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H Esche

Publications and source records attributed to H Esche.

At least 37 records · Page 2Linked to original sources
PubMed1997

Formation of the early-region-2 transcription-factor-1-retinoblastoma-protein (E2F-1-RB) transrepressor and release of the retinoblastoma protein from nuclear complexes containing cyclin A is induced by interferon alpha in U937V cells but not in interferon-alpha-resistant U937VR cells.

We have analysed the different regulation of cell-cycle-relevant proteins by interferon alpha (IFN alpha) in IFN alpha-sensitive and resistant U937 leukemic cell lines. In contrast to the INF alpha-sensitive U937 variant cell line U937V, the IFN alpha-resistant derivative (U937VR) is insensitive to the antiproliferative activity of IFN alpha. As we found no differences between these cell lines concerning the induction by IFN alpha of the pathway involving tyrosine-protein kinases and the signal transducer and activator of transcription (Jak-Stat), we examined whether cell-cycle-regulating proteins are differently affected by IFN alpha in U937VR and U937VR cells. In U937V cells IFN alpha induced the formation of the complex between early-region-2 transcription factor 1 (E2F-1) and retinoblastoma protein (RB) which is known to repress transcription of E2F-1-inducible genes, necessary for cell cycle progression. Formation of this complex was not inducible by IFN alpha in U937VR cells, although the suitable binding partners (E2F-1 and under-phosphorylated RB) were present. Interestingly, treatment of nuclear extracts from logarithmically growing U937V and U937VR cells with an antiserum against cyclin A that disrupts cyclin-A-containing complexes, led to the formation of the E2F-1-RB complex, suggesting the presence of under-phosphorylated (active) RB, trapped in nuclear complexes that contain cyclin A. This suggestion was supported by combined immunoprecipitation/western blot experiments that revealed a physical interaction between phosphorylated as well as under-phosphorylated forms of RB and cyclin A complex(es) in U937V and U937VR cells. RB, especially the under-phosphorylated form, was released by treatment with IFN alpha from this complex(es) in the case of U937V cells but not U937VR cells. We conclude that the missing induction of the E2F-1-RB transrepressor by IFN alpha and the failure to release RB from cyclin-A-containing complexes might contribute to the resistance of U937VR cells to the antiproliferative effects of IFN alpha.

Antibodies↗
PubMed1997

The E1A N terminus (aa 1-29) of the highly oncogenic adenovirus type 12 harbours a trans-activation function not detectable in the non-oncogenic serotype 2.

Early region 1A (E1A) of adenoviruses (Ad) codes for potent activator and repressor molecules which are involved in the regulation of viral and cellular gene expression. Gene regulatory functions of E1A proteins are mainly located in their conserved regions (CR) 1 to 3. In addition to the CRs, specific amino acids (aa) of the N-terminal end play an important role in some gene regulatory functions. We describe here the identification and characterization of a novel trans-activation domain which is located in the non-conserved N-terminal end of Ad12 E1A, namely aa 1-29. Fusion of this region to the DNA-binding domain of the yeast transcription factor Gal4 generates a strong trans-activator which induces gene expression of reporter constructs in dependence on Gal4 DNA-binding sites. Furthermore, transient expression assays using the physiological E1A-responsive adenoviral E2 early promoter revealed that the N terminus is involved in its activation. The gene regulatory function of the N terminus is specific for E1A proteins of the highly oncogenic serotype Ad12, as the respective E1A N terminus of the non-oncogenic serotype Ad2 is unable to activate the expression of the reporter gene as Gal4 fusion protein. Moreover, deletion mutant analyses demonstrate that Ad12 E1A proteins carry three independently acting activation domains: (1) aa 1-29, (2) CR1 and (3) CR3.

Adenoviridae↗
PubMed1997

A cis-acting element 7 bp upstream of the ESF-1-binding motif is involved in E1A 13S autoregulation of the adenovirus 12 TS2 promoter.

