Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes.

Enlarged fat cells exhibit modified metabolic capacities, which could be involved in the metabolic complications of obesity at the whole body level. We show here that sterol regulatory element-binding protein 2 (SREBP-2) and its target genes are induced in the adipose tissue of several models of rodent obesity, suggesting cholesterol imbalance in enlarged adipocytes. Within a particular fat pad, larger adipocytes have reduced membrane cholesterol concentrations compared with smaller fat cells, demonstrating that altered cholesterol distribution is characteristic of adipocyte hypertrophy per se. We show that treatment with methyl-beta-cyclodextrin, which mimics the membrane cholesterol reduction of hypertrophied adipocytes, induces insulin resistance. We also produced cholesterol depletion by mevastatin treatment, which activates SREBP-2 and its target genes. The analysis of 40 adipocyte genes showed that the response to cholesterol depletion implicated genes involved in cholesterol traffic (caveolin 2, scavenger receptor BI, and ATP binding cassette 1 genes) but also adipocyte-derived secretion products (tumor necrosis factor alpha, angiotensinogen, and interleukin-6) and proteins involved in energy metabolism (fatty acid synthase, GLUT 4, and UCP3). These data demonstrate that altering cholesterol balance profoundly modifies adipocyte metabolism in a way resembling that seen in hypertrophied fat cells from obese rodents or humans. This is the first evidence that intracellular cholesterol might serve as a link between fat cell size and adipocyte metabolic activity.

Extensive molecular screening for hereditary non-polyposis colorectal cancer.

The genetic abnormalities underlying hereditary non-polyposis colorectal cancer (HNPCC) are germline mutations in one of five DNA mismatch repair genes or in the TGFbetaRII gene. The aim of our study was to evaluate the significance of simple tests performed on tumours to select appropriate candidates for germline mutational analysis. We studied three groups of patients, HNPCC kindreds fulfilling the International Collaborative Group (ICG) criteria (n = 10), families in which at least one of the criteria was not satisfied (n = 7) and sporadic colorectal cancer (CRC) diagnosed before the age of 50 (n = 17). We searched for microsatellite instability (MSI), presence of hMSH2 and hMLH1 germline mutations, expression of hMSH2, hMLH1 and p53 proteins in tumoural tissue samples by immunostaining. Fifteen out of 17 (88%) of HNPCC and incomplete HNPCC cases were MSI and eight pathogenic germline mutations in hMSH2 or hMLH1 were detected in these two groups (53%). All the 17 early-onset sporadic cases were MSS and no germline mutations were detected among the seven investigated cases. Thirteen out of 15 (81%) familial cases were MSI and p53 protein-negative, whereas 13/14 (93%) sporadic cases were MSS and strongly p53 protein-positive. This extensive molecular investigation shows that simple tests such as MS study combined with hMSH2 and hMLH1 protein immunostaining performed on tumoural tissues may provide valuable information to distinguish between familial, and probably hereditary, and sporadic CRC cases.

Identification and characterization of cvHsp. A novel human small stress protein selectively expressed in cardiovascular and insulin-sensitive tissues.

Starting with computational tools that search for tissue-selective expression of assembled expressed sequenced tags, we have identified by focusing on heart libraries a novel small stress protein of 170 amino acids that we named cvHsp. cvHsp was found as being computationally selectively and highly (0.3% of the total RNA) expressed in human heart. The cvHsp gene mapped to 1p36.23-p34.3 between markers D1S434 and D1S507. The expression of cvHsp was analyzed with RNA dot, Northern blots, or reverse transcription-polymerase chain reaction: expression was high in heart, medium in skeletal muscle, and low in aorta or adipose tissues. In the heart of rat models of cardiac pathologies, cvHsp mRNA expression was either unchanged (spontaneous hypertension), up-regulated (right ventricular hypertrophy induced by monocrotaline treatment), or down-regulated (left ventricular hypertrophy following aortic banding). In obese Zucker rats, cvHsp mRNA was increased in skeletal muscle, brown, and white adipose tissues but remained unchanged in the heart. Western blot analysis using antipeptide polyclonal antibodies revealed two specific bands at 23 and 25 kDa for cvHsp in human heart. cvHsp interacted in both yeast two-hybrid and immunoprecipitation experiments with alpha-filamin or actin-binding protein 280. Within cvHsp, amino acid residues 56-119 were shown to be important for its specific interaction with the C-terminal tail of alpha-filamin.

Overexpression of MDM2, due to enhanced translation, results in inactivation of wild-type p53 in Burkitt's lymphoma cells.

Numerous studies have indicated that inactivation of p53 is one of the essential requirements for the unrestrained growth of tumoral cells. When the status of the p53 gene was examined in various types of lymphoid malignancies, mutations in p53 have been predominantly detected in Burkitt's lymphoma (BL) cells, therefore suggesting that alteration of p53 could specifically contribute to the malignant phenotype of these tumoral cells. In addition to mutations, functional inactivation of p53 can also occur through interaction of the wild-type gene product with various viral or cellular proteins. The cellular MDM2 protein, for example, is able to inhibit p53 tumor suppressor function by concealing its transactivation domain. Mdm2 gene amplification has been described in several types of sarcomas, resulting in overexpression of the MDM2 protein. In this study, we have examined the status of MDM2 and p53 in 20 BL cell lines. Four were found to contain wild-type p53 and to overexpress MDM2 protein. Within these BL cells, both molecules are physically associated since they can be co-precipitated and p53 is inactivated as cells neither arrest in G1 nor enter apoptosis following gamma-radiation. We also report that the high level of the MDM2 protein in BL cells is neither associated with an amplification of the mdm2 gene nor with an elevated level of RNA or an increased protein stability, but is rather due to an enhanced translation ability of the mdm2 RNA. These results indicate that in certain BL cells, overexpression of MDM2 protein regulated at the posttranscriptional level, induces an escape from p53-controlled cell growth.

Primary proliferative T cell response to wild-type p53 protein in patients with breast cancer.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human tumors. There are mainly point mutations that lead to single amino acid substitutions. The mutated proteins have a longer half-life than wild-type p53 and accumulate in the nucleus of tumor cells. Anti-p53 antibodies have been found in sera of patients with several types of cancers including breast cancer. This report describes a T cell immune response in three patients with breast tumors who had mutated p53 gene and accumulated p53 protein. All showed a humoral response to p53 protein and the T cells of these patients recognized the wild-type p53 protein and proliferated in response to it. The data reported here are relevant to the immune processes leading to autoimmunity and have a bearing on anti-p53 vaccine development in tumor immunology.