Comparative clinical and transcriptomal profiles of breast cancer between French and South Mediterranean patients show minor but significative biological differences.

BACKGROUND: In Western countries, breast cancer incidence and mortality are higher than in Mediterranean countries. These differences have been ascribed to environmental factors but also to late-stage diagnostic and biological specific characteristics. PATIENTS AND METHODS: Between September 2002 and September 2005, we collected clinical data by phone counselling 180 French and Mediterranean breast cancer patients and performed microarray experiments. RESULTS: Characteristics of breast cancer in patients from Lebanon, Tunisia and Morocco were more aggressive (more SBR grade III and positive node invasion) and patients were 10 years younger at diagnosis. Sixteen differentially expressed genes such as MMP9, VEGF, PHB1, BRCA1, TFAP2C, GJA1 and TFF1 were also found. Additionally, an up-regulation of cytokeratins KRT8 and KRT18 may indicate a luminal B subtype in "South" (Lebanon, Tunisia and Morocco) tumors while "North" (France) tumors may more frequently be luminal A type. CONCLUSION: This study allowed the identification of specific clinical and transcriptomic parameters in patients from South Mediterranean countries.

Resveratrol and breast cancer chemoprevention: molecular mechanisms.

Despite years of intensive research, breast cancer remains a major cause of death among women. New strategies to combat breast cancer are being developed, one of the most exciting of which is the use of chemopreventive agents. Resveratrol (RES) is a polyphenolic compound found in plants that seems to have a wide spectrum of biological activity. RES has been shown to afford protection against several types of cancer. This review summarizes the chemopreventive effects of RES at the three major stages of breast carcinogenesis: initiation, promotion, and progression. It has anti-oxidant and anti-inflammatory properties, and may induce apoptosis as well as modulate cell cycle and estrogen receptor function in breast cancer cell lines. Although RES has shown remarkable promise as a potent chemopreventive agent in breast cancer, further studies are needed to etablish its usefulness.

Quantitative analysis of BRCA1, BRCA2 and Hmsh2 mRNA expression in colorectal Lieberkühnien adenocarcinomas and matched normal mucosa: relationship with cellular proliferation.

The human DNA mismatch repair gene hMSH2 is involved in the development of sporadic and hereditary nonpolyposis colorectal cancer. An increased risk of colorectal cancer has also been suggested in BRCA1 and BRCA2 mutation carriers. To address the relationship between the expression level of these genes and colorectal tumorigenesis, we studied BRCA1, BRCA2 and hMSH2 mRNA expression by real-time quantitative RT-PCR in 72 colorectal Lieberkühnien adenocarcinomas and matched normal mucosa. We investigated the relationship between mRNA levels and various clinicopathological parameters. The mean expression of BRCA1 3' and BRCA2 3' (mRNA pool), BRCA1 ex11 (with exon 11), BRCA2 ex12 (with exon 12) and hMSH2 mRNAs were increased in tumor samples. BRCA1 and BRCA2 mRNAs expressions were altered according to colon tumor site: BRCA1 3' and BRCA2 3' mRNAs levels were highest, respectively, in the right colon and left colon. No difference in hMSH2 mRNA levels was detected in relation to clinicopathological parameters. The mean SPF value was significantly higher in tumor than in non-tumor colonic tissue, and a high SPF value was correlated with high BRCA2 mRNA levels. BRCA2 3' mRNA levels tended to decrease as the Dukes' stage increased. In conclusion, the mechanisms of colorectal carcinogenesis seem to differ according to the right or left position of the tumor.

Quantification by affinity perfusion chromatography of phosphorylated BRCAl and BRCA2 proteins from tumor cells after lycopene treatment.

