Expression of NG2/human melanoma proteoglycan in human adult articular chondrocytes.
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OBJECTIVE: NG2 is a transmembrane chondroitin sulfate (CS) rich proteoglycan originally identified in rats. It has recently been shown to be identical to human melanoma proteoglycan (HMPG). In rats NG2 has a limited distribution in adult tissues, being expressed predominantly by neuronal and glial cells whereas during development it is also expressed in developing mesenchyme including cartilage. NG2/HMPG has putative roles in interactions between glial and melanoma cells with extracellular matrix (ECM) molecules. This study was undertaken to assess whether NG2/HMPG was expressed by normal and osteoarthritic human articular chondrocytes. DESIGN: Cryostat sections of human fetal knee joints and normal and osteoarthritic articular cartilage were immunostained with antibodies against rat NG2 (N143.8) and HMPG (M28B5, 9.2.27). Immunoprecipitation and Western blotting was carried out on protein extracts of chondrocytes from normal and osteoarthritic cartilage. Immunofluorescence of NG2 and potential ligands was carried out in vitro on cells from normal and osteoarthritic cartilage. RESULTS: Fetal and both normal and osteoarthritic adult cartilage showed strong immunoreactivity for NG2/HMPG. Western blotting showed a smeared component of molecular weight greater than 400 kDa and a faint band at 250 kDa which became predominant upon digestion with chondroitinase ABC. Immunofluorescence of chondrocytes in vitro showed NG2 to be distributed in a punctate pattern without co-localization of actin or several ECM proteins including fibronectin and type VI collagen. CONCLUSION: NG2/HMPG is expressed by human fetal and adult chondrocytes and in adult articular chondrocytes the core protein is chondroitin sulfated. The function of this molecular in human articular cartilage remains to be defined. (+info)
Plasma DNA microsatellites as tumor-specific markers and indicators of tumor progression in melanoma patients.
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Multiple DNA microsatellites with frequent loss of heterozygosity (LOH) in melanomas have been demonstrated. The finding that free DNA is enriched in blood of melanoma patients prompted studies to determine whether tumor-specific DNA, such as DNA microsatellites exhibiting LOH, can be detected in blood and have clinical use. In this study, 57 advanced and 19 early clinically staged melanoma patients were assessed using 10 microsatellite markers on six chromosomes. Matched plasma and melanoma tissues from 40 patients showed significant concordance of LOH (P < 0.0001). The frequency of LOH microsatellite markers detected in plasma significantly increased in more advanced-staged patients. At locus D3S1293, LOH detection showed significant correlation to clinical disease progression (P = 0.02). Additionally, the combination of LOH microsatellite markers D9S157 and D3S1293 (P = 0.01), D9S157 and D1S228 (P = 0.05), and D11S925 and D3S1293 (P = 0.01) were significantly correlated to progression of different clinical stages of disease. These studies indicate that tumor-specific LOH markers in plasma have a potential clinical use as diagnostic and prognostic markers in melanoma patients. (+info)
Hypoxia-induced up-regulation of angiogenin in human malignant melanoma.
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Angiogenesis is essential for tumor progression and metastasis, however, the angiogenesis regulators that are biologically relevant for human melanoma are still unknown. In this study, we analyzed the expression of the potent angiogenic factor angiogenin (ANG) in human melanoma in vitro and in vivo. Four different human melanoma cell lines and two normal melanocytes were kept either under normoxic or hypoxic conditions. After 24 h of hypoxic culture conditions, ANG was up-regulated in the melanoma cell lines but not in normal melanocytes. Induction levels correlated with the metastatic potential of the cell lines. These data were confirmed by Northern blot analysis. In contrast, induction of vascular endothelial growth factor by hypoxia was equally strong in the examined highly aggressive melanoma cell lines and in one nonaggressive cell line. Other angiogenic factors tested as well as the melanoma growth stimulatory activity (Gro-alpha) showed no up-regulation. Thus, in the present study, hypoxia-induced up-regulation in melanoma cells was only observed for ANG and vascular endothelial growth factor. Immunohistochemical studies showed that 8 of 10 melanomas and all 15 metastases were positive for ANG, particularly in the vicinity of small vessels, whereas all benign nevi were negative. Reverse transcription-PCR detected only weak ANG mRNA in nevi but strong signals in primary melanomas and metastases. In conclusion, we demonstrate for the first time enhanced expression of ANG in highly metastatic cell lines as well as in melanomas and metastases in vivo, suggesting that ANG expression is associated with the metastatic potential. (+info)
Molecular analysis of HLA-DR gene expression induced by IFN-gamma in malignant melanoma cell lines.
