ATM has a major role in the double-strand break repair pathway dysregulation in sporadic breast carcinomas and is an independent prognostic marker at both mRNA and protein levels.

BACKGROUND: Ataxia telangiectasia mutated (ATM) is a kinase that has a central role in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of DNA double-strand breaks (DSB). In breast cancer, a low level of ATM was correlated with poor outcome; however, the molecular mechanism of this downregulation is still unclear. METHODS: We used qRT-PCR assay to quantify mRNA levels of ATM gene in 454 breast tumours from patients with known clinical/pathological status and outcome; reverse phase protein arrays (RPPA) were used to assess the levels of ATM and 14 proteins in 233 breast tumours. RESULTS: ATM mRNA was associated with poor metastasis-free survival (MFS) (P=0.00012) on univariate analysis. ATM mRNA and protein levels were positively correlated (P=0.00040). A low level of ATM protein was correlated with poorer MFS (P=0.000025). ATM expression at mRNA or protein levels are independent prognostic factors on multivariate analysis (P=0.00046 and P=0.00037, respectively). The ATM protein level was positively correlated with the levels of six proteins of the DSB repair pathway: H2AX (P<0.0000001), XRCC5 (P<0.0000001), NBN (P<0.0000001), Mre11 (P=0.0000029), Rad50 (P=0.0064), and TP53BP1 (P=0.026), but not with proteins involved in other pathways that are altered in cancer. Low expression of ATM protein was significantly associated with high miR-203 expression (P=0.011). CONCLUSION: We confirmed that ATM expression is an independent prognostic marker at both RNA and protein levels. We showed that alteration of ATM is involved in dysregulation of the DSB repair pathway. Finally, miR-203 may be responsible for downregulation of ATM in breast cancers.

Lack of referral for genetic counseling and testing in BRCA1/2 and Lynch syndromes: a nationwide study based on 240,134 consultations and 134,652 genetic tests.

Based on nationwide data from the French national cancer institute (INCa), we analyzed the evolution of cancer genetics consultations and testing over time, and the uptake of targeted tests in relatives of families with BRCA1/2 or MMR genes mutation. Genetic testing and consultations for familial high-risk individuals are exclusively funded and monitored by the INCa in France. All nationwide cancer genetics centers reported annually standardized parameters of activity from 2003 to 2011. The analysis included a total of 240,134 consultations and 134,652 genetic tests enabling to identify 32,494 mutation carriers. Referral for hereditary breast and ovarian cancer (HBOC) or colorectal cancer predisposition syndromes represented 59 % (141,639) and 23.2 % (55,698) consultations, respectively. From 2003 to 2011, we found a dramatic and steady increase of tests performed for BRCA1/2 (from 2,095 to 7,393 tests/year, P < 0.0001) but not for MMR genes (from 1,144 to 1,635/year, P = NS). The overall percentage of deleterious mutations identified in the probands tested was 13.8 and 20.9 % in HBOC and Lynch syndromes, respectively. Pooled analysis for BRCA1/2 and Lynch syndrome tests showed an inverse relationship between the percentage of mutation detected and the absolute number of tests performed over the time (overall Cochran-Armitage test for trend: P < 0.001). In families with BRCA1/2 or MMR identified mutations, there was an average number of 2.94 and 3.28 relatives performing targeted tests, respectively. This nationwide study shows a lack of referral and genetic testing in Lynch as compared to HBOC syndromes. Only a third of relatives of a proband with a predisposing mutation performed a targeted test. Enhanced information about benefit of genetic testing should be given to clinicians and patients for Lynch syndrome and relatives of a proband carrying an identified predisposing mutation.

Rare germline large rearrangements in the BRCA1/2 genes and eight candidate genes in 472 patients with breast cancer predisposition.

