BRCA1/2 mutations in Swiss patients with familial or early-onset breast and ovarian cancer.

QUESTIONS UNDER STUDY: Germ-line alterations in BRCA1 and BRCA2 genes account for 30-50% of all forms of familial breast and ovarian cancer syndromes. Specific mutations in specific populations and ethnic groups have been identified in BRCA1 and BRCA2. However, it is not known whether such specific mutations prevail in the Swiss population. METHODS: We started to screen patients with primary breast and ovarian cancer and a strong family history of both cancers by sequencing the full-length coding regions of BRCA1 and BRCA2. RESULTS: With the selection criteria used in this study we identified 19 mutations in the first 38 patients screened (50%). These mutations were either defined as deleterious and resulted in a protein truncation (n = 10) or were defined as unclassified variants (n = 9). One novel truncating mutation was found in BRCA2 and two novel unclassified variants were detected in BRCA1. These three mutations are not described in the BIC and HGMD databanks. CONCLUSIONS: We detected three unknown mutations among 38 patients in a Swiss study of BRCA1/2 mutation patterns. One of these novel mutations is clearly deleterious as it leads to protein truncation at nucleotide 133 of BRCA2.

Prognostic and predictive significance of ErbB-2 breast tumor levels measured by enzyme immunoassay.

PURPOSE: A retrospective analysis to assess the prognostic and predictive clinical value of breast tumor ErbB-2 receptor expression quantified by enzyme immunoassay (EIA), to compare levels measured by EIA with ErbB-2 status determined by immunohistochemistry (IHC), and to correlate receptor content with levels of phosphorylated (Y1248-P) ErbB-2, a measure of functional tyrosine kinase activity. MATERIALS AND METHODS: EIA quantification of ErbB-2 was performed on membrane extracts from 3,208 well-characterized primary breast cancers. Overall, relapse-free, distant disease-free, and local/regional-free patient survival data were available on 1,123 of these tumors. IHC scoring for ErbB-2 status (HercepTest; DAKO, Glostrup, Denmark) was performed on adjacent sections of 151 cases, and receptor functionality was measured in 230 tumors by an antibody specific for phosphorylated (Y1248-P) ErbB-2. RESULTS: Unlike nonmalignant breast tissues, breast tumors showed increased ErbB-2 levels in a bimodal distribution, with 12% constituting a distinct set of ErbB-2-overexpressing tumors. The intermodal threshold value for ErbB-2 overexpression distinguished tumors with reduced estrogen and progesterone receptor content, high IHC score for ErbB-2, and significantly increased levels of phosphorylated (Y1248-P) ErbB-2 receptor. By multivariate analysis, EIA-determined ErbB-2 overexpression predicted significantly reduced patient survival that was unaffected by tamoxifen or cyclophosphamide, methotrexate, and fluorouracil adjuvant therapy. CONCLUSION: Determination of ErbB-2 receptor expression by EIA offers a clinically valuable alternative to semiquantitative IHC assessment of breast tumor ErbB-2 overexpression and affords the opportunity to evaluate ErbB-2 phosphorylation, which may represent an important predictive parameter of receptor functionality.

Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins.

To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.

A yeast-based bioassay for the determination of functional and non-functional estrogen receptors.

The response to endocrine therapy of breast cancer is not entirely predictable from hormone receptor status alone since some point mutated or splicing variants of the estrogen receptor (ER) show altered biological activities. In order to characterize the activities of all forms of ER in a heterogeneous breast tumor, a functional assay in Saccharomyces cerevisiae was developed. Total RNA isolated from breast cancer cells and one breast cancer specimen was reverse transcribed and the ER cDNA was amplified by PCR. The products were then cloned into an expression vector by in vivo homologous recombination in yeast. The yeast strain carries a reporter gene ( ADE2 ) coupled to an estrogen response element. Activation of the reporter by ER yielded white colonies whereas lack of ER activity produced red colonies. This permitted the testing for functionality of individual ER molecules and subsequent analysis by rescuing of the ER expression plasmids and complete DNA sequencing. This simple visual test allows discrimination between wild-type ER, constitutively active ER and inactive ER.

Agrin can mediate acetylcholine receptor gene expression in muscle by aggregation of muscle-derived neuregulins.

