International distribution and age estimation of the Portuguese BRCA2 c.156_157insAlu founder mutation.

The c.156_157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement in a total of 5,443 suspected HBOC families from several countries. Whereas the c.156_157insAlu BRCA2 mutation was detected in 11 of 149 suspected HBOC families from Portugal, representing 37.9% of all deleterious mutations, in other countries it was detected only in one proband living in France and in four individuals requesting predictive testing living in France and in the USA, all being Portuguese immigrants. After performing an extensive haplotype study in carrier families, we estimate that this founder mutation occurred 558 ± 215 years ago. We further demonstrate significant quantitative differences regarding the production of the BRCA2 full length RNA and the transcript lacking exon 3 in c.156_157insAlu BRCA2 mutation carriers and in controls. The cumulative incidence of breast cancer in carriers did not differ from that of other BRCA2 and BRCA1 pathogenic mutations. We recommend that all suspected HBOC families from Portugal or with Portuguese ancestry are specifically tested for this rearrangement.

Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability.

Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.

Effect of an immunonutrient mix on human colorectal adenocarcinoma cell growth and viability.

OBJECTIVE: L-Glutamine, L-arginine, RNA, and omega-3 polyunsaturated fatty acids (PUFAs) have been incorporated into nutritional formulas to improve immunity of patients with gastrointestinal cancer. We therefore examined the individual and net effects of these immunonutrients on four different human colorectal adenocarcinoma cell lines. METHODS: LS174T, HT-29, CO112, and Caco-2 cells were exposed to dilutions of 1:50, 1:100, and 1:1000 of a mix or individual components of a mix of 15 g/L of L-glutamine, 16.3 g/L of L-arginine, 1.6 g/L of RNA, and 2.7 g/L of omega-3 PUFAs. Cell growth kinetic was assessed using cell count with a flow cytometer. Cell cycle and apoptosis were evaluated with double fluorescence-activated cell sorter analyses using bromodeoxyuridine labeling index and annexin V staining, respectively. One-way analysis of variance and Student's t tests were used for comparison. RESULTS: Evaluation of the cell growth kinetic over an 18-d period showed that the immunonutrient mix stimulated cancer cell growth only when diluted > or =100 times. Individual component evaluation indicated that the cell growth stimulation was mainly due to the presence of L-glutamine and to a lesser extent RNA in the mix. L-Arginine had no effect. At a lower dilution of 1:50, omega-3 PUFA concentrations were sufficient to induce cell cycle arrest and massive cell death in part through apoptosis. CONCLUSION: These results suggest that cancer cell growth stimulation by current immunonutrient formulas is unlikely due to predominant cytotoxic effect of omega-3 PUFAs.

Omega-3 polyunsaturated fatty acids and ionizing radiation: combined cytotoxicity on human colorectal adenocarcinoma cells.

OBJECTIVE: This study evaluated whether omega-3 polyunsaturated fatty acids (PUFAs) could enhance the radiosensitivity of three different human colorectal adenocarcinoma cell lines. To understand the underlying mechanisms, the effects of omega-3 PUFAs on the cell growth, survival, and apoptosis were evaluated alone or in combination with an antioxidant (vitamin E) and compared with the effects of omega-6 PUFAs. METHODS: LS174T, CO112, and Caco-2 cell survival was assessed by clonogenic assay after a 3-d pretreatment with omega-3/omega-6 PUFAs and/or vitamin E before a single X-ray exposure to 4 Gy. Cell growth and viability were measured by double fluorescence-activated cell sorter analyses using propidium iodide and fluorescein isothiocyanate-conjugated annexin V. Student's t test or multivariable linear regression analyses were used for comparison. RESULTS: Preincubation with 30 to 100 micromol/L of omega-3 PUFAs induced a dose-dependent additive decrease in cell survival after irradiation (P < 0.05). Evaluation of the underlying mechanisms indicated that omega-3 PUFAs mainly decreased the cell number via apoptosis induction. Moreover, formation of lipid peroxidation products and modulation of cyclooxygenase II activity seemed to be involved, because coincubation with 10 micromol/L vitamin E abolished the effect of 50 micromol/L of omega-3 PUFAs (P < 0.05), whereas omega-6 PUFAs could partly mimic omega-3 PUFA effects. CONCLUSION: These observations suggest that omega-3 PUFAs may be potential candidates as nutritional adjuvants to enhance the efficacy of human colorectal cancer radiotherapy.

BRCA1 and BRCA2 germline mutations screening in Algerian breast/ovarian cancer families.

BACKGROUND: Breast cancer is the leading cause of cancer death in women in Algeria. The contribution of BRCA1 and BRCA2 mutations to hereditary breast/ovarian cancer in Algerian population is largely unknown. Here, we describe analysis of BRCA1 and BRCA2 genes in 86 individuals from 70 families from an Algerian cohort with a personal and family history suggestive of genetic predisposition to breast cancer. METHODS: The approach used is based on BRCA1 and BRCA2 mutations screening by High-Resolution Melting (HRM) curve analysis followed by direct sequencing. All samples for which no pathogenic mutation was found were analyzed by MLPA for large deletions or duplications. RESULTS: Three distinct pathogenic mutations c.83_84delTG, c.181T>G, c.798_799delTT and two large rearrangements involving deletion of exon 2 and exon 8 respectively, were detected in BRCA1 gene. Moreover 17 unclassified variants and polymorphisms were detected in BRCA1 gene (6 described for the first time). Two pathogenic mutations, c.1310_1313delAAGA and c.5722_5723delCT and 40 unclassified variants and polymorphisms (14 never described before) were identified in BRCA2 gene. CONCLUSIONS: For the first time, we used HRM and MLPA to identify BRCA1 and BRCA2 mutations in Algerian patients with a personal and family history suggestive of genetic predisposition to breast cancer. The implications of these new findings in regard to genetic testing and counseling are substantial for the Algerian population.