Hereditary breast cancers account for up to 5-10 % of breast cancers and a majority are related to the BRCA1 and BRCA2 genes. However, many families with breast cancer predisposition do not carry any known mutations for BRCA1 and BRCA2 genes. We explored the incidence of rare large rearrangements in the coding, noncoding and flanking regions of BRCA1/2 and in eight other candidate genes--CHEK2, BARD1, ATM, RAD50, RAD51, BRIP1, RAP80 and PALB2. A dedicated zoom-in CGH-array was applied to screen for rearrangements in 472 unrelated French individuals from breast-ovarian cancer families that were being followed in eight French oncogenetic laboratories. No new rearrangement was found neither in the genomic regions of BRCA1/2 nor in candidate genes, except for the CHEK2 and BARD1 genes. Three heterozygous deletions were detected in the 5' and 3' flanking regions of BRCA1. One large deletion introducing a frameshift was identified in the CHEK2 gene in two families and one heterozygous deletion was detected within an intron of BARD1. The study demonstrates the usefulness of CGH-array in routine genetic analysis and, aside from the CHEK2 rearrangements, indicates there is a very low incidence of large rearrangements in BRCA1/2 and in the other eight candidate genes in families already explored for BRCA1/2 mutations. Finally, next-generation sequencing should bring new information about point mutations in intronic and flanking regions and also medium size rearrangements.

Hedgehog pathway activation in human transitional cell carcinoma of the bladder.

BACKGROUND: The Hedgehog (Hh) signalling pathway functions as an organiser in embryonic development. Recent studies have shown constitutive activation of this pathway in various malignancies, but its role in bladder cancer remains poorly studied. METHODS: Expression levels of 31 genes and 9 microRNAs (miRNAs) involved in the Hh pathway were determined by quantitative real-time RT-PCR in 71 bladder tumour samples (21 muscle-invasive (MIBC) and 50 non-muscle-invasive (NMIBC) bladder cancers), as well as in 6 bladder cancer cell lines. RESULTS: The SHH ligand gene and Gli-inducible target genes (FOXM1, IGF2, OSF2, H19, and SPP1) were overexpressed in tumour samples as compared with normal bladder tissue. SHH overexpression was found in 96% of NMIBC and 52% of MIBC samples, as well as in two bladder cancer cell lines. Altered expression of miRNAs supported their oncogene or tumour-suppressor gene status. In univariate analysis, high expression levels of PTCH2, miRNA-92A, miRNA-19A, and miRNA-20A were associated with poorer overall survival in MIBC (P=0.02, P=0.012, P=0.047, and P=0.036, respectively). CONCLUSION: We observed constitutive activation of the Hh pathway in most NMIBC and about 50% of MIBC. We also found that some protein-coding genes and miRNAs involved in the Hh pathway may have prognostic value at the individual level.

A refined molecular taxonomy of breast cancer.

The current histoclinical breast cancer classification is simple but imprecise. Several molecular classifications of breast cancers based on expression profiling have been proposed as alternatives. However, their reliability and clinical utility have been repeatedly questioned, notably because most of them were derived from relatively small initial patient populations. We analyzed the transcriptomes of 537 breast tumors using three unsupervised classification methods. A core subset of 355 tumors was assigned to six clusters by all three methods. These six subgroups overlapped with previously defined molecular classes of breast cancer, but also showed important differences, notably the absence of an ERBB2 subgroup and the division of the large luminal ER+ group into four subgroups, two of them being highly proliferative. Of the six subgroups, four were ER+/PR+/AR+, one was ER-/PR-/AR+ and one was triple negative (AR-/ER-/PR-). ERBB2-amplified tumors were split between the ER-/PR-/AR+ subgroup and the highly proliferative ER+ LumC subgroup. Importantly, each of these six molecular subgroups showed specific copy-number alterations. Gene expression changes were correlated to specific signaling pathways. Each of these six subgroups showed very significant differences in tumor grade, metastatic sites, relapse-free survival or response to chemotherapy. All these findings were validated on large external datasets including more than 3000 tumors. Our data thus indicate that these six molecular subgroups represent well-defined clinico-biological entities of breast cancer. Their identification should facilitate the detection of novel prognostic factors or therapeutical targets in breast cancer.

Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases.

BACKGROUND: It was shown recently on the level of gene expression that UGT8, coding UDP-galactose:ceramide galactosyltransferase, is one of six genes whose elevated expression correlated with a significantly increased the risk of lung metastases in breast cancer patients. In this study primary tumours and their lung metastases as well as breast cancer cell lines were analysed for UGT8 expression at the protein level. METHODS: Expression of UGT8 in breast cancer tissue specimens and breast cancer cell lines was analysed using IHC, real-time PCR and Western blotting. RESULTS: Comparison of the average values of the reaction intensities (IRS scale) showed a significant difference in UGT8 expression between (1) primary and metastatic tumours (Mann-Whitney U, P<0.05), (2) tumours of malignancy grades G3 and G2 (Mann-Whitney U, P<0.01) as well as G3 and G1 (Mann-Whitney U, P<0.001) and (3) node-positive and node-negative tumours (Mann-Whitney U, P<0.001). The predictive ability of increased expression of UGT8 was validated at the mRNA level in three independent cohorts of breast cancer patients (721). Similarly, breast cancer cell lines with the 'luminal epithelial-like' phenotype did not express or weakly expressed UGT8, in contrast to malignant, 'mesenchymal-like,' cells forming metastases in nude mice. CONCLUSION: Our data suggest that UGT8 is a significant index of tumour aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer.

The TP53 Arg72Pro and MDM2 309G>T polymorphisms are not associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers.

BACKGROUND: The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T>G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance. METHODS: To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T>G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework. RESULTS: No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR)=1.01, 95% confidence interval (CI): 0.93-1.10, P(trend)=0.77; MDM2: HR=0.96, 95%CI: 0.84-1.09, P(trend)=0.54) or for BRCA2 mutation carriers (TP53: HR=0.99, 95%CI: 0.87-1.12, P(trend)=0.83; MDM2: HR=0.98, 95%CI: 0.80-1.21, P(trend)=0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association. CONCLUSION: There was no evidence that TP53 Arg72Pro or MDM2 309T>G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers.

Inhibition of the Wnt/beta-catenin pathway by the WWOX tumor suppressor protein.

The WWOX gene encodes a candidate tumor suppressor protein (WWOX) implicated in a variety of human diseases such as cancer. To better understand the molecular mechanisms of WWOX action, we investigated novel partners of this protein. Using the two-hybrid system and a coimmunoprecipitation assay, we observed a physical association between WWOX and the Dishevelled protein (Dvl) family signaling elements involved in the Wnt/beta-catenin pathway. We found that enforced WWOX expression inhibited, and inhibition of endogenous WWOX expression stimulated the transcriptional activity of the Wnt/beta-catenin pathway. Inhibition of endogenous WWOX expression also enhanced the effect of Wnt-3a on beta-catenin stability. Moreover, we observed the sequestration of Dvl-2 wild type and Dvl-2NESm, a mutated form of Dvl-2 predominantly localized in the nucleus, in the cytoplasm compartment by WWOX. Our results indicate that WWOX is a novel inhibitor of the Wnt/beta-catenin pathway. WWOX would act, at least in part, by preventing the nuclear import of the Dvl proteins.

Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene.