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Human leukocyte antigen (HLA) class II molecules are polymorphic cell surface glycoproteins that are crucial for the cellular interaction in immune response. The expression of class II molecules is regulated in a tissue-specific and cytokine-inducible manner, and is mainly restricted to the antigen presenting cells. However, some tumor cells also express class II molecules, and in some class-II-negative tumor cells, class II expression is inducible by interferon (IFN)-gamma. However, their expression varies, even though the tumor cells originate from the same histological origin; some tumor cells show strong expression, others show weak or no expression. To determine whether this differential expression of class II molecules on tumor cells is transcriptionally regulated, FACS analysis and Northern hybridization were performed using a panel of melanoma cell lines, IGR3, Malme-3M, SK-Mel-24, and SK-Mel-28 to analyze the cell surface expression and mRNA transcription rate of HLA-DR before and after treatment with IFN-gamma. FACS analysis showed that before IFN-gamma treatment, IGR3 and Malme-3M cells barely expressed HLA-DR. On the contrary, almost all of the SK-Mel-24 cells (> 90%) and a relatively high rate (> 50%) of SK-Mel-28 cells expressed HLA-DR. After IFN-gamma treatment, HLA-DR expression was induced in Malme-3M cells and SK-Mel-28 cells which displayed elevated levels of HLA-DR expression in a time-dependent manner. However, IGR3 cells never responded to IFN-gamma. Northern analysis showed that treatment with IFN-gamma led to the steady-state mRNA augmentation of the HLA-DR gene in Malme-3M and SK-Mel-28, whereas in IGR3, IFN-gamma did not augment the transcriptional rate of the HLA-DR gene. To further clarify this differential modulation, sequencing analysis of PCR product of the HLA-DR proximal promoter region was done, since the transcription rate of the class II gene is controlled by the well-conserved proximal promoter region. Six independent clones from PCR products of the HLA-DRA proximal promoter region and 16 clones from PCR products of the HLA-DRB proximal promoter region were isolated from the above cell lines and sequenced. Comparison of the nucleotide sequences of all 6 clones of DRA promoter showed that the sequences are extremely similar in both regulatory sequences and their intervening sequences. Sixteen clones of HLA-DRB promoter showed sequence variations such as substitution and insertion/deletion, and these 16 clones could be further grouped into 6 homologues with sequence homology. These data established that the melanoma cell lines studied here showed a differential susceptibility to IFN-gamma on the modulation of HLA-DR molecules, that this modulation is transcriptionally regulated, and that the difference in promoter activity by sequence variation might contribute to such a differential transcriptional regulation at the promoter level. (+info)
Neurology and the skin.
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As knowledge of pathophysiology grows, so does the refinement of diagnoses. Sometimes increased knowledge permits consolidation and unification. Unfortunately, at our present level of understanding, it usually demands proliferation of diagnostic categories. As tedious as this diagnostic splintering may seem, such is the price currently exacted of both the investigator and the clinician who seek to optimise management. Increased diagnostic refinement often requires inquiry into matters outside the bounds of one's specialty. Most often we turn to the radiologist or to the laboratory to narrow the differential diagnosis generated from the history and neurological examination. As we have shown, a useful intermediate step is extension of the physical examination to organs such as the skin, which are not the traditional preserve of the neurologist. That any text could confer the sophistication required for expert dermatological diagnosis is an unrealistic expectation. However, we hope that this review will encourage careful examination of the skin, hair, and nails by the neurological practitioner, with consideration of referral to a dermatologist when greater expertise is required. (+info)
Somatic mutations in the Peutz-Jeghers (LKB1/STKII) gene in sporadic malignant melanomas.
