Narayan K. Bhat

Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http://mgc.nci.nih.gov).

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

Ewings 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.

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.

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.

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 E1a 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.

The ets Family of Genes: Molecular Biology and Functional Implications

To better understand the process of the cellular transformation and conversion of proto-oncogenes to the transforming retroviral oncogenes, our laboratory has pursued the study of the normal cellular genes comparing these to the homologous viral oncogemes. We have focused on the ets gene family which are related to sequences originally identified as a second cellular derived genes transduced by the avian leukemia virus, E26 (Watson et al. 1985). In mammals there are three ets genes located on two different chromosomes, termed ets-1, ets-2 and erg. The ETS-1 gene most related to the cellular ets captured by the virus (v-ets) is located in chromosome 11; the ETS-2 and ERG genes are located on chromosome 21. In higher mammals as well as humans these ets genes, are dispersed in separate chromosomes; but retain their homologous syntenic groups with respect to other genetic markers (Watson et. al. 1986). The ets genes are transcriptionally active, their gene products are homologous to one another and they are differentially regulated. Figure 1 presents a diagramatic comparison of the amino acid sequences of a wide variety of species ranging from Drosophila to humans.

Expression of ETS Family of Genes in Systemic Lupus Erythematosis

Publisher Summary Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown etiology. SLE is characterized by the involvement of multiple organ systems and the production of autoantibodies directed against nuclear components, including ssDNA, ds- DNA, and histones. The hallmark of autoimmunity is the activation and proliferation of lymphocytes directed against self-antigen. Defect in the signal transduction cascade mechanisms in lymphocytes from SLE patients result in the aberrant expression of many genes and some of which encode DNA sequence specific transcription factors. These in turn regulate the transcription of genes contributing towards disease process. The chapter briefly describes the ETS gene family, the expression pattern of ETSl, ETS2, and ERGB/FLIl genes in lymphocytes from SLE patients, and the possible role of ERGB/FLIl transcription factor in the autoimmune disease process. The ETS genes encode sequence-specific DNA-binding proteins and function as transcription factors. ETS family of proteins is involved in lymphoid cell development, differentiation, maturation, and activation and therefore, they are important regulators of lymphoid gene expression. Inappropriate expression of ETS family of genes may have a detrimental effect on lymphoid cell differentiation, activation, and function. The chapter discusses the role of ETS in cancer and disease. This can occur by by activation through retroviral transduction of cellular sequences, retroviral promoter integration, or by chromosomal translocation, dysregulation of ETS. The chapter describes the expression of ETSl, ETS2 AND FLI1 genes during normal development, in lymphocytes from SLE patients, and in lymphocytes from autoimmune prone mice.

c-ets-2 and the Mitogenic Signal Pathway

The feature that defines a cancer cell is its ability to propagate under conditions that typically inhibit the growth of the normal cell. In cells where the genetic constitution remains unaltered, restraints on growth are imposed by regulatory activities that take place at the cell surface. These activities, which may be due to either cell contact or a decrease in the production and availability of growth factors, are communicated through the cytoplasm to the nucleus, thus regulating the synthesis of messenger RNAs essential for the unique proteins needed to initiate subsequent DNA synthesis and mitosis. Cells can be relieved of this barrier to mitosis by cell dispersal or addition of growth factors; this molecular environmental change is similarly recognized at the surface of the cell, and is transmitted through the cytoplasm to the nucleus. This signal transduction process activates the transcription and synthesis of specific proteins and other macromolecules that trigger the mitotic process. The process and events leading to cell division are called here the “mitogenic signal pathway”.

Immunologic detection of protein antigens after phenol-chloroform denaturation

The study of gene expression in cells and tissues often begins with phenol-chloroform extraction of the biologic material of interest for the isolation of intact mRNA. In most cases, the proteins denatured by phenol-chloroform are discarded. However, we found that the proteins recovered from phenol-chloroform extractions maintain their antigenicity. Therefore a method was developed for recovering the proteins from phenol-chloroform-denatured extracts that could be saved in lyophilized form until immunologic analysis. In this way, the RNA and the protein analysis can utilize exactly the same sample, and the biologic material can be saved. This is important because often these materials are available only in limited quantities. The method has been used to examine the sea urchin ets-related antigen and sea urchin ets-2 mRNA.

The ets genes in cells and viruses: implications for leukemias and other human diseases.

One of the most interesting scientific challenges of our time has been to understand the spectrum of oncogenic changes necessary to commit normal cells to a neoplastic life-style. On the molecular level, this enigmatic quest centers upon our understanding the essential ingredients needed to control cell growth (and its coordinate processes), as well as to maintain the normal differentiated pattern of expression for specific genes. Another, perhaps simpler, approach to this difficult challenge would be to examine the genetic changes resulting from the introduction of a known acute retroviral transforming sequence capable of eliciting a malignant cellular phenotype in vitro and neoplastic disease in vivo. Through the application of the powerful technique of recombinant technology application in the study of model retrovirus systems, the precise DNA sequences responsible for cellular transformation have been identified and studied. Thus, the employment of acute retroviruses as tools to identify a select cohort of genes capable of malignant transformation has been most fruitful. More important, from this study came a realization that this category of genes, the viral oncogenes, were capable of establishing and maintaining the transformed state, as well as the recognition that these genes were derivatives of a limited population of normal cellular genes, the proto-oncogenes, that can be captured and modified by the viral transduction process. These homologous genes, now numbering several dozen, are found distributed in the genomes of almost all vertebrates and of some invertebrate species, as well as of a few more primitive single-cell species.