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Biomedical subjects

H C Hurst

Publications and source records attributed to H C Hurst.

At least 37 records · Page 2Linked to original sources
PubMed1996

Chromosomal mapping of the human and mouse homologues of two new members of the AP-2 family of transcription factors.

The AP-2 transcription factor has been shown to play an important role in the development of tissues of ectodermal origin and has also been implicated in mammary oncogenesis. It has recently been found that AP-2 is encoded by a family of related genes, AP-2alpha, AP-2beta, and AP-2gamma. As a further step in understanding the role each of these genes has in development, we have used fluorescence in situ hybridization to map the chromosomal locations of the mouse and human homologues of the newly isolated AP-2beta and AP-2gamma genes. Tcfap2b and Tcfap2c map to mouse chromosomes 1A2-4 and 2H3-4, respectively, while TFAP2B and TFAP2C map to human chromosomes 6p12 and 20q13.2, the latter being a region that is frequently amplified in breast carcinoma.

Animals↗
PubMed1996

Copurification of casein kinase II with transcription factor ATF/E4TF3.

We have developed a simple method to purify sequence-specific DNA-binding proteins directly from crude cell extracts by using DNA affinity latex beads. The method enabled us to purify not only DNA-binding proteins, but also their associated proteins. Using beads bearing the ATF/E4TF3 site from the adenovirus E4 gene promoter, a protein kinase activity was copurified with the ATF/E4TF3 family. We found that the kinase interacted with ATF1 in vitro efficiently. The kinase did not bind directly to DNA. The kinase mainly phosphorylated ATF1 on serine 36, which was one of target amino acids for casein kinase (CK) II. Biological features of the kinase were the same as those of CKII and an anti-CKII serum reacted with the kinase, indicating that the kinase was CKII. Moreover, it was clearly shown that one of CKII subunits, the CKII alpha protein bound to glutathione-S-transferase (GST) fusion ATF1 but not GST in vitro. It has been reported that a specific CKII inhibitor, 5,6-dichloro-1-beta-D-ribo-furanosylbenzimidazole (DRB) inhibits transcription by RNA polymerase II [Zandomeni et al., (1986) J. Biol. Chem. 261, 3414-3419]. Taken together, these results suggest that ATF/E4TF3 may recruit the CKII activity to a transcription initiation machinery and stimulate transcription.

Activating Transcription Factors↗
PubMed1996

Transcription factors as drug targets in cancer.

Within multicellular organisms, cells are continually signalling to each other to keep in tune with their environment. The ultimate targets for the majority of these signal pathways are upstream transcription factors, whose activity is thereby modulated, resulting in a new pattern of gene expression suitably coupled to the needs of the cell. It has been estimated that up to 10% of human genes may encode transcription factors, thus emphasising how fundamental the control of gene expression is to the processes of cellular division and differentiation during normal development. As a corollary of this, transcriptional regulation can also profoundly affect the course of growth-related diseases such as cancer. Of course it has been realised for some time that the normal counterparts of many oncogenes are transcription factors whose proper role is in the control of normal cell growth. More recent work has begun to identify several other transcription factors which may play a role in cancer, and strategies are now being developed which are designed to use our growing knowledge of transcriptional control mechanisms in the development of novel cancer therapies.

Antineoplastic Agents↗
PubMed1996

Suicide gene expression induced in tumour cells transduced with recombinant adenoviral, retroviral and plasmid vectors containing the ERBB2 promoter.

In order to exploit the tumour-specific nature of ERBB2 expression for genetic prodrug-activation therapy, we have generated recombinant adenoviral, retroviral and plasmid vectors containing an expression cassette consisting of the ERBB2 promoter and herpes simplex virus thymidine kinase coding sequence. In the case of the adenoviral vectors, the expression cassette was introduced into the E1 or E3 region of the genome. All of the vectors were capable of sensitizing ERBB2-positive cells to the action of ganciclovir. In contrast to the retroviral and plasmid vectors, however, transduction with the adenoviral vectors also resulted in sensitization of ERBB2-negative cells to ganciclovir, infection of cell lines with a beta-galactosidase expressing adenovirus showed that the sensitizing effect was not due to adenoviral infection per as in all but one of the cell lines tested. This study demonstrates that the ERBB2 promoter can be used to induce ERBB2-dependent sensitization to ganciclovir when in the context of retroviral and plasmid vectors. Observations made in this study do, however, suggest that adenoviral vectors may not be the ideal system to engineer conditional expression, and possible explanation for this phenomenon are discussed.