A new procedure for the quantification of phosphorylated BRCA1 (P-BRCA1) and BRCA2 (P-BRCA2) proteins in breast cell lines after different treatments was carried out. Cells were cultivated with [35S]-methionine and extracts subjected to three perfusion chromatographies. First heparin affinity chromatography purified cellular DNA-binding proteins. Subsequent specific immunoprecipitation of BRCA1 and BRCA2 proteins was performed with antibodies raised against BRCA1 or BRCA2. The immune complexes were isolated by protein A affinity chromatography. Phosphorylated BRCA1 or BRCA2 proteins were then purified with a Poros 20 AL column where anti-phosphothreonine and anti-phosphoserine antibodies were previously bound. The percentage of phosphorylated BRCA1 or BRCA2 proteins was calculated as follows: 100 x dpm of P-BRCA1 or P-BRCA2 eluted from the POROS 20AL column/total dpm eluted from POROS 20AL column. Treatment with 10 microM lycopene increased P-BRCA1 and P-BRCA2 in the breast tumor cell line MCF7 but not in MDA-MB-231 or MCF-10a, breast tumor or fibrocystic cell lines, respectively.

The effects of lycopene on the proliferation of human breast cells and BRCA1 and BRCA2 gene expression.

The purpose of this study was to demonstrate the effects of lycopene, the major tomato carotenoid, on the expression of the BRCA1 and BRCA2 genes in three breast tumour cell lines, MCF-7, HBL-100, MDA-MB-231 and the fibrocystic breast cell line MCF-10a. Flow cytometry analysis showed a G(1)/S phase cell cycle-arrest after treatment of the cells with 10 microM lycopene for 48 h. mRNA expression was studied by quantitative reverse transcription-polymerase chain reaction using the Taqman method. We observed an increase of BRCA1 and BRCA2 mRNA in the oestrogen receptor (ER)-positive cell lines (MCF-7 and HBL-100), and a decrease (MDA-MB-231) or no change (MCF-10a) in the ER-negative cell lines. BRCA1 and BRCA2 proteins were quantified by perfusion affinity chromatography. No variation in their expression was observed. These preliminary results on the effects of lycopene on the expression of BRCA1 and BRCA2 oncosuppressor genes in breast cancer may reflect cross-talk between the oestrogen and retinoic acid receptor (RAR) pathways.

Ultrastructural localization of binding sites for PNA and VVA-B(4) lectins in human breast cancer cell lines detected by confocal fluorescence microscopy.

Three cancer cell lines (MCF-7, HBL-100, MDA-MB 231) and subnormal breast epithelial cell line MCF-10A were labeled with FITC-conjugated VVA-B4 lectin, specific for D-GalNAcalpha-O-ser/thr, matching the structure of Tn antigen sugar residues, and with RTIC-conjugated PNA lectin, specific for DGalbeta1-3GalNAc-O-ser/thr, corresponding to the structure of T antigen. Simultaneous expression of Tn and T antigens on the same cells (but in widely differing proportions) led to their large heterogeneity and occurrence of numerous cell subpopulations within each of the studied cell lines. This observation proved that the changes leading to the formation of Tn antigen are not caused by an irreversible genetic mutation of beta1-3-galactosyltransferase. Expression of Tn antigen on MCF-10A cells with normal (or subnormal) karyotype suggests that the process of malignant transformation of the cell begins with the changes in molecular structure of glycoconjugates.

Subcellular localization of BRCA1 protein in sporadic breast carcinoma with or without allelic loss of BRCA1 gene.

The localization of BRCA1 protein was studied in 49 sporadic breast carcinomas for which allelic losses of BRCA1 have been investigated. One group consisted of 15 breast carcinomas having one allelic loss of BRCA1 and the other group of 34 breast carcinomas with no allelic loss of BRCA1. The localization of BRCA1 in the 2 groups was performed using polyclonal antibodies (K-18; C-20; D-20; I-20) raised against BRCA1 and by comparing frozen and paraffin-embedded tissues. We show that no correlation was found between the expression of BRCA1 protein and allelic loss of BRCA1. But, the nuclear detection of BRCA1 in frozen samples was improved when compared to paraffinized ones.

Brca1 and Brca2 protein expression patterns in different tissues of murine origin.