Transcription of the E1A gene of the highly oncogenic adenovirus 12 (Ad12) initiates at two start sites (TS1 and TS2). We have previously shown that the E2F and ATF motifs distal of TS1 co-operatively participate in E1A autostimulation from the TS1 promoter region. Here we report the identification of a second E2F-like target region (E2DFII) immediately upstream of the E1A-stimulating factor 1 binding site (ESF-1), important for 13S-mediated autoactivation from TS2. Reporter constructs lacking distinct TS2 cis-acting elements were analysed for their levels of CAT expression in the absence and presence of the E1A 13S protein in transient expression assays. In the absence of 13S, full promoter activity was observed only for a construct containing all elements (the E2F-like motif, and E-Box and the TATA element). Promoter activation increased significantly in Ad12 E1A-co-transfected cells. Induction by the 13S protein was also detected for the construct containing a non-functional ESF-1 sequence. Our results indicate that the E2F-like motif is responsible for activation medicated by the 13S protein from TS2, while ESF-1-or TATA-binding protein activity were not involved. Additionally, the TATA sequence appeared to be dispensable for transactivation. Gel-shift experiments using the E2F-like promoter element as a probe indicated the binding of an E2F-5 or E2F-5-like transcription factor to this region. We conclude that transcription through the TS1 as well as the TS2 promoter region is stimulated by the Ad 12 13S protein. Moreover, transfection of the construct including both TS1 and TS2 indicates an E2F-site-mediated synergism between both regions with respect ot E1A-induced transactivation.

Adenoviridae↗
PubMed1997

E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter.

The transformation-defective Vero cell host range mutant CS-1 of the highly oncogenic adenovirus type 12 (Ad12) (Ad12-CS-1) has a 69-bp deletion in the early region 1A (E1A) gene that removes the carboxy-terminal half of conserved region 2 and the amino-terminal half of the Ad12-specific so-called spacer that seems to play a pivotal role in the oncogenicity of the virus. Despite its deficiency in immortalizing and transforming primary rodent cells, we found that the E1A 13S protein of Ad12-CS-1 retains the ability to bind p105-RB, p107, and p130 in nuclear extract binding assays with glutathione S-transferase-E1A fusion proteins and Western blot analysis. Like wild-type E1A, the mutant protein was able to dissociate E2F from retinoblastoma-related protein-containing complexes, as judged from gel shift experiments with purified 12S and 13S proteins from transfection experiments with an E1A expression vector or from infection with the respective virus. Moreover, in transient expression assays, the 12S and 13S products of wild-type Ad12 and Ad12-CS-1 were shown to transactivate the Ad12 E1A promoter containing E2F-1 and E2F-5-motifs, respectively, in a comparable manner. The same results were obtained from transfection assays with the E2F motif-dependent E2 promoter of adenovirus type 5 or the human dihydrofolate reductase promoter. These data suggest that efficient infection by Ad12 and the correlated virus-induced reprogramming of the infected cells, including the induction of cell cycle-relevant mechanisms (e.g. E2F activation), can be uncoupled from the transformation properties of the virus.

Adenoviridae↗
PubMed1996

Repression of c-Jun-induced mouse major histocompatibility class I promoter (H-2Kb) activity by the Adenovirus type 12-unique 52R E1A protein.

Down-regulation of major histocompatibility (MHC) class I gene expression by protein products of the early region 1A (E1A), which might allow transformed cells to escape the host immune system, is discussed as one cause for the oncogenicity of Adenovirus (Ad) subtype 12-transformed cells. The MHC class I promoter is activated through several cellular-transcription factors among them AP-1, whose target sequences are located in the enhancers A and B, and NF kappa B. In this report we present evidence that the Ad12-unique 52R E1A protein inhibits c-Jun-induced activation of MHC class I gene expression. Repression occurs through both AP-1 recognition sequences with the AP-1 binding site of Enhancer A, which can be bound by c-Jun dimers in vitro, being the main target for c-Jun activation as well as 52R-mediated down-regulation. Furthermore our data revealed that both promoter elements of Enhancer A, the AP-1 and NF kappa B binding sites, are necessary for full promoter activity. As NF kappa B is down-regulated by the 266R protein of Ad12 E1A our results suggest a model in which two Ad12 E1A proteins co-operate in the repression of MHC class I gene expression.