Adenoviridae↗
PubMed1995

The developmentally regulated transcription factor AP-2 is involved in c-erbB-2 overexpression in human mammary carcinoma.

Overexpression of the c-erbB-2/HER2 protooncogene in breast carcinoma is controlled not only by the degree of amplification of the gene but also at the level of gene transcription. Thus, whether or not the gene is amplified, the activity of the c-erbB-2 promoter is enhanced in overexpressing cells through the binding of an additional transcription factor, OB2-1, whose activity is increased in these lines. Here we describe further characterization of OB2-1 and show that it is identical to the developmentally regulated transcription factor AP-2. Functional assays confirm that AP-2 is able to regulate c-erbB-2 expression in mammary-derived cell lines. Furthermore, although AP-2 is barely detectable in cells with the low c-erbB-2 expression phenotype, protein levels are clearly elevated in a panel of c-erbB-2-overexpressing lines. These findings demonstrate an important role for this transcription factor in human cancer.

Base Sequence↗
PubMed1995

Targeting gene transcription: a new strategy to down-regulate c-erbB-2 expression in mammary carcinoma.

Overexpression of the c-erbB-2 proto-oncogene in mammary carcinoma is frequently associated with amplification of the c-erbB-2 gene, but it also occurs from single-copy gene. Studies in mammary-derived cell lines have shown that, whether or not the gene is amplified, there is a 6- to 8-fold increase in the accumulation of c-erbB-2 mRNA per gene copy in overexpressing cells. We have recently shown that this phenomenon is due to increased activity of the c-erbB-2 promoter mediated by the binding of a novel transcription factor, OB2-1, which is present at higher levels in overexpressing cells than in low expressors. OB2-1 activity therefore represents a novel therapeutic target for the down-regulation of c-erbB-2 levels in human cells. As a prototype for this strategy, we show here that the drug sodium aurothiomalate is able to inhibit the DNA-binding activity of OB2-1 in vitro and also to interfere with c-erbB-2 promoter activity in cell-based transfection assays. In addition, endogenous c-erbB-2 immunoreactivity was reduced in cells treated with aurothiomalate as compared with the levels observed in control cells.

Breast Neoplasms↗
PubMed1995

A novel cyclic AMP response element-binding protein-1 (CREB-1) splice product may down-regulate CREB-1 activity.

In this report we identify novel spliced forms of cyclic AMP (cAMP) response element-binding protein-1 (CREB-1) mRNA. These forms contained an additional 17 nucleotide insert, which we refer to as the beta exon, located between exons 4 and 7 of the delta, and 5 and 7 of the alpha forms of CREB-1 transcript (nomenclature of Ruppert et al. 1992; EMBO Journal 11, 1503-1512). The inclusion of the beta exon led to the generation of mRNAs in which the frame of CREB-1 sequences 3' to the exon was shifted such that the encoded proteins terminate after the transactivation domain, but before the target serine for cAMP-dependent protein kinase. The beta exon-containing CREB-1 mRNAs were more abundant in tissues that respond poorly to cAMP, suggesting that the generation of beta CREB-1 mRNAs may contribute to the down-regulation of CREB-1 activity and cAMP responsiveness.

Amino Acid Sequence↗
PubMed1994

Identification of a distinctive P-glycoprotein-mediated resistance phenotype in human ovarian carcinoma cells after their in vitro exposure to fractionated X-irradiation.

BACKGROUND: Clinical drug resistance is recognized in patients previously treated with radiotherapy and after chemotherapy. In vitro exposure of mammalian tumor cells to fractionated X-irradiation also resulted in the expression of drug resistance. Analysis of the resistance phenotype of irradiated Chinese hamster ovary sublines revealed P-glycoprotein overexpression, without any concomitant increase in P-glycoprotein messenger RNA, under posttranslational regulation. This study aimed to determine whether this distinctive resistance phenotype could also be identified in irradiated human tumor cells. METHODS: Irradiated sublines established from two human ovarian tumor cell lines, SK-OV-3 and JA-T, which showed resistance to vincristine and to etoposide, were studied. Protein and RNA expression were quantitated by Western and Northern blotting or RNase protection assays. P-glycoprotein turnover was measured after immunoprecipitation of metabolically labelled cells. RESULTS: Significant P-glycoprotein overexpression was detected using the C219 and C494 monoclonal antibodies in the two irradiated human ovarian tumor sublines. No concomitant increase in P-glycoprotein messenger RNA was detectable in the SK-OV-3/DXR10 subline, contrasting with the increased message characteristic of vincristine-selected SKVCR sublines. In addition, turnover of P-glycoprotein was significantly reduced in these DXR10 cells when compared with that measured in a vincristine-selected subline. These irradiated sublines showed reduced levels of epidermal growth factor receptors and unchanged levels of topoisomerase II, but they overexpressed c-erbB2 marginally and heat shock protein 27 significantly. These latter elevations in protein levels, however, were associated with concomitant increases in their respective messenger RNAs, implicating regulation at the transcriptional level. CONCLUSIONS: Exposure of human ovarian tumor cells to fractionated X-irradiation in vitro resulted in the expression of a distinctive multiple drug resistance phenotype unusually involving posttranslational regulation of P-glycoprotein. Monitoring tumor biopsies for P-glycoprotein-associated drug resistance in patients treated with radiotherapy should evaluate protein levels rather than, or as well as, MDR1 mRNA expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Transcriptional repression by the human bZIP factor E4BP4: definition of a minimal repression domain.