BACKGROUND: Many unclassified variants (UV) of BRCA1 or BRCA2 may have an effect on pre-mRNA splicing. Patient blood samples suitable for RNA extraction are not always available for testing UVs at the RNA level. METHODS: Analyses of RNA from patient peripheral blood were performed, using a one-step reverse transcriptase-PCR (RT-PCR) protocol, and were compared with an ex vivo splicing assay based on PCR-amplified patient DNA inserted into a splicing reporter minigene. Using both methods 20 variants found in 17 patients were examined. RESULTS: Data from patient RNA and from the minigene assay were fully concordant, but the ex vivo splicing assay, which is monoallelic, clarified several ambiguities in the patient RNA data. Two intronic variants induced strong splicing defects: BRCA1 c.4987-5T-->A (IVS16-5T-->A) induced exon 17 skipping and BRCA2 c.316+5G-->C (IVS3+5G-->C) induced complete skipping of exon 3. Of the exonic variants, BRCA2 c.7805G-->C (p.Arg2602Thr), at the last base of exon 16, induced both exon skipping and activation of a cryptic exonic donor site, and BRCA2 c.8023A-->G (p.Ile2675Val) generated a strong donor site within exon 18. These four variants were thus classified as pathogenic, because of the total absence of a normal transcript from the corresponding allele. Variant BRCA2 c.9501+3A-->T (IVS25+3A-->T) induced incomplete skipping of exon 25, suggesting a mutation with incomplete penetrance, and BRCA2 c.8257_8259del (p.Leu2753del) modified the alternative splicing of exons 17 and 18. CONCLUSIONS: We show that functional analysis using a splicing reporter minigene is sensitive and specific, and should be used for initial screening of potential splicing defects, especially when patient RNA is not readily available.

Oligonucleotide microarray analysis of estrogen receptor alpha-positive postmenopausal breast carcinomas: identification of HRPAP20 and TIMELESS as outstanding candidate markers to predict the response to tamoxifen.

The estrogen receptor alpha (ER alpha) status of breast tumors is used to identify patients who may respond to endocrine agents such as tamoxifen. However, ER alpha status alone is not perfectly predictive, and there is a pressing need for more reliable markers of endocrine responsiveness. In this aim, we used a two-step strategy. We first screened genes of interest by a pangenomic 44 K oligonucleotide microarray in a series of ten ER alpha-positive tumors from five tamoxifen-treated postmenopausal patients who relapsed (distant metastasis) and five tamoxifen-treated postmenopausal patients who did not relapse, matched with respect to age, Scarff-Bloom-Richardson grade, lymph node status, and macroscopic tumor size. Genes of interest (n=24) were then investigated in an independent well-characterized series of ER alpha-positive unilateral invasive primary breast tumors from postmenopausal women who received tamoxifen alone as adjuvant hormone therapy after primary surgery. We identified four genes (HRPAP20, TIMELESS, PTPLB, and MGC29814) for which high mRNA levels were significantly associated with shorter relapse-free survival (log-rank test). We also showed that hormone-regulated proliferation-associated 20 kDa protein (HRPAP20) and TIMELESS are 17beta-estradiol-regulated in vitro and are ectopically expressed in OH-Tam-resistant cell lines. In conclusion, these findings point to HRPAP20 and TIMELESS as promising markers of tamoxifen resistance in women with ER alpha-positive breast tumors.

High-resolution oligonucleotide array-CGH applied to the detection and characterization of large rearrangements in the hereditary breast cancer gene BRCA1.

We have developed a new method for detecting and characterizing large rearrangements in the BRCA1 gene based on high-resolution oligonucleotide array-CGH technology. We designed a specific CGH array for the BRCA1 gene and its flanking regions. We then used this approach to analyze nine DNA samples known to contain large deletions and large duplications. When possible, the deleted or duplicated region was sequenced to identify the break point. All the large rearrangements were detected by the new method, and their size was estimated to be within 1--2 kb. This enabled us to develop a simple polymerase chain reaction screening test for other family members. A refined choice of oligonucleotides should improve the precision of the breakpoint determination. Finally, the high resolution of oligonucleotide array-CGH should help to detect new large rearrangements missed by other current methods.

Transcriptome analysis reveals an osteoblast-like phenotype for human osteotropic breast cancer cells.

