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Biomedical subjects

N K Bhat

Publications and source records attributed to N K Bhat.

At least 19 recordsLinked to original sources
PubMed2004

Expressions of hepatic genes, especially IGF-binding protein-1, correlating with serum corticosterone in microarray analysis.

Microarray technology was evaluated for usefulness in assessing relationships between serum corticosterone and hepatic gene expression. Nine pairs of female Swiss mice were chosen to provide a wide range of serum corticosterone ratios; cDNA microarray analysis (approximately 8000 genes) was performed on their livers. A statistical method based on calculation of 99% confidence intervals discovered 32 genes which varied significantly among the livers. Five of these ratios correlated significantly with serum corticosterone ratio, including tyrosine aminotransferase, stress-induced protein, pleiotropic regulator 1 and insulin-like growth factor-binding protein-1; the latter has a potential role in cancer development. Secondly, linear regression of gene expression vs corticosterone ratios was screened for those with r> or =0.8 (P<0.01), yielding 141 genes, including some known to be corticosterone regulated and others of interest as possible glucocorticoid targets. Half of these significant correlations involved data sets where no microarray ratio exceeded +/- 1.5. These results showed that microarray may be used to survey tissues for changes in gene expression related to serum hormones, and that even small changes in expression can be of statistical significance in a study with adequate numbers of replicate samples.

Animals↗
PubMed2001

Physical interaction and functional synergy between glucocorticoid receptor and Ets2 proteins for transcription activation of the rat cytochrome P-450c27 promoter.

We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor. Ets2 protein binding to a "weak" Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and chemical cross-linking experiments show physical interaction between GR and Ets2 proteins. Mutational analyses show synergistic effects of Ets2 and GR in dexamethasone-mediated activation of the cytochrome P-450c27 promoter. The DNA-binding domain of GR, lacking the transcription activation and ligand-binding domains, was fully active in synergistic activation of the promoter with intact Ets2. The DNA-binding domain of Ets2 lacking the transcription activation domain showed a dominant negative effect on the transcription activity. Finally, a fusion protein consisting of the GR DNA-binding domain and the transcription activation domain of Ets2 fully supported the transcription activity, suggesting a novel synergy between the two proteins, which does not require the transactivation domain of GR. Our results also provide new insights on the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators.

3T3 Cells↗
PubMed2000

Ets1 oncogene induction by ELF-modulated 50 MHz radiofrequency electromagnetic field.

We have analyzed gene expression in hemopoietic and testicular cell types after their exposure to 50 MHz radiofrequency (RF) non-ionizing radiation modulated (80%) with a 16 Hz frequency. The exposure system generates a 0.2 microT magnetic field parallel to the ground and a 60 V/m electric field orthogonal to the earth's magnetic field. Exposure conditions were selected so as to interfere with the calcium ion flow. Under these electromagnetic field (EMF) conditions, we observed an overexpression of the ets1 mRNA in Jurkat T-lymphoblastoid and Leydig TM3 cell lines. This effect was observed only in the presence of the 16 Hz modulation, corresponding to the resonance frequency for calcium ion with a DC magnetic field of 45.7 microT. We have also identified a putative candidate gene repressed after EMF exposure. The experimental model described in this paper may contribute to the understanding of the biological mechanisms involved in EMF effects.

Animals↗
PubMed1999

Prevalence of disease-related DNA polymorphisms among participants in a large cancer prevention trial.