The bZIP factor E4BP4 overlaps in DNA binding site specificity with the transcriptional activator CREB and members of the ATF family of transcription factors, but is an active transcriptional repressor. In this study we have mapped the repressing activity of E4BP4 to a small 'domain' of 65 amino acids that retains its ability to repress transcription when transferred to the heterologous DNA binding domain of the yeast transcriptional activator GAL4. This segment of the E4BP4 polypeptide contains a high proportion of charged amino acids and does not resemble the repression domains that have been characterized so far from other active transcriptional repressors such as the Drosophila Krüppel, Engrailed or Even-skipped proteins. A mutation which changes the charge configuration of this repression module resulted in a complete loss of repressor activity. The E4BP4-GAL4 fusion protein is able to repress the residual transcription from minimal promoters containing the adenovirus E4 or E1b TATA box. This is consistent with a mechanism of action whereby E4BP4 interacts with some component of the general transcription machinery to cause repression of basal and activated transcription. Although a number of nuclear proteins are able to interact with the E4BP4 repression domain in vitro, these proteins do not appear to include the general transcription factors TFIIB or TBP.

Amino Acid Sequence↗
PubMed1994

Gene therapy for cancer using tumour-specific prodrug activation.

Current treatments for metastatic malignant disease are often ineffective. One of the most promising of the selective genetic strategies against cancer is VDEPT (virally directed enzyme prodrug therapy). This uses a viral vector to carry a prodrug-activating enzyme gene into both tumour and normal cells. By linking the foreign gene downstream of tumour-specific transcription units, tumour-specific expression of the foreign enzyme gene can be achieved. We have developed a genetic therapy strategy using VDEPT against cancers that overexpress the oncogene ERBB2. This occurs in approximately one-third of breast and pancreatic tumours (and in a smaller proportion of other tumours) and involves transcriptional up-regulation of the ERBB2 gene with or without gene amplification. We have constructed a chimeric minigene consisting of the proximal ERBB2 promoter linked to the gene encoding cytosine deaminase, an enzyme that can deaminate the prodrug 5-fluorocytosine (5-FC) to form cytotoxic 5-fluorouracil (5-FU). We have constructed a double-copy recombinant retrovirus to deliver the enzyme gene under the control of the ERBB2 promoter into a panel of ERBB2 expression-positive (ERBB2+) and -negative (ERBB2-) pancreatic and breast cell lines. Cytosine deaminase activity was high in ERBB2+ transduced cells but was not detected in ERBB2- transduced cells. Significant cell death was observed in ERBB2+ transduced cells treated with 5-FC whereas ERBB2- cells were not affected. Hence we present a novel gene therapy strategy that is potentially tumour-specific and could be used against a range of tumour types that overexpress the ERBB2 oncogene.

Base Sequence↗
PubMed1993

Identification of proteins that interact with CREB during differentiation of F9 embryonal carcinoma cells.

The mammalian transcription factor CREB is thought to activate cAMP-inducible genes in a variety of differentiated cell types and is probably involved in other signalling pathways. Undifferentiated F9 embryonal carcinoma (UF9) cells are refractory to cAMP and become cAMP-responsive following differentiation to endoderm like cells. It has been proposed that UF9 cells contain a negative regulator(s) of the cAMP-response that might act through direct interaction with CREB. We have used a protein blotting assay and 32P-labelled CREB to probe for CREB-binding proteins in nuclear extracts from F9 cells and to examine their abundance during differentiation. We find that ATF1 (a protein that is highly homologous to CREB) and a novel polypeptide(s) of approximately 100 kDa (CBP100) are the major CREB-binding proteins in extracts from UF9 cells. As expected ATF1 is detected due to leucine zipper-dependent heterodimerisation with CREB. In contrast CBP100 interacts with CREB independently of the leucine zipper. The total amount of ATF1 and the amount of ATF1 that is complexed with CREB are substantially reduced following differentiation. In addition, ATF1 mRNA levels are lower in differentiated F9 cells indicating that a pretranslational mechanism contributes to the decreased ATF1 protein levels observed. CBP100 levels are also reduced or CBP100 is modified upon differentiation. We discuss the potential roles of ATF1 and CBP100 in regulating CREB activity during differentiation of F9 embryonal carcinoma cells.