Metastatic breast cancer cells exhibit the selective ability to seed and grow in the skeleton. We and others have previously reported that human breast tumors which metastasize to the skeleton overexpress bone matrix extracellular proteins. In an attempt to reveal the osteoblast-like phenotype of osteotropic breast cancer cells, we performed a microarray study on a model of breast cancer bone metastasis consisting of the MDA-MB-231 human cell line and its variant B02 selected for its high capacity to form bone metastases in vivo. Analysis of B02 cells transcriptional profile revealed that 11 and 9 out of the 50 most up- and down-regulated mRNAs, respectively, corresponded to genes which expression has been previously associated with osteoblastic differentiation process. Thus, osteoblast specific cadherin 11 which mediates the differentiation of mesenchymal cells into osteoblastic cells is up-regulated in B02. While S100A4, recently described as a key negative regulator of osteoblast differentiation, is the most down-regulated gene in B02 cells. RT-PCR and western blotting experiments allowed the validation of the modulation of several genes of interest. Using immunohistochemistry, performed on human breast primary tumors and their matched liver and bone metastases, we were able to confirm that the osteoblast-like pattern of gene expression observed in our model holds true in vivo. This is the first report demonstrating a gene-expression pattern corresponding to the acquisition of an osteomimetic phenotype by bone metastatic breast cancer cells.

Identification of novel genes that co-cluster with estrogen receptor alpha in breast tumor biopsy specimens, using a large-scale real-time reverse transcription-PCR approach.

The estrogen receptor alpha (ERalpha) plays a critical role in the pathogenesis and clinical behavior of breast cancer. To obtain further insights into the molecular basis of estrogen-dependent forms of this malignancy, we used real-time quantitative reverse transcription (RT)-PCR to compare the mRNA expression of 560 selected genes in ERalpha-positive and ERalpha-negative breast tumors. Fifty-one (9.1%) of the 560 genes were significantly upregulated in ERalpha-positive breast tumors compared with ERalpha-negative breast tumors. In addition to well-known ERalpha-induced genes (PGR, TFF1/PS2, BCL2, ERBB4, CCND1, etc.) and genes recently identified by cDNA microarray-based approaches (GATA3, TFF3, MYB, STC2, HPN/HEPSIN, FOXA1, XBP1, SLC39A6/LIV-1, etc.), an appreciable number of novel genes were identified, many of, which were weakly expressed. This validates the use of large-scale real-time RT-PCR as a method complementary to cDNA microarrays for molecular tumor profiling. Most of the new genes identified here encoded secreted proteins (SEMA3B and CLU), growth factors (BDNF, FGF2 and EGF), growth factor receptors (IL6ST, PTPRT, RET, VEGFR1 and FGFR2) or metabolic enzymes (CYP2B6, CA12, ACADSB, NAT1, LRBA, SLC7A2 and SULT2B1). Importantly, we also identified a large number of genes encoding proteins with either pro-apoptotic (PUMA, NOXA and TATP73) or anti-apoptotic properties (BCL2, DNTP73 and TRAILR3). Surprisingly, only a small proportion of the 51 genes identified in breast tumor biopsy specimens were confirmed to be ERalpha-regulated and/or E2-regulated in vitro (cultured cell lines). Therefore, this study identified a limited number of genes and signaling pathways, which better delineate the role of ERalpha in breast cancer. Some of the genes identified here could be useful for diagnosis or for predicting endocrine responsiveness, and could form the basis for novel therapeutic strategies.

The contribution of germline rearrangements to the spectrum of BRCA2 mutations.