Adenovirus E1A Proteins↗
PubMed1996

Selectively enhanced cellular signaling by Gi proteins in essential hypertension. G alpha i2, G alpha i3, G beta 1, and G beta 2 are not mutated.

Recent studies have shown an enhanced signaling capacity of receptors coupled to pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G proteins) in immortalized B lymphoblasts from patients with essential hypertension. In the present study, we analyzed (1) whether such alterations would also be expressed in nontransformed cells of these individuals and (2) whether other G protein-mediated signaling pathways were also altered. Therefore, we established primary cultures of skin fibroblasts from previously characterized normotensive and hypertensive individuals (NT and HT cells, respectively). [Ca2+]i rises induced by lyso-phosphatidic acid (LPA), thrombin, and sphingosine-1-phosphate as well as the formation of inositol 1,4,5-trisphosphate and [3H]thymidine incorporation evoked by LPA were PTX sensitive and enhanced twofold in HT fibroblasts. In contrast, cellular responses induced by bradykinin, endothelin-1, and angiotensin II (all PTX insensitive) were similar in NT and HT cells. Formation of cAMP induced by stimulation of Gs with isoproterenol was identical in NT and HT cells. Western blot analysis yielded no evidence for an overexpression of G alpha i2, G alpha i3, G beta 2, and G beta 4. Furthermore, sequencing of cDNAs encoding for the ubiquitously expressed PTX-sensitive G protein subunits G alpha i2, G alpha i3, G beta 1, and G beta 2 from NT and HT cell lines yielded no evidence for mutations in these genes. Although the molecular mechanisms remain to be defined, these data support the concept of a selective enhancement of signal transduction via PTX-sensitive G proteins in essential hypertension.

Base Sequence↗
PubMed1995

G protein regulation of the Na+/H+ antiporter in Xenopus laevis oocytes. Involvement of protein kinases A and C.

We have characterized the regulation of the endogenous Na+/H+ exchanger in Xenopus laevis oocytes by G proteins and protein kinases by measuring the ethylisopropylamiloride-sensitive Li+ uptake. Injection of oocytes with the stable GTP analog GTP gamma S stimulated Li+ uptake up to almost 4-fold, an effect blocked by coinjection with the GDP analog, guanyl-5'-yl thiophosphate. Injection into oocytes of beta gamma subunits of the heterotrimeric G protein transducin enhanced Li+ uptake by about 3-fold. This stimulation was blocked by transducin alpha subunits, which by themselves did not influence Li+ uptake. Using various activators and inhibitors of protein kinases, it is demonstrated that the X. laevis oocyte Na+/H+ antiporter can be stimulated by activation of both protein kinase A and C. Stimulation of Na+/H+ exchanger activity by GTP gamma S but not that induced by transducin beta gamma subunits was blocked by the protein kinase A inhibitor H-89. On the other hand, transducin beta gamma subunit-stimulated activity was prevented by the protein kinase C inhibitor, calphostin C. The non-selective protein kinase inhibitor H-7 blocked both GTP gamma S- and transducin beta gamma subunit-stimulated Na+/H+ exchanger activity. The results suggest that the Na+/H+ exchanger of X. laevis oocytes can be activated by G proteins and that this activation is not direct but mediated by protein kinase A- and/or protein kinase C-dependent pathways.

Animals↗
PubMed1995

Repression of the c-Jun trans-activation function by the adenovirus type 12 E1A 52R protein correlates with the inhibition of phosphorylation of the c-Jun activation domain.

