Nuclear location and cell cycle regulation of the BRCA2 protein.

Women carrying a germ-line mutation in the BRCA1 or BRCA2 genes have a high risk of developing breast cancer, and loss of the wild-type allele in tumors suggests that these genes function as tumor suppressor genes. The BRCA2 gene encodes a 3418-amino acid protein with no significant sequence similarity to any known protein. To begin to elucidate the cellular role of BRCA2, we have raised antibodies to the BRCA2 protein and used these to study its subcellular localization and expression. We show that BRCA2 is a nuclear protein expressed in response to cell proliferation and that BRCA2 expression is initiated before DNA synthesis.

Expression of the BRK tyrosine kinase in mammary epithelial cells enhances the coupling of EGF signalling to PI 3-kinase and Akt, via erbB3 phosphorylation.

A high proportion of human breast cancers, in contrast with normal mammary tissue, express the intracellular tyrosine kinase BRK. BRK expression enhances the mitogenic response of mammary epithelial cells to epidermal growth factor, and conferment of a proliferative advantage through this mechanism may account for the frequent elevation of BRK expression in tumours. Here we report that BRK expression in mammary epithelial cells, at pathologically relevant levels, results in an enhanced phosphorylation of the epidermal growth factor receptor-related receptor erbB3 in response to epidermal growth factor. As a consequence, erbB3 recruits increased levels of phosphoinositide 3-kinase, and this is associated with a potentiated activation of Akt. This effect of BRK on the regulation of phosphoinositide 3-kinase and Akt activity may account for BRK's ability to enhance mammary cell mitogenesis, and raises the possibility that breast tumours expressing BRK may acquire a resistance to pro-apoptotic signals.

BRK tyrosine kinase expression in a high proportion of human breast carcinomas.

BRK is a recently described non receptor protein tyrosine kinase whose mRNA was found to be expressed in human breast tumours and breast cancer cell lines. Expression of BRK in fibroblasts and mammary epithelial cells has been shown to enhance their ability to grow anchorage independently, and mammary epithelial cells expressing BRK acquire a potentiated mitogenic response to epidermal growth factor. In order to investigate further the expression of BRK in breast cancers, we have isolated monoclonal antibodies specifically recognising the protein. Whereas BRK expression was low or undetectable in normal mammary tissue and benign lesions, approximately two-thirds of breast tumours expressed appreciable levels, and 27% of tumours over expressed BRK by fivefold or more (up to 43x). This expression pattern was mirrored in a comparison of cell lines derived either from normal mammary epithelial cells or from carcinomas. BRK expression was found to be constant throughout the cell cycle, and did not vary with cell proliferation rate. A consideration of this expression data, in conjunction with BRK's demonstrated ability to deregulate the proliferation of mammary epithelial cells, supports the hypothesis that the over expression of BRK in a high proportion of breast carcinomas is a functionally important factor in their evolution.

Characterisation and chromosome mapping of the human non receptor tyrosine kinase gene, brk.

The brk gene encodes a non-receptor protein tyrosine kinase that consists of single SH3, SH2 and catalytic domains. Although BRK shows strongest sequence similarity to members of the SRC family of PTKs, there are several key structural and regulatory differences that place it on its own amongst non-receptor PTKs. In this study we have isolated genomic DNA clones corresponding to the human brk locus and used these to determine the intron-exon structure of the brk gene. The genomic structure of brk consists of 8 exons, whose boundaries are distinct from other non-receptor PTK family members, again indicating a structural and functional divergence. Alternate splicing of the primary brk transcript generates a distinct mRNA which encodes a truncated protein consisting of an SH3 domain and a novel C-terminal proline rich sequence. Using an antiserum raised to the SH3 domain, we have demonstrated that the product of this alternate brk transcript is expressed in the human breast tumour cell line T-47D. We have previously reported that expression of a tumour derived brk cDNA in mouse embryonic fibroblasts and human mammary epithelial cells supports anchorage independent growth, and in the latter potentiates the mitogenic response to epidermal growth factor. The protein encoded by the genomic sequence derived from normal human tissue is identical to that encoded by the tumour derived cDNA, and therefore the altered growth regulation is not associated with mutations within brk. In addition, we have identified a 5' genomic region that has promoter activity. The brk gene has been assigned to chromosome 20q 13.3 [corrected] using fluorescence in situ hybridisation (FISH).