BACKGROUND: Few germline BRCA2 rearrangements have been described compared with the large number of germline rearrangements reported in the BRCA1 gene. However, some BRCA2 rearrangements have been reported in families that included at least one case of male breast cancer. OBJECTIVE: To estimate the contribution of large genomic rearrangements to the spectrum of BRCA2 defects. METHODS: Quantitative multiplex PCR of short fluorescent fragments (QMPSF) was used to screen the BRCA2 gene for germline rearrangements in highly selected families. QMPSF was previously used to detect heterozygous deletions/duplications in many genes including BRCA1 and BRCA2. RESULTS: We selected a subgroup of 194 high risk families with four or more breast cancers with an average age at diagnosis of < or = 50 years, who were recruited through 14 genetic counselling centres in France and one centre in Switzerland. BRCA2 mutations were detected in 18.6% (36 index cases) and BRCA1 mutations in 12.4% (24 index cases) of these families. Of the 134 BRCA1/2 negative index cases in this subgroup, 120 were screened for large rearrangements of BRCA2 using QMPSF. Novel and distinct BRCA2 deletions were detected in three families and their boundaries were determined. We found that genomic rearrangements represent 7.7% (95% confidence interval 0% to 16%) of the BRCA2 mutation spectrum. CONCLUSION: The molecular diagnosis of breast cancer predisposition should include screening for BRCA2 rearrangements, at least in families with a high probability of BRCA2 defects.

Structural organization and expression of human MTUS1, a candidate 8p22 tumor suppressor gene encoding a family of angiotensin II AT2 receptor-interacting proteins, ATIP.

The Mitochondrial Tumor suppressor 1 (MTUS1) gene is a newly identified candidate tumor suppressor gene at chromosomal position 8p22. We report here that MTUS1 encodes a family of proteins whose leader member (ATIP1) was previously isolated in our laboratory as a novel interacting partner of the angiotensin II AT2 receptor involved in growth inhibition (Nouet, JBC 279: 28989-97, 2004). The MTUS1 gene contains 17 coding exons distributed over 112 kb of genomic DNA. Alternative exon usage generates three major transcripts (ATIP1, ATIP3 and ATIP4), each showing different tissue distribution. ATIP polypeptides are identical in their carboxy-terminal region carrying four coiled-coil domains. In their amino-terminal portion, ATIP polypeptides exhibit distinct motifs for localisation in the cytosol, nucleus or cell membrane, suggesting that MTUS1 gene products may be involved in a variety of intracellular functions in an AT2-dependent and independent manner. ATIP1 is ubiquitous and highly expressed in the brain. ATIP3 is the major transcript in tissues (prostate, bladder, breast, ovary, colon) corresponding to cancer types with frequent loss of heterozygosity at 8p22. Interestingly, ATIP4 is a brain-specific transcript highly abundant in the cerebellum and fetal brain. High evolutionary conservation of ATIP amino-acid sequences suggests important biological roles for this new family of proteins in tumor suppression and/or brain function.

Trimodal GSTT1 and GSTM1 genotyping assay by real-time PCR.

The GSTT1 and GSTM1 genes are characterized by the existence of a GST*0 null allele responsible for a lack of enzyme activity, with the respective null genotypes GSTT1*0/0 and GSTM1*0/0. The three resulting genotypes (GSTs*1/1, *1/0 and *0/0) are associated with a trimodal distribution of glutathione-conjugator activity. Previous epidemiological studies have only evaluated the cancer risk associated with the GST null genotype relative to the two GST carrier genotypes (GSTs1*1/1 and *1/0). We developed GSTT1 and GSTM1 TaqMan real-time quantitative PCR assays to discriminate each of the three genotypes, with the albumin gene (ALB) as reference. The mean N(GSTT1*1/1) value was 1.0 (95% confidence interval 0.80-1.20). The mean N(GSTT1*1/0) value was 0.48 (95% CI 0.36-0.60). One (3.4%) of the 29 DNA samples yielded the GSTM1*1/1 genotype (N(GSTM1*1/1) = 1), a frequency in keeping with the Hardy-Weinberg distribution. The mean N(GSTM1*1/0) value was 0.50 (95% CI 0.42-0.58). All GSTT1*0/0 and GSTM1*0/0 samples yielded N(GST) values of 0 (Ct = 40); the frequencies of these genotypes (27.6% and 55.2%, respectively) were in keeping with published data. The GSTT1 and GSTM1 real-time PCR assays described here unambiguously discriminate each of the three existing genotypes which should be valuable for assessing the relative risk of cancer associated with each of the three GST genotypes.

