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M R Crompton

Publications and source records attributed to M R Crompton.

At least 19 recordsLinked to original sources
PubMed2001

Binding of the merlin-I product of the neurofibromatosis type 2 tumour suppressor gene to a novel site in beta-fodrin is regulated by association between merlin domains.

The mechanism underlying the tumour-suppressor activity of the neurofibromatosis type 2 (NF2) gene product, merlin, is largely undefined but there is evidence that the biological function of the protein might be mediated partly through interactions with the cytoskeleton. Merlin is expressed predominantly as two isoforms that differ at their C-termini owing to alternative splicing of exon 16. By expressing merlin isoform I as bait in a yeast two-hybrid screen, we isolated a clone encoding a region of the cytoskeletal protein beta-fodrin. Confirmation of the merlin-fodrin interaction was provided by using the mammalian two-hybrid system and binding assays in vitro. In addition, these assays and co-immunoprecipitation from mammalian cells revealed that the binding site for fodrin is located in the C-terminal half of merlin at a site that is masked in the native protein. Co-expression of the N-terminus of merlin decreased the interaction of its C-terminus with fodrin, implicating homophilic interactions of merlin isoform I in masking the fodrin-binding site. The effect of three disease-associated mutations on the merlin-fodrin interaction and merlin dimerization was also investigated. The mutation L535P, but not L360P or K413E, significantly decreased the merlin-fodrin interaction but not dimerization, indicating that the tumour suppressor ability of merlin might reside partly in its ability to interact with the cytoskeleton via fodrin.

Amino Acid Sequence↗
PubMed2000

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.

Breast↗
PubMed2000

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

Adaptor Proteins, Signal Transducing↗
PubMed1999

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.

Animals↗
PubMed1999

Cytotoxic response of ovarian cancer cell lines to IFN-gamma is associated with sustained induction of IRF-1 and p21 mRNA.

Interferon-gamma (IFN-gamma) has some anti-tumour activity in human ovarian cancer. This cytokine inhibited proliferation in three of four ovarian cancer cell lines in vitro. We then compared the action of IFN-gamma in two cell lines, one sensitive and one resistant to its growth inhibitory effects. IFN-gamma signalling appeared normal in both cell lines, with stat1 DNA binding activity detectable at 30 min. Continuous exposure to IFN-gamma for 2-3 days was necessary for an irreversible effect on cell growth and apoptosis in cells sensitive to growth inhibition. During this time there was an increase in mRNA for the CKI p21, but no alterations in mRNA levels for other members of the CKI family. Maintenance of p21 mRNA required continuous mRNA synthesis. mRNA for the transcription factor IRF-1 was also induced in growth inhibited cells with similar kinetics to those observed for p21. Maximal induction of both p21 and IRF-1 mRNA was observed after 2-3 days IFN-gamma exposure as the cells became committed to cell death. There was also a rapid increase in p21 and IRF-1 mRNA in cells resistant to the growth inhibitory effects of IFN-gamma, but this increase was not maintained. Thus, continuous interaction with the IFN-gamma receptor, together with a sustained induction of p21 and IRF-1, is associated with growth inhibitory and apoptotic effects of IFN-gamma in ovarian cancer cells.

Adenocarcinoma↗
PubMed1998

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.

BRCA1 Protein↗
PubMed1998

Expression of v-src in mammary epithelial cells induces transcription via STAT3.

Transgenic mouse models of mammary tumorigenesis and analyses of human breast tumour samples have indicated a role for Src proteins in the tumorigenic process. The downstream effectors of Src function in mammary epithelial cells are less well understood. STAT proteins constitute a family of transcription factors whose activation by cytokine and non-cytokine receptors leads to tyrosine phosphorylation, dimerization and translocation from the cytoplasm to the nucleus. In the nucleus they activate the transcription of specific genes by binding to consensus DNA elements. STATs 1 and 3 can be activated by both cytokine and non-cytokine receptors, and bind as homodimers or heterodimers to viral simian sarcoma virus (sis)-inducible elements such as that found in the c-fos promoter. Here we report that one of the downstream effectors of Src function in mammary epithelial cells is STAT3. We demonstrate that v-src expression in mammary epithelial cells induces Tyr-705 phosphorylation, nuclear translocation and DNA binding of STAT3. Furthermore, we demonstrate that v-src can induce STAT3-dependent transcription. These observations are the first direct evidence that v-src can regulate transcription through the activation of STAT proteins, and add a further level of complexity to the understanding of the mode of action of v-src.