Genetic susceptibility polymorphisms may be of substantial importance in the modulation of cancer risk. The prevalence for an array of polymorphic genes was determined in a cohort of male smokers who participated in a cancer prevention trial in Finland. A random sample of 120 individuals was selected from the trial cohort and the prevalence of variant alleles for nine genes was determined using a polymerase chain reaction-based approach. The prevalence values from this study were also compared with those of other populations derived from previous studies. Our results show that, with the exception of cytochrome P450-1A1 (CYP1A1) and cytochrome P450-2E1 (CYP2E1), all genes tested were sufficiently polymorphic to warrant an investigation of gene-environment studies. Most of the variant alleles, including alcohol dehydrogenase 3 (ADH3), glutathione-S-transferase (GSTM1), methionine synthase (MS), methylene tetrahydofolater reductase (MHTFR), CYP2E1 and CYP1A1, exhibited similar frequencies to other Caucasian populations. Interestingly, the prevalence of androgen receptor-CAG repeat (AR-CAG) and vitamin D receptor (VDR) polymorphisms differed significantly between the alpha-tocopherol, beta-carotene (ATBC) Study and other Caucasian populations. We present herein results from this survey and conclude that the ATBC study population in Finland is sufficiently heterogeneous to facilitate analysis of genetic polymorphisms and disease associations.

Adult↗
PubMed1999

Effect of vitamin intervention on the relationship between GSTM1, smoking, and lung cancer risk among male smokers.

The GSTM1 (glutathione S-transferase mu-1) null genotype is suspected of increasing an individual's susceptibility to tobacco smoke carcinogens because of impaired carcinogen detoxification. We were interested in whether there were differences in lung cancer susceptibility to smoking within the GSTM1 genotypes and the impact of antioxidant supplementation on this. For this purpose, we conducted a nested lung cancer case-control study and evaluated the role of GSTM1 within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. GSTM1 genotype status was determined for 319 cases and 333 controls using a PCR-based approach. GSTM1 was evaluated as an independent risk factor and as an effect modifier of smoking using logistic regression analyses. The GSTM1 null genotype itself was unrelated to risk of lung cancer, odds ratio (OR) = 1.09 and 95% confidence interval (CI), 0.79-1.50, but it may have modified the effect of smoking. There was a suggestion for a stronger association between years of smoking and lung cancer among the GSTM1 null genotype, but the differences between GSTM1 null and present genotypes were not statistically significant (P = 0.12). Furthermore, the smoking association was strongest among those with the GSTM1 null genotype not receiving alpha-tocopherol supplementation, whereas among those receiving alpha-tocopherol, there was no modification by GSTM1 on the association between smoking duration and lung cancer risk. Beta-carotene supplementation did not modify the relationship between GSTM1, smoking years, and lung cancer risk. In conclusion, GSTM1 is not associated with lung cancer risk in male smokers but may confer a higher susceptibility to cumulative tobacco exposure. This association may be attenuated by alpha-tocopherol but not by beta-carotene supplementation.

Adult↗
PubMed1997

Characterization of human N8 protein.

We have shown before that the N8 mRNA is expressed at higher levels in lung tumor and lung tumor-derived cell lines than normal lung cells. In this paper, we have characterized the N8 protein, and studied its properties. The N8 gene encodes a major 24 kDa protein and its expression correlates well with the N8 mRNA expression pattern observed in different cell lines. N8 protein is capable of forming a homodimer or multimeter in vitro. It is a phosphorylated cytoplasmic protein and phosphorylation occurs mainly at serine residues. N8 protein is expressed at higher levels in epithelial cells than in mesenchymal cells. N8 protein expression is induced in a fibroblast cell line expressing adenoviral Ela protein, which acquired epithelial-like characteristics. Furthermore, ectopic expression of N8 protein in NIH3T3 cells converts them into a spheroid form. These spheroids also have some of the characteristic features of epithelial cells. Taken together, these results suggest that the N8 protein may be associated with the development or maintenance of epithelial cell phenotype.

3T3 Cells↗
PubMed1997

A variant form of ETS1 induces apoptosis in human colon cancer cells.

We have previously shown that the human ETS1 protein (p51-ETS1), when ectopically expressed in colon cancer cell lines, is able to reduce its tumorigenicity without affecting its growth properties. To understand the mechanism of tumor reduction, we have expressed two different forms of ETS1 in colon cancer cell lines. Data presented in this paper indicate that the naturally occurring spliced variant protein, p42-ETS1, lacking the region encoded by ETS1 exon VII, represses the tumorigenicity, while p51-ETS1 reduces the tumorigenicity. Repression of tumorigenicity mediated by p42-ETS1 appears to be caused by its ability to induce apoptosis in epithelial cancer cells. This work can have profound medical significance in that it may open up new insights into the potential role of the p42-ETS1 in the induction of apoptosis in epithelial cell cancers and may provide a rationale for its use for potential gene therapy experiments to initiate cell death in cancer cells.

