To evaluate the mutational profiles associated with BRAF mutations in human melanoma, we have studied BRAF, RAS, PTEN, TP53, CDKN2A and CDK4 genes and their expression in melanoma lesions. Owing to the lack of sufficient material from fresh specimens, we employed short-term cell lines obtained from melanoma biopsies. In all, 41 melanoma obtained from eight primary lesions, 20 nodal, 11 cutaneous and two visceral metastases from patients with sporadic (n ¼ 31), familial (n ¼ 4) and multiple melanoma (n ¼ 2) were analysed. The results revealed novel missense mutations in the BRAF, PTEN, CDKN2A and CDK4 genes. Overall, activating mutations of BRAF and loss of functional p16 and ARF were detected in the majority of melanomas (29/41, 36/41 and 29/41, respectively), while PTEN alterations/loss, NRAS and TP53 mutations occurred less frequently (6/41, 6/41 and 10/41, respectively). In the resulting 12 mutational profiles, p16/ARF loss associated with mutated BRAF V599E was the most represented (n ¼ 15). In addition, TP53 and PTEN mutations were always accompanied with BRAF alterations, while PTEN loss was found in association with CDKN2A or TP53 mutations in the absence of BRAF activation. The p16/ARFD þ BRAF/ RAS profile was significantly associated with a longer survival, while complex mutational profiles were detected in highly aggressive disease and poor survival. These data support the existence of several molecularly defined melanoma groups which likely reflect different clinical/ biological behaviour, thus suggesting that a more extensive molecular classification of melanoma would significantly impact its clinical management.

The DHCR24 gene encoding for the 3β‐hydroxysterol Δ24‐reductase, an oxidoreductase involved in cholesterol biosynthesis, was isolated by subtractive hybridization as highly expressed in a short‐term melanoma cell line derived from a cutaneous metastases (S/M2) compared to that obtained from the autologous primary tumor (S/P). DHCR24 (alias seladin‐1, diminuto/dwarf1 homolog) has been reported to act as an antiapoptotic factor in neurons. Gene expression analysis by Northern blot confirmed that DHCR24 was 5‐fold upregulated in S/M2 compared to S/P cells. High levels of DHCR24 gene expression were detected in 13/25 melanoma metastases and in 1/7 primary melanomas by real‐time PCR, indicating that upregulation of this gene may occur in melanoma progression. In S/M2 cells, high DHCR24 gene expression associated with resistance to apoptosis triggered by oxidative stress induced by exposure to hydrogen peroxide. DHCR24 gene transfer was shown to protect melanoma cells from H2O2‐induced cytotoxicity. Although higher cholesterol levels were shown in S/M2 cells compared to S/P cells, DHCR24 gene transfer did not increase cholesterol content. To evaluate whether DHCR24 acts as an antiapoptotic factor in melanoma metastases, the cytotoxic effect of chemotherapeutic agents was tested in DHCR24 transfectants and in the presence of a DHCR24 inhibitor, U18666A. High DHCR24 gene expression in transfectants did not result in a higher resistance to cytotoxic agents; treatment with U18666A was cytotoxic in S/P cells with a lower DHCR24 content and showed additive cytotoxic effect only when associated with H2O2 and not with cysplatin or etoposide, indicating that the DHCR24 protective effect is exerted through an oxidative stress‐specific mechanism. © 2005 Wiley‐Liss, Inc.

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Objective:Lipid accumulation in skeletal muscle and the liver is strongly implicated in the development of insulin resistance and type 2 diabetes, but the mechanisms underpinning fat accrual in these sites remain incompletely understood. Accumulating evidence of muscle mitochondrial dysfunction in insulin-resistant states has fuelled the notion that primary defects in mitochondrial fat oxidation may be a contributory mechanism. The purpose of our study was to determine whether patients with congenital lipodystrophy, a disorder primarily affecting white adipose tissue, manifest impaired mitochondrial oxidative phosphorylation in skeletal muscle.Research Design and Methods:Mitochondrial oxidative phosphorylation was assessed in quadriceps muscle using 31P-magnetic resonance spectroscopy measurements of phosphocreatine recovery kinetics after a standardized exercise bout in nondiabetic patients with congenital lipodystrophy and in age-, gender-, body mass index-, and fitness-matched controls.Results:The phosphocreatine recovery rate constant (k) was significantly lower in patients with congenital lipodystrophy than in healthy controls (P < 0.001). This substantial (∼35%) defect in mitochondrial oxidative phosphorylation was not associated with significant changes in basal or sleeping metabolic rates.Conclusions:Muscle mitochondrial oxidative phosphorylation is impaired in patients with congenital lipodystrophy, a paradigmatic example of primary adipose tissue dysfunction. This finding suggests that changes in mitochondrial oxidative phosphorylation in skeletal muscle could, at least in some circumstances, be a secondary consequence of adipose tissue failure. These data corroborate accumulating evidence that mitochondrial dysfunction can be a consequence of insulin-resistant states rather than a primary defect. Nevertheless, impaired mitochondrial fat oxidation is likely to accelerate ectopic fat accumulation and worsen insulin resistance.