Amino Acid Sequence↗
PubMed1993

A novel transcription factor, OB2-1, is required for overexpression of the proto-oncogene c-erbB-2 in mammary tumour lines.

The c-erbB-2 receptor tyrosine kinase proto-oncogene product is overexpressed in 20-30% of breast carcinomas and this has been shown to correlate with poor prognosis. Previous analysis of tumour-derived lines has demonstrated that although the c-erbB-2 gene is often amplified, overexpression can occur from a single-copy gene. Moreover, whether or not the gene is amplified, overexpressing cells produce 6- to 8-fold more mRNA per gene copy than low-expressing cells. In this paper, we examine the possible mechanisms causing this deregulation of c-erbB-2 mRNA accumulation. Nuclear run-on studies indicated that the extra mRNA accumulation was due to increased transcription of the gene in overexpressing cells. Promoter analyses using c-erbB-2 5' flanking sequences linked to CAT showed that the promoter is more active in overexpressing cells. Coupling promoter deletion functional studies with footprinting experiments, using nuclear extracts derived from both low and overexpressing cells, allowed the identification of a DNA-binding protein, OB2-1, which is considerably more abundant in a range of overexpressing lines. We discuss the possible role of OB2-1 in c-erbB-2 overexpression in breast tumour lines.

Base Sequence↗
PubMed1993

Specific short transmembrane sequences can inhibit transformation by the mutant neu growth factor receptor in vitro and in vivo.

The neu oncogene is activated by a point mutation within its transmembrane domain that results in the substitution of glutamic acid for valine at position 664, and is associated with constitutive activation of the tyrosine kinase. It has been proposed that the mutation allows for stabilization of homodimers of the receptor that are necessary for transduction of the mitogenic signal. To investigate the role of the alpha-helical transmembrane sequence in the function of neu, we constructed an expression vector to produce a variety of short transmembrane neu proteins, lacking ligand binding or intracellular kinase domains. Such sequences should interact with full-length receptors and prevent receptor dimerization and thus act as specific inhibitors of function. These small proteins all included a pentapeptide from position 661-665, which has been proposed to be necessary for packing. We show that the short transmembrane molecules are expressed at the cell surface and can retard the growth of neu-transformed cells in monolayers, as colonies in soft agar and as tumours in animals. As predicted by molecular modelling, the magnitude of inhibition depended on the nature of the packing surface, suggesting that the neu transmembrane domain is directly involved in neu protein dimerization.

3T3 Cells↗
PubMed1993

Transcriptional regulation of the c-erbB-3 gene in human breast carcinoma cell lines.

The c-erbB-3 gene is a member of the EGF receptor family of membrane tyrosine kinases. Previous work has shown that the expression of this gene is frequently unregulated in human breast carcinoma-derived cell lines and in tumour biopsy material. As there is no evidence of gene amplification, we decided to examine the control of c-erbB-3 transcription in overexpressing cells. In this paper we describe the cloning of the c-erbB-3 promoter and its functional analysis within mammary-derived cell lines. We show that the promoter is more active in overexpressing cells and this is largely due to the action of the OB2-1 transcription factor which we have recently identified as mediating overexpression of the c-erbB-2 promoter in similar cell lines.

Amino Acid Sequence↗
PubMed1992

Expression of the ERBB3 gene product in breast cancer.

Abnormalities of the EGF receptor and/or the related ERBB2 receptor occur in a significant proportion of cases of human breast cancer and are important influences in the behaviour of this tumour type. In this report we demonstrate by nucleic acid analysis and immunohistochemistry that the recently recognised third member of this gene family, ERBB3, shows a wide range of expression in breast cancer, and shows stronger immunoreactivity than that observed in normal tissue in 43 out of 195 cases (22%) of primary breast cancer. Overexpression of ERBB3 appears to result from increased levels of gene transcription since in none of the cell lines or primary cancers analysed did we find evidence of gene amplification. High expression of ERBB3 is positively associated with the presence of lymph node metastases, but there was no demonstrable relationship with patient survival in this series.

Blotting, Southern↗