Animals↗
PubMed1997

Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides.

Ewing's sarcoma family of tumors (EFT) contain reciprocal translocations, of which approximately 90% occur between the long arm of chromosomes 11 and 22,t(11;22)(q24;q12) resulting in the formation of chimeric proteins generated by a fusion of the EWS and FLI-1 genes. To determine if EWS-FLI-1 protein is responsible for the Ewing sarcoma phenotype we have used sequence-specific antisense oligodeoxynucleotides (ODN) to block its expression. We have evaluated a series of antisense ODN directed toward the breakpoint region in an effort to prevent translation of the fusion messenger RNA. ODN were first evaluated in a cell-free in vitro translation system. Exogenously added RNase H was found to be required for translation inhibition. ODN that showed complete inhibition of translation were electroporated into TC-32 cells, a EFT cell line. Fusion protein and EWS protein levels were evaluated by Western blot analysis. A 40-60% decrease in the fusion protein was observed in TC-32 cells with antisense ODN directed toward the breakpoint region. Cell viability was reduced with antisense sequences in TC-32 cells but not in a prostate cancer cell line. Since inhibition of t(11:22) gene product is correlated to effects on cell viability reduction of the fusion protein may thus offer insight into the biology of EFT.

Bone Neoplasms↗
PubMed1996

Isolation and characterization of a novel gene expressed in multiple cancers.

Using differential display method, we have isolated and characterized a novel gene, N8, encoding an approximately 24 kDa protein. It is located on human chromosome 8q13 region. N8 gene is expressed at high levels in tumor derived cell lines from multiple cancers. It is also expressed at higher levels in lung tumors than normal lung tissue. N8 is also differentially expressed in fetal and adult tissues. In adult, N8 is expressed at high levels in brain, kidney, prostate, pancreas and intestine and at very low levels in lung, liver, hematopoietic cells and gonads. During murine embryonic development N8 is expressed in the epithelium of the intestine, stomach, olfactory epithelium, neuronal layers of retina, kidney and salivary gland. Taken together, these results suggest that N8 may play different roles during embryogenesis and in the adult animals.

Amino Acid Sequence↗
PubMed1995

ETS1 suppresses tumorigenicity of human colon cancer cells.

We have ectopically expressed transcription factor ETS1 in two different highly tumorigenic human colon cancer cell lines, DLD-1 and HCT116, that do not express endogenous ETS1 protein and have obtained several independent clones. The expression of wild-type ETS1 protein in these colon cancer cells reverses the transformed phenotype and tumorigenicity in a dose-dependent manner. By contrast, expression in DLD-1 cells of a variant form of ETS1, lacking transcriptional activity, did not alter the tumorigenic properties of the cells, suggesting that the reduction in tumorigenicity in these clones was specific for the wild-type ETS1 gene products. Since these colon cancer cells have multiple genetic alterations, the system described in this paper could be a good model to study the suppression of tumorigenicity at a transcriptional level, which could lead to the design and development of novel drugs for cancer treatment.

Base Sequence↗
PubMed1995

Role of ETS1 in IL-2 gene expression.