A novel adaptor-like protein which is a substrate for the non-receptor tyrosine kinase, BRK.

The brk gene encodes a non-receptor tyrosine kinase that has been found to be overexpressed in approximately two thirds of breast tumours. Using a yeast two-hybrid based screen, we have cloned cDNAs encoding a novel protein, BKS, that is a substrate for the kinase activity of BRK and has the characteristics of an adaptor protein. BKS possesses an N-terminal PH-like domain followed by an SH2-like domain. In co-transfection experiments, high levels of phosphotyrosine were observed on BKS and BRK was found to be associated with BKS, both of which were dependent on the catalytic activity of BRK. The phosphorylation of and association with BKS by BRK was also dependent on the SH2-like domain present within BKS. In addition, BKS recruited an unidentified 100 kDa protein that was also phosphorylated on tyrosine residues in the presence of BRK. We have determined that the BKS protein is expressed in most adult human tissues. Oncogene (2000) 19, 4273 - 4282

Cloning and characterisation of cDNAs encoding a novel non-receptor tyrosine kinase, brk, expressed in human breast tumours.

Using a polymerase chain reaction based differential screening approach, we have isolated and characterised a cDNA from a human metastatic breast tumour representing a novel protein tyrosine kinase (brk). Sequencing of brk cDNAs isolated from T-47D and MCF-7 human breast tumour cell lines indicate that they encode a protein with the features of a novel nonreceptor tyrosine kinase, including amino terminal SH3 and SH2 domains. When synthesised in recombinant baculovirus and bacterial expression systems, brk protein products are capable of autophosphorylation on tyrosine residues. Initial expression studies have detected low levels of brk transcripts in some human breast tumours and breast tumour cell lines, but not in normal breast tissue.

Expression patterns of the novel receptor-like tyrosine kinase, DDR, in human breast tumours.

Using a reverse transcriptase-polymerase chain reaction based differential screening procedure, we have identified the discoidin domain receptor as a protein tyrosine kinase that is expressed in lymph nodes containing breast tumour metastases. By Northern blotting and in situ hybridisation we have demonstrated the expression of the discoidin domain receptor in human primary breast tumour samples, metastasis-containing lymph nodes and a number of normal tissues. Direct comparison of malignant breast and adjacent normal epithelial tissue revealed over expression in the tumour cells.

Novel p53 mutants selected in BRCA-associated tumours which dissociate transformation suppression from other wild-type p53 functions.

Inheritance of germ-line mutant alleles of BRCA1 and BRCA2 confers a markedly increased risk of breast cancer and we have previously reported a higher incidence of p53 mutations in these tumours than in grade matched sporadic tumours. We have now characterized these p53 mutants. The results of these studies identify a novel class of p53 mutants previously undescribed in human cancer yet with multiple occurrences in BRCA-associated tumours which retain a profile of p53-dependent activities in terms of transactivation, growth suppression and apoptosis induction which is close or equal to wild-type. However, these mutants fail to suppress transformation and exhibit gain of function transforming activity in rat embryo fibroblasts. These mutants therefore fall into a novel category of p53 mutants which dissociate transformation suppression from other wild-type functions. The rarity of these mutants in human cancer and their multiple occurrence in BRCA-associated breast tumours suggests that these novel p53 mutants are selected during malignant progression in the unique genetic background of BRCA1- and BRCA2-associated tumours.

p53 mutation with frequent novel condons but not a mutator phenotype in BRCA1- and BRCA2-associated breast tumours.