The neural isoforms of agrin can stimulate transcription of the acetylcholine receptor (AChR) epsilon subunit gene in electrically active muscle fibers, as does the motor neuron upon the formation of a neuromuscular junction. It is not clear, however, whether this induction involves neuregulins (NRGs), which stimulate AChR subunit gene transcription in vitro by activating ErbB receptors. In this study, we show that agrin- induced induction of AChR epsilon subunit gene transcription is inhibited in cultured myotubes overexpressing an inactive mutant of the ErbB2 receptor, demonstrating involvement of the NRG/ErbB pathway in agrin- induced AChR expression. Furthermore, salt extracts from the surface of cultured myotubes induce tyrosine phosphorylation of ErbB2 receptors, indicating that muscle cells express biological NRG-like activity on their surface. We further demonstrate by RT-PCR analysis that muscle NRGs have Ig-like domains required for their immobilization at heparan sulfate proteoglycans (HSPGs) of the extracellular matrix. In extrasynaptic regions of innervated muscle fibers in vivo, ectopically expressed neural agrin induces the colocalized accumulation of AChRs, muscle-derived NRGs, and HSPGs. By using overlay and radioligand-binding assays we show that the Ig domain of NRGs bind to the HSPGs agrin and perlecan. These findings show that neural agrin can induce AChR subunit gene transcription by aggregating muscle HSPGs on the muscle fiber surface that then serve as a local sink for focal binding of muscle-derived NRGs to regulate AChR gene expression at the neuromuscular junction.

Increased expression of estrogen-receptor exon-5-deletion variant in relapse tissues of human breast cancer.

A substantial percentage (30-70%) of human breast carcinomas that initially respond to endocrine therapy acquire resistance during the treatment. Many patients with tumor progression despite treatment with anti-estrogen tamoxifen show continued expression of estrogen receptors (ER) and/or progesterone receptors (PgR) in the relapse tissue. This indicates that, in these tumors, mechanisms other than loss of ER expression are responsible for treatment failure. We have investigated the occurrence and frequency of the exon-5-deletion variant (d5) of ER in human breast-cancer biopsies and in normal tissues. In all normal and tumor tissues tested, both wild-type (wt) and d5 were detected, indicating that expression of the d5 variant is a naturally occurring polymorphism. However, the primary tumors of patients who relapse within 15 months (n = 13) express higher ratios of d5 than do those of patients with no relapse during the same period (p = 0.4, n = 19), though this difference is statistically not significant. A significant increase in the expression level of d5 was determined in relapse as compared with the respective primary tumor (p = 0.02). These data indicate that increased expression of the ER exon-5-deletion variant in relapse tissues might be due to clonal selection of cells resistant to anti-estrogen treatment.

Selective regulation of steroid receptor expression in MCF-7 breast cancer cells by a novel member of the heregulin family.

A 52 kDa heregulin secreted by estrogen receptor (ER)-negative human breast cancer cells induced rapid growth of ER-positive MCF-7 breast cancer cells with a stimulatory effect observed at 10(-11)M. This heregulin down-regulated the message for ER in MCF-7 cells within 24 hours after stimulation. Similarly the ER protein was down-regulated within 24 to 48 hours after stimulation of cells. However, this down-regulation occurred without activation of the ER, since the progesterone receptor (PR) level of cells stimulated with the 52 kDa heregulin did not increase over the time period measured. As a control, estradiol down-regulated and activated ER as shown by a pronounced increase in PR content of MCF-7 cells. This finding indicates an important role of this heregulin in the down-regulation of ER in estrogen-dependent human breast cancer cells.

The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome.

The VirD2 protein of Agrobacterium tumefaciens was shown to pilot T-DNA during its transfer to the plant cell nucleus. We analyze here its participation in the integration of T-DNA by using a virD2 mutant. This mutation reduces the efficiency of T-DNA transfer, but the efficiency of integration of T-DNA per se is unaffected. Southern and sequence analyses of integration events obtained with the mutated VirD2 protein revealed an aberrant pattern of integration. These results indicate that the wild-type VirD2 protein participates in ligation of the 5'-end of the T-strand to plant DNA and that this ligation step is not rate limiting for T-DNA integration.

Mapping of the RNA-binding domain of the alfalfa mosaic virus movement protein.

In-frame contiguous deletions were created in the movement protein gene of alfalfa mosaic virus by site-directed mutagenesis. The mutated movement proteins were expressed in Escherichia coli, extracted and then purified by denaturing gel electrophoresis and then renatured. Their binding ability with RNA was assayed by electrophoretic retardation and u.v.-crosslinking. Results indicated that a domain included within amino acids 36 to 81 was necessary for RNA binding.

Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation.

As an early stage of plant transformation by Agrobacterium tumefaciens, the Ti plasmid is nicked at the border sequences that delimit the T-DNA. Cleavage results in covalent attachment of VirD2 to the 5' terminal of the nicked strand by a process resembling initiation of DNA transfer that occurs in the donor cell during bacterial conjugation. We demonstrate that this cleavage can be reproduced in vitro: VirD2 protein, the border-cleaving enzyme, was overproduced and purified. Cleavage assays were performed with single-stranded oligodeoxyribonucleotides encompassing the Ti plasmid border region or the transfer origin's nick region of the conjugative plasmid RP4. VirD2 of pTiC58 cleaves both border- and nick region-containing oligonucleotides. However, the relaxase TraI of RP4 can cut only the cognate nick regions. The respective proteins remain covalently bound to the 5' end of the cleavage sites, leaving the 3' termini unmodified. VirD2-mediated oligonucleotide cleavage was demonstrated to be an equilibrium reaction that allows specific joining of cleavage products restoring border and nick regions, respectively. The possible role of VirD2 in T-DNA integration into the plant cell's genome is discussed in terms of less stringent target-sequence requirements.

Binding of RNA by the alfalfa mosaic virus movement protein is biphasic.

The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography. The purified protein bound single-stranded RNA cooperatively in a biphasic manner. At protein saturation, RNA/protein complexes (designated 'primary complexes') were detected by a nitrocellulose-retention assay within 1 min of mixing, both at 4 and 22 degrees C. In contrast, an incubation of 30 min at 22 degrees C was necessary to obtain electrophoretically retarded complexes ('stabilized complexes'), containing a large number of protein molecules bound stably to each molecule of RNA. Stabilization did not take place at 4 degrees C. The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a bimolecular interaction. In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules. In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation. The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed.

An N-proximal sequence of the alfalfa mosaic virus movement protein is necessary for association with cell walls in transgenic plants.

We have made transgenic tobacco plants (Nicotiana tabacum, cv. Xanthi nc) expressing the movement protein (P3, 300 amino acids) of alfalfa mosaic virus (A1MV) and two N-terminally deleted proteins lacking respectively 12 and 77 amino acids of the P3 sequence (P3 delta[1-12] and P3 delta[1-77]). The same proteins were expressed in recombinant yeast. By subcellular fractionation, the full-length P3 protein expressed by transgenic plants was found to be associated with cell walls as well as with cytoplasmic particulate material, as was the wild type movement protein expressed by A1MV-infected tobacco plants. P3 delta[1-12] behaved similarly but P3 delta[1-77] was found only in the cytoplasm. It thus appears that a polypeptide domain located between amino acids 13 and 77 of the P3 sequence is necessary for association of the protein with cell walls.

Nucleic acid-binding properties of the alfalfa mosaic virus movement protein produced in yeast.

The movement protein of alfalfa mosaic virus (P3) was purified from yeasts transformed with an expression vector containing the P3 gene. Its nucleic acid-binding properties were tested by electrophoretic retardation, nitrocellulose retention, and RNA-protein cross-linking. The recombinant protein had a higher affinity for single-stranded RNA and DNA than for double-stranded nucleic acids. Each nucleic acid molecule bound several protein molecules without sequence specificity. The binding was 80% inhibited by 0.2 M NaCl. These properties are qualitatively similar, but not strictly identical, to those of two other viral movement proteins, the 30-kDa protein of tobacco mosaic virus and the gene I product of cauliflower mosaic virus.

Nucleic acid binding properties of the 92-kDa replicase subunit of alfalfa mosaic virus produced in yeast.

The 92-kDa non-structural protein of alfalfa mosaic virus (one of the replicase subunits) was synthesized by Saccharomyces cerevisiae transformed with a recombinant expression vector. The yeast-expressed protein had the immunological and size characteristics of the naturally made viral protein. It was partially purified and its nucleic acid binding properties were tested by gel-retardation electrophoresis and nitrocellulose adsorption. The protein interacted with single-stranded RNA, double-stranded RNA and double-stranded DNA in a salt-dependent manner, with a slight preference for RNA. These properties may be related to its putative function as a core RNA polymerase.