We have analyzed by immunohistochemistry Brca1 and Brca2 protein expression in mouse during embryonic development, in adult tissues, and during postnatal mammary gland development. Our observations confirm previous localization of Brca1 and Brca2 mRNA on frozen sections by in situ hybridization, and demonstrate that Brca1 and Brca2 proteins are expressed in rapidly proliferating cell types undergoing differentiation. These results imply that Brca1 and Brca2 proteins are involved in the process of proliferation and differentiation in multiple tissues, notably in the mammary gland during pregnancy and lactation.

Loss of heterozygosity of BRCA1, BRCA2 and ATM genes in sporadic invasive ductal breast carcinoma.

The present study was undertaken to analyse the loss of heterozygosity (LOH) of the three genes, BRCA1, BRCA2 and ATM, and their correlation to clinicopathological parameters in sporadic breast cancer. We studied 59 sets of invasive ductal carcinoma, compared to matched normal control DNA. Microsatellite markers intragenic to BRCA1 (D17S1323, D17S1322, D17S855), BRCA2 (D13S1699, D13S1701, D13S1695) and ATM (D11S2179) were simultaneously used. In addition, one marker telomeric to BRCA2 (D13S1694) and four markers flanking ATM were analysed (D11S1816, D11S1819, D11S1294, D11S1818). Thirty-one per cent of the informative cases showed loss of heterozygosity for the BRCA1 gene, 22.8% for BRCA2 gene and 40% for ATM. LOH of BRCA1 correlated with high grade tumors (p=0.0005) and negative hormone receptors (p=0.01). LOH of ATM correlated with higher grade (p=0.03) and a younger age at diagnosis (p=0.03) in our set of tumors. No correlations were detected between BRCA2 LOH and any of the analysed clinicopathological parameters. However, a correlation was detected between allelic loss of the D13S1694 marker, telomeric to BRCA2, and larger tumor sizes and negative estrogen receptors, favoring the hypothesis of the presence of another putative tumor suppressor gene, telomeric to BRCA2, in the 13q12-q14 region. Only 11 tumors had LOH at more than one of the three genes, most of them (6/11) associated LOH of BRCA1 and ATM. One tumor only combined loss of the three genes BRCA1, BRCA2 and ATM.

Regulation of BRCA1 and BRCA2 transcript in response to cisplatin, adriamycin, taxol and ionising radiation is correlated to p53 functional status in ovarian cancer cell lines.

Recent studies suggest that BRCA1 and BRCA2 expression, in response to cytotoxic agents, may be dependent on p53 status. To investigate this possibility, we quantified their transcripts in ovarian cancer cells, PA1 (wild-type p53), CaOV-3 (mutated p53) and SKOV-3 (null p53) exposed to four cytotoxic agents. In PA1, taxol and cisplatin had no effect, while adriamycin and ionising radiation (IR) induced both genes. In SKOV-3, expression decreased in response to taxol and cisplatin, and initially decreased then increased in response to adriamycin while IR had no effect. CaOV-3 responded similarly to SKOV-3, except for both genes being increased by cisplatin. We speculate that this regulation may be part of the survival response to certain cytotoxic agents and may be dependent in part on p53 status of cells.

Characterization of anti-BRCA2 antibodies in cell lines by Western blot analysis.

BRCA2 is a tumor suppressor gene associated with familial predisposition to breast and ovarian cancer. BRCA2 has been implicated in response to DNA damage, cell cycle control and transcription. However, the mechanisms by which the BRCA2 protein suppresses tumor cell growth are largely unknown. To begin to understand the contribution of BRCA2 protein to tumorigenesis, we evaluated the specificity of 4 anti-BRCA2 antibodies directed against several different epitopes using immunoblotting techniques. The two monoclonal antibodies (3E6 and 5F6) detected a specific 384-kDa protein in human breast cancer cell lines (MCF7 and MDA-MB 231) and in a human colon carcinoma cell line (CCL 221). The two polyclonal antibodies (9433 and 9434) recognized the 384-kDa BRCA2 protein respectively in MCF7 and in CCL 221 cells, but both BRCA2 polyclonal antibodies also cross-reacted with smaller proteins.

Evaluation of the 5' spliced form of human cathepsin B mRNA in colorectal mucosa and tumors.