The early region 1A 52R polypeptide, a protein expressed exclusively by the in vivo oncogenic adenovirus subtype 12, represses the trans-activating function of the cellular transcription factor complex AP-1 consisting of c-Jun-c-Jun homodimers. In this report we demonstrate that the repression in vivo correlates with a direct physical interaction of the adenovirus protein with c-Jun in vitro. Interestingly, the 52R protein binds to the bZIP domain of c-Jun essential for dimerization and DNA binding but not to the c-Jun activation domain. This interaction does not prevent the promoter binding of c-Jun/AP-1. Moreover, the physical association between c-Jun and the TATA box-binding protein TBP is not disturbed by the 52R polypeptide. In fact, we show evidence that down-regulation of c-Jun activity by the adenoviral protein is due to the inhibition of phosphorylation of the c-Jun trans-activation domain. In vivo phosphorylation of the c-Jun activation domain is necessary for the interaction of c-Jun with specific cofactors such as CBP and therefore a prerequisite for the activation of target genes. Due to these results we propose a model in which the 52R protein represses the trans-activating function of c-Jun by preventing its phosphorylation through a specific kinase necessary for the activation of the cellular transcription factor.

Adenovirus E1A Proteins↗
PubMed1994

Adenovirus type 12 early region 1A expresses a 52R protein repressing the trans-activating activity of transcription factor c-Jun/AP-1.

Oncoproteins of the early transcription unit 1A (E1A) of adenoviruses (Ad) are known to modulate the expression of all viral and of a variety of cellular genes. In this communication we present data demonstrating for the first time an activity associated with a protein (the 52R protein) expressed exclusively from E1A of the highly oncogenic subtype Ad12. The 52R protein, which does not contain any of the conserved regions (CR1-CR3) necessary for the trans-regulatory functions of the larger E1A proteins (266R, 235R), represses the trans-activating activity of the transcription factor c-Jun/AP-1. The repression of c-Jun/AP-1 activity is neither due to a down-regulation of the c-jun transcription rate nor due to an inhibition of binding of the transcription factor complex to its target sequence, at least under the conditions of in vitro band shift assays. As the 52R protein is most probably not associated with DNA-bound c-Jun/AP-1, we suppose that it represses c-Jun activity either by a squelching mechanism sequestering a cofactor, which is an essential link between c-Jun/AP-1 and the components of the basal transcriptional machinery or by modifying post-translational modifications necessary for the trans-activation function of c-Jun.

Adenoviridae↗
PubMed1993

Deletion of the beta-turn/alpha-helix motif at the exon 2/3 boundary of human c-Myc leads to the loss of its immortalizing function.

The protein product (c-Myc) of the human c-myc proto-oncogene carries a beta-turn/alpha-helix motif at the exon2/exon3 boundary. The amino acid (aa) sequence and secondary structure of this motif are highly conserved among several nuclearly localized oncogene products, c-Myc, N-Myc, c-Fos, SV40 large T and adenovirus (Ad) Ela. Removal of this region from Ad E1a results in the loss of the transforming properties of the virus without destroying its known transregulatory functions. In order to analyse whether deletion of the above-mentioned region from c-Myc has a similar effect on its transformation activity, we constructed a deletion mutant (c-myc delta) lacking the respective aa at the exon2/exon3 boundary. In contrast to the c-myc wild-type gene product, constitutive expression of c-myc delta does not lead to the immortalization of primary mouse embryo fibroblast cells (MEF cells). This result indicates that c-Myc and Ad El a share a common domain which is involved in the transformation process by both oncogenes.

3T3 Cells↗
PubMed1990

Cloning of adenovirus type 12 E1 genes into a retroviral vector and their differential splicing in mouse cells.

Early region 1 (E1) of adenovirus type 12 (Ad12) genome is able to transform nonpermissive primary rodent cells in vivo and in vitro. To analyse the role of the E1a gene products alone or in connection with the 58-kDa protein encoded by E1b during oncogenic transformation, we have cloned genomic fragments of both subregions into the retroviral vector, pZIP-NeoSV(X)1. Both constructs are expressed in mouse 3T3 cells, but, in contrast to E1b, the amount of genomic retroviral RNA carrying E1a-specific sequences was low in transfected psi2 cells and not detectable in infected NIH3T3 cells. Nevertheless, we could demonstrate the integration of the complete E1a-carrying provirus into the NIH3T3 genome. However, after infection of primary mouse embryo fibroblasts, high retrovirus-mediated expression of E1a leads to the immortalization of these cells. In the derived cell line, only the 13S transcript and the unspliced form of E1a RNA could be demonstrated, but not the 12S transcript. These results demonstrate that the ratio of genomic vs. subgenomic retroviral RNAs of Ad12 E1-carrying vectors is dependent on the cloned insert and the cell system used.