The status of p53 was investigated in breast tumours arising in germ-line carriers of mutant alleles of BRCA1 and BRCA2 and in a control series of sporadic breast tumours. p53 expression was detected in 20/26 (77%) BRCA1-, 10/22 (45%) BRCA2-associated and 25/72 (35%) grade-matched sporadic tumours. Analysis of p53 sequence revealed that the gene was mutant in 33/50 (66%) BRCA-associated tumours, whereas 7/20 (35%) sporadic grade-matched tumours contained p53 mutation (P<0.05). A number of the mutations detected in the BRCA-associated tumours have not been previously described in human cancer databases, whilst others occur extremely rarely. Analysis of additional genes, p16INK4, Ki-ras and beta-globin revealed absence or very low incidence of mutations, suggesting that the higher frequency of p53 mutation in the BRCA-associated tumours does not reflect a generalized increase in susceptibility to the acquisition of somatic mutation. Furthermore, absence of frameshift mutations in the polypurine tracts present in the coding sequence of the TGF beta type II receptor (TGF beta IIR) and Bax implies that loss of function of BRCA1 or BRCA2 does not confer a mutator phenotype such as that found in tumours with microsatellite instability (MSI). p21Waf1 was expressed in BRCA-associated tumours regardless of p53 status and, furthermore, some tumours expressing wild-type p53 did not express detectable p21Waf1. These data do not support, therefore, the simple model based on studies of BRCA-/- embryos, in which mutation of p53 in BRCA-associated tumours results in loss of p21Waf1 expression and deregulated proliferation. Rather, they imply that proliferation of such tumours will be subject to multiple mechanisms of growth regulation.

cDNA cloning of a homeobox-containing gene expressed in avian myeloblastic virus-transformed chicken monoblastic leukaemia cells.

By combining the polymerase chain reaction and differential library screening, a cDNA for an mRNA expressed in chicken avian myeloblastosis virus (AMV)-transformed monoblasts was isolated. This mRNA is not expressed in erythroblast or T-lymphoblast cell lines. Induced differentiation of the cells of the AMV-transformed BM2 line was associated with reduced levels of this transcript. The predicted protein product of Chox M was a homeodomain factor similar to murine Hox-4.3.

Functional consequences of cyclin D1 overexpression in human mammary luminal epithelial cells.

The proliferation of eukaryotic cells is primarily regulated by a decision made during the G1 phase of the cell cycle as to remain in the cycle and divide, or to withdraw from the cycle and adopt a different cell fate. During this time, environmental signals, which regulate the synthesis of the G1 cyclins, are coupled to cell division. In this context, mammalian D-type cyclins have been shown to control progression through the G1 phase of the mammalian cell cycle. Specifically, cyclin D1 has been reported frequently to be amplified, over-transcribed and overexpressed in human breast carcinomas. Although the effects of cyclin D1 overexpression have been examined in human breast carcinoma cell lines, the biological consequences of cyclin D1 expression in normal human mammary epithelial cells remain to be elucidated. In this study we have stably over expressed cyclin D1 in human mammary luminal epithelial cells in order to more directly address the role of cyclin D1 in cell cycle control and tumorigenesis of the human breast. Here, we demonstrate that the effect of cyclin D1 overexpression in these cells is to reduce their growth factor dependency, as well as shorten the duration of G1 and correspondingly reduce the mean generation time. Collectively, our data indicate that deregulation of cyclin D1 expression in human mammary epithelial cells can provide a growth advantage and hence contribute to the oncogenic potential of these cells.

Brain beta-adrenoceptor binding sites in depressed suicide victims: effects of antidepressant treatment.

beta-Adrenoceptor binding sites were measured by saturation binding of [3H]CGP 12177 in nine brain regions from 13 suicides, with a firm retrospective diagnosis of depression, who had been receiving antidepressant drugs, and 11 matched controls. Significantly lower numbers of beta-adrenoceptor binding sites were found in thalamus and temporal cortex (Brodmann area 38), but not in other brain regions, of antidepressant-treated suicides compared to controls. The lower number of beta-adrenoceptor binding sites in thalamus appeared to be related to drug treatment, whereas lower numbers of beta-adrenoceptors in temporal cortex were also found in antidepressant-free suicides.

Brain 5-HT1 binding sites in depressed suicides.