The ETS1 gene encodes a sequence-specific transcription factor binding to purine-rich DNA sequences (-GGAA-) present in the transcriptional regulatory regions of many cellular and viral promoters/enhancers, including many lymphokine genes. The ETS1 gene is expressed at high levels in resting T cells and at very low levels after T cell activation, suggesting it may suppress the expression of genes induced during T cell activation. To find out if ETS1 regulates expression of the IL-2 gene, we have ectopically expressed antisense (AS) ETS1 in Jurkat T cells to block the formation of ETS1 proteins. AS ETS1 transfectants produce higher levels of IL-2 compared with sense ETS1 transfectants. Expression of ETS1 DNA binding domain in Jurkat T cells also decreased the production of IL-2. In AS ETS1 transfectants, IL-2 formation was completely inhibited by cyclosporin A and FK590. The IL-2 promoter linked to a chloramphenicol acetyl transferase reporter gene has high activity in AS ETS1 transfectants, indicating that increased IL-2 production seems to be a result of transcriptional induction. Taken together, these results suggest the possibility that ETS1 may act as a negative regulator of IL-2 gene transcription and provide a rational approach toward engineering the endogenous expression of IL-2 in T cells.

Cell Line↗
PubMed1995

Generation and characterization of monoclonal antibodies against the ERGB/FLI-1 transcription factor.

Five monoclonal antibodies were produced from mice immunized with recombinant full length human ERGB protein. Among these monoclonal antibodies, four clones did not cross react with other ets family proteins and thus are specific for the ERGB protein; however, one clone did react with the ERG protein, which has high amino acid identity with the ERGB protein. The epitope location of these antibodies was studied using bacterially expressed fragments of the human, ERGB protein. These monoclonal antibodies recognized 51 kDa (p51) and 48 kDa (p48), two ERGB gene-encoded proteins, from human, mouse, and rat cell lines. These results suggest that the monoclonal antibodies can be used in human, mouse, or rat cell lines and will be useful for the biochemical and functional analysis of the ERGB protein.

Animals↗
PubMed1994

Epitope mapping of human ETS1 monoclonal antibody.

The epitope for E44 monoclonal antibody (mAb) was mapped using mutated ETS1 proteins lacking different carboxy-terminal regions and by the employment of synthetic oligopeptides spanning the epitope region. This epitope lies around Arg211 of the human ETS1 protein since substitution of Arg211 by Gln211 in the epitope region results in the loss of recognition of the mouse ETS1 protein by E44 mAb. Substitution of Leu214 by valine214 in the epitope region (as is found in the chicken ETS1 and viral Ets proteins) does not alter the capacity of the E44 mAb to recognize this antigen. Taken together, these results suggest that a specific ionic interaction is able to play a pivotal role in the recognition of the ETS1 protein by the E44 mAb.

Amino Acid Sequence↗
PubMed1993

HTLV-1 gene expression by defective proviruses in an infected T-cell line.

We have examined human T-lymphotropic virus type I (HTLV-I) gene expression in the human T-cell line, C8166-45 (C81), as a model to define the gene products expressed from defective proviruses. C81 cells contain one complete and two different deleted proviral genomes. The internal deletions of the latter encompass most of the gag to env region. All three proviruses are transcriptionally active as evidenced by the presence of three unspliced nuclear mRNAs. Unspliced genomic mRNAs and singly spliced env mRNAs were present in the nucleus, but were not detected in the cytoplasm, suggesting a defect in Rex function. Three small cytoplasmic mRNAs were observed and are likely to correspond to the normal 1.8-kb Tax1/Rex1 mRNA, a 1.6-kb mRNA formed by splicing the 5'LTR to the pX region, and a 2.1-kb mRNA of unknown origin. Consistent with the subcellular mRNA distribution pattern, the viral structural proteins encoded by gag and env genes were not detected. The transcriptional transactivator protein, Tax1 (p40), was abundantly expressed in C81 cells; in addition, a 42-kDa Tax1 protein, unique to this cell line, was also detected. Although Tax1 and Rex1 (p27) are translated from overlapping open reading frames in the same mRNA, Rex1 was not detected in C81 cells. The presence of a premature termination codon in the Tax1/Rex1 mRNA encoded by the full-length provirus was inferred from the presence of small Rex1-related polypeptides lacking C-terminal sequences and confirmed by sequence analysis. Furthermore, a p21X protein lacking the N-terminus of Rex1 was expressed at high levels; our data indicate that p21X is translated from the 1.6-kb mRNA which is derived primarily from deleted proviruses.