Adenoviridae↗
PubMed1990

Isolation and characterization of novel adenovirus type 12 E1A mRNAs by cDNA PCR technique.

The technique of cDNA polymerase chain reaction was used to study the heterogeneity of adenovirus type 12 early region 1A mRNAs in infected and transformed cells. In addition to 13 S and 12 S mRNAs, three transcripts of 10 S, 9.5 S, and 9 S could be isolated from infected HeLa cells. A fourth transcript of 11 S was found in transformed cells. The 9 S mRNA is generated by removing one intron (nucleotide (nt) 589 to nt 1143) from the RNA precursor. For the 11 S and 10 S mRNAs the primary transcript is spliced twice. The first splicing event removes a common intron from nt 589 to nt 715; the second splicing event removes introns deleted also during the processing of either the 13 S mRNA (leading to the 11 S mRNA) or the 12 S mRNA (leading to the 10 S mRNA). The 9.5 S mRNA is also generated by removing two introns (first splice junction: nt 588/852; the second splice junction is identical to that of the 12 S mRNA). In comparison to the 13 S and 12 S transcripts, the four smaller mRNAs alter the translational reading frames with the beginning of their second exons. In vitro translation of these mRNAs resulted in protein products with molecular weights of 10,100 (11 S and 10 S mRNAs), 10,500 (9.5 S mRNA), and 6200 (9 S mRNA) kDa.

Adenovirus Early Proteins↗
PubMed1989

[Does the duration of work disability in patients with uncomplicated duodenal ulcer affect risk of recurrence the following year? A multicenter study].

At present there is no therapeutic approach conducive to success, with the help of which the inclination to recidivation of duodenal ulcer might be restricted. In a prospective multicentric study with the help of two randomized groups of patients (group A: 13 days of disablement, group B: 37 days of disablement) was tried to prove a possible influence of the duration of the disablement on the inclination to recidivation in ulceration. In comparable groups of patients out of 99 evaluable documentations of patients in group A were found 62.5% of recidivations, in group B 68.6%. The difference was not significant. No connection could be found between duration of the disablement in ulceration and inclination to recidivation. Smokers have a distinctly higher inclination to recidivation than non-smokers.

Absenteeism↗
PubMed1987

Expression of adenovirus type 12 E1b 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::beta-galactosidase fusion protein.

DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli.

Adenovirus Early Proteins↗
PubMed1985

Structure and expression of adenovirus type 12 E1B 58K protein in infected and transformed cells: studies using antibodies directed against a synthetic peptide.

We have studied the kinetics of synthesis of the early adenovirus type 12 (Ad12) E1B 58K tumor antigen during lytic infection and analysed its half-life, intracellular localization and phosphorylation in infected KB and transformed hamster (HA12/7) cells. Our analysis has been based on immunoprecipitations using antibodies directed against a synthetic peptide corresponding to the carboxy-terminal end of the E1B 58K protein. Its synthesis was first detectable approximately 8 h after infection and reached a maximum at about 20 h. There is a slight decrease of synthesis late after infection although its level of production is rather high throughout the infectious cycle. The half-life of the Ad12 E1B 58K polypeptide is 2-3 h in infected cells, but strikingly higher (less than 10 h) in the Ad12-transformed cell line HA12/7. Pulse-chase experiments combined with cell fractionation and immunofluorescence studies suggested that about 50% of the amount of the 58K polypeptide accumulates in the nucleus of infected KB cells at least at late times after infection, but only approximately 10% in Ad12-transformed cells. The 58K polypeptide is phosphorylated in both infected and transformed cells. Analysis of the products of acid hydrolysis indicates phosphorylation to equal amounts of serine and threonine. The implications of all these findings for possible roles of the E1B 58K tumor antigen in lytic infection and transformation are discussed.

Adenoviruses, Human↗