5-HT1 and 5-HT1A binding sites were measured in brain tissue obtained at postmortem from 19 suicides, with definite evidence of depression, and 19 sex and age-matched controls. Thirteen of the depressed suicides had not been prescribed psychoactive drugs recently (drug-free suicides); six had been receiving antidepressant drugs, alone or in combination with other drugs (antidepressant-treated suicides). No significant differences were found in the number or affinity of 5-HT1 and 5-HT1A binding sites in frontal or temporal cortex between drug-free suicides and controls. The number of 5-HT1 sites was significantly lower (by 20%), affinity unaltered, in hippocampus and the affinity significantly lower (by 33%), number unaltered, in amygdala of drug-free suicides than controls. The number of 5-HT1 binding sites tended to be higher and the affinity lower in the antidepressant-treated compared to drug-free suicides, and significantly so in hippocampus. The present results, together with our previous studies, provide no evidence of altered cortical 5-HT markers in depressed suicides, but further emphasise abnormalities in the hippocampus.

Corticotropin-releasing factor binding sites in cortex of depressed suicides.

Corticotropin-releasing factor (CRF) receptors were measured by saturation binding in frontal and motor cortex of suicides with a firm retrospective diagnosis of depression, and matched controls. The suicides were divided into those who were free of antidepressant drugs, and those in whom prescription of antidepressant drugs was clearly documented. There were no differences in the number or affinity of CRF receptors between antidepressant-free or antidepressant-treated suicides and matched controls in either brain region. When suicides were divided according to violence of death, again there were no differences between violent or non-violent suicides and controls.

Brain [3H]cAMP binding sites are unaltered in depressed suicides, but decreased by antidepressants.

Saturation binding of [3H]cAMP to the regulatory subunit of cAMP-dependent protein kinase (PKA) was measured in the soluble fraction of brain samples, obtained at post-mortem, from suicides with a firm retrospective diagnosis of depression and individually matched controls. Suicides were subdivided into those who had been free of antidepressant drugs for at least 3 months and those in whom prescription of antidepressants was clearly documented. In antidepressant-free suicides, we found no significant differences in the number or affinity of [3H]cAMP binding sites in the five regions studied. In antidepressant-treated suicides however, Bmax values were lower in all regions, reaching statistical significance in parietal cortex and amygdala. Kd values for antidepressant-treated suicides were significantly higher in parietal cortex, temporal cortex and amygdala. These results suggest the regulatory subunit of PKA is unaltered in depression, but is influenced by antidepressant drugs.

Brain beta-adrenoceptor binding sites in antidepressant-free depressed suicide victims.

beta-Adrenoceptor binding sites were quantitated by saturation binding of [3H]CGP 12177 in 9 brain regions from 21 suicide victims, with a firm retrospective diagnosis of depression, who had not recently received antidepressant drugs, and 20 age- and sex-matched controls. In depressed suicides the number of total beta-adrenoceptors was significantly lower in temporal cortex (Brodmann area 38, by 19%) and beta 1-adrenoceptors (Brodmann area 21/22, by 17%) compared to controls. Suicides who died by violent means had significantly lower numbers of total beta- and beta 1-adrenoceptors in frontal cortex than matched controls (by 23 and 25%, respectively) and than non-violent suicides (by 20 and 22%, respectively) and lower numbers of beta 1-adrenoceptors in temporal cortex (Brodmann area 21/22) than matched controls (by 16%). Depressed suicides who died by non-violent means had lower numbers of total beta-adrenoceptors in occipital cortex than matched controls (by 24%) and than violent suicides (by 18%), and lower numbers of total beta- and beta 1-adrenoceptors in temporal cortex (Brodmann area 38) than matched controls (by 27 and 24%, respectively). Depression in suicide victims is associated with deficits in beta-adrenoceptor binding sites, largely restricted to cortical areas.

Brain GABAA/benzodiazepine binding sites and glutamic acid decarboxylase activity in depressed suicide victims.