Blotting, Northern↗
PubMed1993

Characterization of the human interleukin-2 receptor beta-chain gene promoter: regulation of promoter activity by ets gene products.

The interleukin-2 receptor (IL-2R) beta chain (IL-2R beta) is an essential signaling component of high- and intermediate-affinity IL-2Rs. Our laboratory previously reported that a DNA fragment containing 857 bp of 5'-flanking sequence of the human IL-2R beta gene exhibited promoter activity. We have now further characterized the promoter and delineated cis-acting regulatory regions. The region downstream of -363 is critical for basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and contains at least three enhancer-like regions. Among them, the -56 to -34 enhancer was the most potent and had high-level activity in two T-cell lines but not in nonlymphoid HeLaS3 and MG63 cells. This enhancer contains a GGAA Ets binding site which bound two Ets family proteins, Ets-1 and GA-binding protein in vitro. Mutation of the Ets motif strongly diminished both promoter and enhancer activities. We conclude that this Ets binding site plays a key role in regulating basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and may also contribute to tissue-specific expression of the IL-2R beta gene.

Amino Acid Sequence↗
PubMed1993

Human ERG-2 protein is a phosphorylated DNA-binding protein--a distinct member of the ets family.

We describe the identification of the ERG-2 gene products using an antibody raised against recombinant human ERG-2 protein. ERG-2 is a nuclear phosphoprotein and binds to purine-rich sequences (C/G)(C/a)GG-AA(G/a)T. ERG-2 protein, with a half-life of 21 h, is considerably more stable than the short-lived ETS-1 or ETS-2 proteins. Its phosphorylation is stimulated by phorbol myristate acetate (PMA), but not by Ca2+ ionophore treatment. ETS-1 protein is phosphorylated by Ca(2+)-dependent events, whereas ERG-2 protein is phosphorylated by activation of protein kinase C, suggesting their involvement in distinct signal transduction mechanisms. The expression of ERG-2 protein is restricted to few cell types and is high in early myeloid cells, indicating that it may function at an early stage of hematopoietic lineage determination. The DNA-binding sequence for ERG-2 protein is identified by using a random oligonucleotide selection procedure. The selected sequence is very similar to the binding sequence determined for human ETS-1 using the same method. Like other ets proteins, ERG-2 is a sequence-specific DNA-binding protein and is expressed at higher levels in early myeloid cells than in mature lymphoid cells. These results suggest that it may act as a regulator of genes required for maintenance and/or differentiation of early hematopoietic cells.

Base Sequence↗
PubMed1992

The basal promoter elements of murine cytochrome c oxidase subunit IV gene consist of tandemly duplicated ets motifs that bind to GABP-related transcription factors.

DNA sequences required for expression of the mouse cytochrome c oxidase subunit IV (COXIV) promoter were identified by transient expression of recombinant COXIV-chloramphenicol acetyltransferase constructs in COS and NIH-3T3 cells. Activity of the COXIV promoter is shown to depend upon upstream Sp1 binding sequences and two tandemly repeated 21-base pair sequence elements each mapping to sites of mRNA initiation. Each initiation region repeat contains a binding site for an ets-related transcription factor which demonstrates specificity for the characteristic GGAA ets sequence motif and reactivity with an ets domain-directed monoclonal pan ets antibody. The two 21-base pair repeats are sufficient for transcriptional activity suggesting that the ets-related factor may be involved in both transcriptional activation and start site positioning. The ets-related protein found in COS nuclear extracts is shown to be identical or closely related to the GA-binding protein (GABP) by comparison of electrophoretic mobilities and immunological reactivities of DNA-protein complexes formed with purified recombinant expressed GABP alpha and beta subunits. Sp1 and the GABP-related factors also bind to another mouse cytochrome oxidase subunit gene COXVb. The similar promoter features of these two genes suggests a possible means of coordinate transcriptional regulation among such respiratory proteins.

3T3 Cells↗