Molecular changes associated with the agonist activity of hydroxy-tamoxifen and the hyper-response to estradiol in hydroxy-tamoxifen-resistant breast cancer cell lines.

The aim of this study was to explore the pharmacological response to 4-hydroxy-tamoxifen (OH-Tam) and to estradiol (E2) in three cell lines: MVLN, a human breast carcinoma cell line derived from MCF-7, and two MVLN-derived OH-Tam-resistant (OTR) cell lines, called CL6.8 and CL6.32. The OH-Tam response in the OTR cells was associated with the development of both an agonist activity of the drug on cell proliferation and the resistance of the cells to OH-Tam-induced apoptosis. The OTR cells also developed an increased sensitivity to the E2 growth-stimulating activity. To delineate the genes that determine such responses, we combined a mini-array-based gene-selection approach and an extensive real-time quantitative PCR exploration in the MVLN and OTR cell lines exposed to three pharmacological conditions: a 4-day treatment with E2, OH-Tam or both E2 and OH-Tam. Compiled data revealed a hyper-response to E2 and a modification of the OH-Tam pharmacological response (loss of antagonist action and agonist activity) at the gene-expression level. The proteins encoded by the genes selected in this study have been reported to be involved in the regulation of cell proliferation, cell transformation, DNA repair and apoptosis, or belong to the ErbB/epidermal growth factor receptor-driven pathway. Our data also provide evidence of changes in transcriptional co-regulator expression, elevated mitogen-activated protein kinase activity and increase in the phosphorylation status of estrogen receptor alpha on serine residue 118 in the OTR cell lines, suggesting the possible involvement of such mechanisms in the agonist activity of OH-Tam and/or the hyper-response of cells to E2. Taken together, our study should enhance our knowledge of the multifactorial events associated with the development of Tam resistance in two independent cell lines issued from the same selection process and should help in the identification of potential molecular targets for diagnosis or therapy.

Trimodal GSTT1 and GSTM1 genotyping assay by real-time PCR.

The GSTT1 and GSTM1 genes are characterized by the existence of a GST*0 null allele responsible for a lack of enzyme activity, with the respective null genotypes GSTT1*0/0 and GSTM1*0/0. The three resulting genotypes (GSTs*1/1, *1/0 and *0/0) are associated with a trimodal distribution of glutathione-conjugator activity. Previous epidemiological studies have only evaluated the cancer risk associated with the GST null genotype relative to the two GST carrier genotypes (GSTs1*1/1 and *1/0). We developed GSTT1 and GSTM1 TaqMan real-time quantitative PCR assays to discriminate each of the three genotypes, with the albumin gene (ALB) as reference. The mean NGSTT1*1/1 value was 1.0 (95% confidence interval 0.80-1.20). The mean NGSTT1*1/0 value was 0.48 (95% CI 0.36-0.60). One (3.4%) of the 29 DNA samples yielded the GSTM1*1/1 genotype (NGSTM1*1/1 = 1), a frequency in keeping with the Hardy-Weinberg distribution. The mean NGSTM1*1/0 value was 0.50 (95% CI 0.42-0.58). All GSTT1*0/0 and GSTM1*0/0 samples yielded NGST values of 0 (Ct = 40); the frequencies of these genotypes (27.6% and 55.2%, respectively) were in keeping with published data. The GSTT1 and GSTM1 real-time PCR assays described here unambiguously discriminate each of the three existing genotypes which should be valuable for assessing the relative risk of cancer associated with each of the three GST genotypes. (Int J Biol Markers 2005; 20: 81-6).