To evaluate the 5' spliced form of human cathepsin B mRNA in colorectal mucosa and tumors, we have determined the ratio of the spliced form for the exon 2/the complete form of cathepsin B mRNAs obtained by RT-PCR. Such ratio is significantly higher in colorectal tumors than in colorectal mucosa (p < 0.05, Kruskal-Wallis test) or in skeletal muscle (p < 0.05). Moreover, 2-fold more complete form than the spliced mRNA was found in the tumors than in the colorectal mucosa. Our data indicate that the alternative splicing of human cathepsin B mRNA in the 5'UTR may be considered as an indicator of the cellular transformation, in colorectal cancer.

Electrophoresis of BRCA1 oncosuppressor.

BRCA1 is a familial breast and ovarian cancer susceptibility gene and encodes proteins that function as tumor suppressors in human breast cancer cells. To elucidate the biological function of BRCA1, knowledge of cellular localization is needed. This can be achieved by using specific antibodies, so in a first step, we characterized by Western blot analysis the rabbit polyclonal antibodies (K-18) and (D-20) raised against the amino-terminus of human BRCA1 protein, and the polyclonal antibodies (C-20) and (I-20) raised against the carboxy terminus of human BRCA1 protein. The 220-kDa band corresponding to BRCA1 protein was recognized by the four tested antibodies in two mammary carcinoma cell lines (HBL100 and MCF7).

Association between H-ras minisatellite and colorectal cancer risk.

BACKGROUND: The H-ras locus has been suggested to play an important role in susceptibility to cancer. However, the results remain controversial. METHODS: In order to elucidate the potential role of the H-ras locus in colorectal carcinogenesis, 142 colorectal tumors with matched normal samples were studied for genomic instability and loss of heterozygosity (LOH), and 143 healthy samples of white blood cell DNA were examined by PCR, for H-ras allelic polymorphism. RESULTS: Nine percent of colorectal cancer patients constitutionally presented at least one rare allele versus 1.4% of healthy individuals (P = 0.0034). The risk of developing colorectal cancer increased significantly with the presence of rare H-ras alleles (odds ratio = 7.10 and 95% confidence interval = 1.92-26.35). The genotype associating one common allele and one rare allele was overrepresented in cancer patients (P < 0.01). No associations were observed between the rare alleles and tumor site or with the aggressiveness of cancer. Low frequencies of LOH (5%) and genetic instability (0.7%) at the H-ras locus were found in our colorectal cancer set. CONCLUSIONS: Consequently, the presence of uncommon alleles at the H-ras locus appeared to be an informative genetic marker.

Specific H-Ras minisatellite alleles in breast cancer susceptibility.

Mutations in BRCA1 and BRCA2 genes account for the majority of familial aggregation of breast and ovarian cancers but other common genes in the population with low penetrance should be also involved in susceptibility to breast cancer. The H-ras minisatellite, located downstream of H-ras oncogene, is considered to be a likely candidate. Previous findings have estimated that as many as 1 in 11 cancers of the breast might be attributed to this region, but other studies observed inconsistent results. We propose to elucidate the potential role of H-ras locus in breast cancer, by looking at somatic alterations occurring in tumor DNAs such as the instability or the loss of heterozygosity (LOH) and by determining a potential correlation between constitutional specific H-ras alleles and clinical and/or pathological characteristics. DNA was extracted from 123 sporadic breast tumors and matched peripheral blood lymphocytes. 143 DNA samples from of peripheral blood lymphocytes from healthy donors served as a control population. The allelic diversity was determined by polymerase chain reaction analysis. Rare H-ras alleles were found to be present in about 9% of breast cancer patients while they were detected in only 1.4% of lymphocytes from healthy donors (P = 0.0044). Therefore, the risk of breast cancer is increased in patients with one or two rare alleles (odd ratio = 7.14 and 95% confidence interval = 1.94-22.27). Analyses of somatic alterations in tumor DNA have shown the lost of one allele, in general the longest, in 6.7% informative cases and an instability to H-ras locus in 6.5% tumors that appeared as a size increase of one of the two alleles. No correlation of rare H-ras alleles with clinicopathological parameters was found. Our results demonstrated an association of rare H-ras alleles with breast cancer and suggest that minisatellite H-ras may be considered as an informative marker for the breast cancer risk.

