlation existed between the amount of RNA and the number of cells with nuclear or cytoplasmic expression of G-catenin. In conclusion, nuclear expression of beta-catenin is seen in cutaneous melanoma but, in contrast to the case of many other cancers, does not correlate with tumor stage or mutation status. A combination of immunohistochemistry and RTPCR showed that down-regulation of membranous beta-catenin was associated with an increased amount of beta-catenin RNA in primary or metastatic melanoma. Our results suggest that posttranslational events, rather than CTNNB1 mutations, are responsible for the altered distribution of beta-catenin in cutaneous melanoma