We have investigated the involvement of gamma-aminobutyric acid (GABA) in depression by quantitating benzodiazepine (BZ) binding sites, the ability of GABA to stimulate BZ binding and glutamic acid decarboxylase activity in frontal and temporal cortex obtained at postmortem examination from 21 suicide victims and 21 age- and sex-matched controls. We limited our study to suicide victims with clear evidence of depression, in the absence of symptoms of other psychiatric disorders. Thirteen of the depressed suicide victims had not been prescribed psychoactive drugs recently and none were found in their blood at postmortem; of the remaining 8 suicides, 6 were receiving antidepressant drugs, alone or in combination with other drugs. The number of BZ binding sites was significantly greater (by 18%) in the frontal cortex of the total group of depressed suicides compared to controls, but did not differ in the temporal cortex. The increase in the number of BZ binding sites in the frontal cortex was of similar magnitude when drug-free (16%), drug-treated (21%) and antidepressant-treated suicides (16%) were compared to matched controls, although the increase was only statistically significant for the drug-treated suicides. The Kd of BZ binding and the ability of GABA to stimulate BZ binding did not differ significantly between controls and the total, drug-free, drug-treated or antidepressant-treated suicides in either cortical area. Glutamic acid decarboxylase activity did not differ significantly between control and suicide groups, but was markedly reduced in subjects dying by carbon monoxide poisoning. The present study provides evidence for a greater number of BZ binding sites in the frontal cortex of depressed suicide victims, which could not clearly be attributed to drug treatment.

Brain 5-HT2 receptor binding sites in depressed suicide victims.

5-HT2 receptor binding sites were measured (by saturation binding of [3H]ketanserin) in brain tissue obtained at postmortem from 19 suicide victims with definite evidence of depression and 19 sex and age-matched control subjects. Five of the suicide victims were receiving antidepressant drugs prior to death; 13 suicide victims had not been prescribed antidepressant or other psychoactive drugs recently and none were found in their blood at postmortem. The number of serotonin-2 (5-HT2) binding sites in frontal, temporal and occipital cortex and amygdala did not differ significantly between the depressed suicide victims and controls, either in the total suicide group or in the antidepressant drug-free suicides. The number of 5-HT2 binding sites in the hippocampus did not differ from controls in the total suicide group but was significantly lower (by 23%) in the antidepressant-free suicide group. The affinity of [3H]ketanserin binding did not differ from controls in the antidepressant-free suicides but was lower (increased Kd) in those subjects receiving antidepressant drugs. No correlation was found between the time of death and storage of tissue or the duration of tissue storage prior to assay and the number or affinity of 5-HT2 binding sites. A significant negative correlation was found between age of the subject and the number of 5-HT2 binding sites in the frontal and occipital cortex. The present study of suicide victims with definite evidence of depression do not confirm previous studies of increased numbers of 5-HT2 binding sites in suicide victims and suggest that these previous findings may be related to factors other than depression.

Brain 5-HT2 receptors in suicide victims: violence of death, depression and effects of antidepressant treatment.

5-HT2 binding sites were quantitated, by saturation binding with [3H]ketanserin, in six brain regions from 73 subjects who died by suicide and 70 sudden death controls. There were no significant differences in the number of 5-HT2 binding sites between suicides and controls in any brain region within the total suicide group or when suicides were divided on the basis of violence of death. Similar results were found when suicides were divided into those with a firm retrospective diagnosis of depression, whether they had been receiving antidepressants or not, and those who were heterogeneous in respect of psychiatric diagnosis and drug treatment. The present findings contrast with previous reports of higher cortical 5-HT2 binding sites in suicides; possible reasons for these differences are discussed.

Reduced dopamine turnover in the basal ganglia of depressed suicides.

We have measured the concentrations of dopamine, and the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), in five brain regions from suicide victims with a firm retrospective diagnosis of depression, and matched controls. The suicides were divided into those free of antidepressant drugs and those in whom prescription of antidepressant drugs was clearly documented. DOPAC concentrations were significantly lower in caudate, putamen and nucleus accumbens of antidepressant-free suicides compared to controls. In antidepressant-treated suicides, lower concentrations of DOPAC were observed in the basal ganglia, reaching statistical significance in caudate. Lower DOPAC concentrations were largely restricted to those suicides who died by non-violent methods. There were no significant differences in dopamine and HVA concentrations in either suicide group compared to controls, although there was a trend for HVA concentrations to be lower in suicides. This study provides evidence for reduced dopamine turnover, as judged from reduced DOPAC levels, in depressed suicides, although we cannot exclude the possibility that this may be due to ingestion of toxic agents.