Cyclosporine A inhibition of prolactin-dependent up-regulation of BRCA1 protein expression in human breast cell lines.

BACKGROUND: Previously, we reported experimental evidence that BRCA1, breast and ovarian cancer susceptibility gene is up-regulated in response to Prolactin stimulation. In this work, we studied the effects of Cyclosporine A and the competition with Prolactin on BRCA1 protein expression in vitro. METHODS: The expression of BRCA1 was monitored in a human breast cancer cell line (MCF7) by comparison with a normal breast epithelial one (MCF10a) treated with Cyclosporine A and ovine Prolactin. The amount of BRCA1 protein expression was quantified using a strategy of two successive affinity perfusion chromatographies. RESULTS AND CONCLUSION: We showed that Prolactin in presence of Cyclosporine A has no effect on BRCA1 protein expression in human breast cell lines. This emphasized the hypothesis that BRCA1 may be stimulated by Prolactin.

Localization of human BRCA1 and BRCA2 in non-inherited colorectal carcinomas and matched normal mucosas.

We characterized the expression of BRCA1 and BRCA2 in 38 sporadic colorectal carcinomas and matched normal mucosas with 9 anti-BRCA1 antibodies and 4 anti-BRCA2 antibodies, raised against several different epitopes, using immunohistochemical technique. We demonstrated an increased BRCA1 and BRCA2 staining in the apical cell pole of epithelial malignant cells and we also revealed a significant increase in BRCA1 and BRCA2 nuclear foci in tumor colorectal specimens in comparison with corresponding normal tissues. These increases in BRCA1 and BRCA2 expression may be explained by the fact that colorectal tissue is subject to very active proliferation and differentiation.

Determination of BRCA2 oncosuppressor protein expression in human mammary cells by affinity perfusion high-performance chromatography.

In this work, we report a method for the determination of BRCA2 oncosuppressor protein in human mammary cells by affinity perfusion chromatography. This method involves labeling proteins with [(35)S]-methionine. The isolation and purification of DNA-binding proteins was performed by affinity chromatography on Heparin POROS 20HE. BRCA2 proteins, known to act in the transcriptional control and in DNA repair activity, are included in the DNA-binding proteins. Specific immunoprecipitation was performed with anti-BRCA2 antibodies, and the immune complex [(35)S-BRCA2 proteins/anti-BRCA2 antibodies] was isolated by affinity chromatography on a Protein A POROS column. This procedure allows the determination of the percentage of BRCA2 among the DNA-binding proteins and the quantitation of the difference of expression of BRCA2 oncosuppressor protein in breast carcinoma cells and in normal breast cells treated or untreated with phytoestrogens, such as daidzein or genistein.

[Mutation by deletion-insertion in BRCA-1 gene in three unrelated French breast/ovarian cancer families: possible implication of a mobile element]. / Mutation par délétion-insertion dans le géne BRCA-1 dans trois families françaises non apparentées de cancers du sein et/ou de l'ovaire: possible implication d'un elément mobile.

A new type of mutation by deletion-insertion in BRCA-1 gene is found in three unrelated French breast/ovarian cancer families. Surprisingly, deletion and insertion occurred at the same nucleotide position at the end of exon 11 (3958del5ins4), thus generating a truncated protein. This original mutation consists in a deletion of 5 bp (CTCAG) and in an insertion of 4 different bp (AGGC). Here, we proposed two hypothesis to explain this phenomenom. The first hypothesis is the formation of a hairpin stem-loop structure comprising the mutational site and the sequence corresponding to the duplication insertion 2 nucleotides before the mutation. The second hypothesis, more speculative, consists in an abortive integration of a human mobile element as a human transposon (tigger 1) which involved a deletion of 5 bp during its excision and an insertion of 4 bases corresponding to the 5' extremity of the transposon.