Biological Transport↗
PubMed1998

[3H] imipramine binding in brain samples from depressed suicides and controls: 5-HT uptake sites compared with sites defined by desmethylimipramine.

Using desmethylimipramine (DMI) defined and Na+ dependent [3H]imipramine binding, we have examined both 5-HT uptake sites and sites unrelated to 5-HT uptake, in frontal cortex, putamen and substantia nigra of suicides with a firm retrospective diagnosis of depression and matched controls. No differences were seen between antidepressant-free suicides and controls, although [3H]imipramine binding sites were significantly lower in putamen of the subgroup of non-violent suicides. The number of DMI defined [3H]imipramine binding sites was also significantly lower in putamen of antidepressant-treated suicides.

Adolescent↗
PubMed1998

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.

Breast↗
PubMed1998

Ras-related TC21 is activated by mutation in a breast cancer cell line, but infrequently in breast carcinomas in vivo.

Activating ras mutations are found in many types of human tumour. Mutations in Harvey (H-), Kirsten (K-) and neuronal (N-) ras are, however, rarely found in breast carcinomas. TC21 is a ras family member that shares close homology to H-, K- and N-ras, and activating mutations have been found in ovarian carcinoma and leiomyosarcoma cell lines. We have examined panels of cDNAs from breast, ovarian and cervical cell lines, and primary and metastatic breast tumours for mutations in TC21 using a single-strand conformational polymorphism (SSCP)-based assay. One breast cancer cell line, CAL51, exhibited an altered SSCP pattern, compared with normal tissue, which was due to an A-T base change in codon 72, causing a predicted Gln-Leu activating mutation. Of nine primary and 15 metastatic breast tumour cDNAs analysed, none exhibited an altered pattern by SSCP. The apparently wild-type pattern by SSCP analysis was confirmed by sequence analysis of some of the cDNAs assayed. Thus, we conclude that mutations in TC21 are uncommon in breast carcinomas.

Breast Neoplasms↗
PubMed1997

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.

Animals↗
PubMed1997

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.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1997

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).

Amino Acid Sequence↗
PubMed1997

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.

Antibodies, Monoclonal↗
PubMed1997

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.

Adult↗
PubMed1997

Brain alpha-adrenoceptors in depressed suicides.

alpha1-Adrenoceptors and alpha2-adrenoceptors were measured by radioligand binding to homogenates of brain samples obtained at post-mortem from suicides with a retrospective diagnosis of depression, and age and gender-matched controls. Suicides were subdivided into those who had been free of antidepressant drugs for at least three months, and those in whom prescription of antidepressant drugs was clearly documented. The number of alpha1-adrenoceptors (or alpha1A + alpha1D-adrenoceptors) did not differ significantly between antidepressant-free or antidepressant-treated suicides and controls. In antidepressant-free suicides, the number of alpha2-adrenoceptors was significantly higher in temporal cortex (Ba 21/22). alpha2A-Adrenoceptors did not differ significantly from controls in this brain region, suggesting the involvement of other alpha2-adrenoceptor subtypes. In antidepressant-treated suicides, significantly lower numbers of alpha2-adrenoceptors were found in occipital cortex and hippocampus (and for alpha2A-adrenoceptors in caudate and amygdala) compared to controls.

Adolescent↗
PubMed1997

Dopamine D1 and D2 receptor binding sites in brain samples from depressed suicides and controls.

Dopamine D1 and D2 receptors were measured (by saturation binding of [3H]SCH23390 and [3H]raclopride) in caudate, putamen and nucleus accumbens, obtained at post-mortem from suicide victims with a firm retrospective diagnosis of depression, and matched controls. There were no differences in the number or affinity of D1 or D2 receptors between suicides who had been free of antidepressants for at least three months prior to death, and controls. Increased numbers and decreased affinity of D2 receptors were however found in each brain region of antidepressant-treated suicides. We argue that these increases are related to concurrent treatment with neuroleptics rather than a direct effect of antidepressants. Increased numbers of D1 receptors in antidepressant-treated suicides were seen only in nucleus accumbens. This increase could not be clearly attributed to neuroleptics and may be related to antidepressant treatment.

Adolescent↗