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Germline mutations in the LKB1/STK11 gene cause characteristic hamartomas and freckling to develop in patients with Peutz-Jeghers syndrome (PJS). The hamartomas arise as a result of somatic "second hits" at LKB1/STK11 and therefore contain a neoplastic element. The origin of the pigmented lesions in PJS is unknown and difficult to test, as these are hardly ever biopsied. PJS patients are at increased risk of benign and malignant tumors, particularly of the colon, breast, pancreas, testis, and ovary, although the increased risk for any one of these sites may be quite modest. Somatic LKB1/STK11 mutations have been found, albeit at a low frequency, in sporadic tumors of the colon, stomach, ovary, and testis. Although PJS patients are not known to have an excess of skin tumors, if the freckles of PJS patients are actually small, benign tumors, LKB1/STK11 mutations must provide these lesions with a selective advantage, and similar mutations might also give a selective advantage to related malignant tumors, such as melanomas. We have therefore screened 16 melanoma cell lines, 15 primary melanomas, and 19 metastases for LKB1/STK11 mutations. Two LKB1/STK11 mutations were found: a missense change (Y49D) accompanied by allele loss in a cell line; and a missense change (G135R), without a detected mutation in the other allele, in a primary tumor. Both these mutations are highly likely to be pathogenic. Novel polymorphisms, including an unusual heptanucleotide repeat, were also found in introns 2 and 3. LKB1/STK11 mutations occur in a significant minority of tumors of several sites, including malignant melanomas. (+info)
Color Doppler sonography of focal lesions of the skin and subcutaneous tissue.
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We evaluated with color Doppler sonography 71 visible and palpable nodules of the skin and subcutaneous tissue from 51 patients. The nodules were classified as avascular (type I), hypovascular with a single vascular pole (type II), hypervascular with multiple peripheral poles (type III), and hypervascular with internal vessels (type IV). Of the 32 malignant nodules, 9% showed a type I pattern, 50% had a type III pattern, and 41% had a type IV pattern; of the 39 benign nodules, 86% showed a type I pattern and 14% had a type II pattern. The sensitivity and specificity of hypervascularity in malignant lesions were 90% and 100%, respectively, whereas the sensitivity and specificity of hypovascularity in benign lesions were 100% and 90%, respectively. The authors conclude that color Doppler sonography is able to increase the specificity of ultrasonography in the evaluation of nodular lesions of the skin. (+info)
Somatic mutation of the Peutz-Jeghers syndrome gene, LKB1/STK11, in malignant melanoma.
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Mutations in LKB1/STK11, a gene mapping to chromosome 19p13.3 and encoding a widely expressed serine/threonine kinase, were recently identified as the cause of Peutz-Jeghers syndrome. Despite the hamartomatous polyps and increased cancer risk associated with this syndrome, somatic alterations in LKB1/STK11 have not been identified in human tumours. Prompted by another feature of the syndrome, lentigines of the lips and oral mucosa, we evaluated the status of LKB1/STK11 expression, deletion, and mutation in cell lines and tumour samples from 35 patients with sporadic malignant melanoma. Two somatic mutations were identified, a nonsense mutation (Glu170Stop) causing exon skipping and intron retention, and a missense mutation (Asp194Tyr) affecting an invariant residue in the catalytic subunit of LKB1/STK11. Our data suggest that LKB1/STK11 may contribute to tumorigenesis in a small fraction of malignant melanomas. (+info)