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Biomedical subjects

T S Papas

Publications and source records attributed to T S Papas.

At least 37 records · Page 2Linked to original sources
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

P53 gene mutations in women with breast cancer and a previous history of benign breast disease.

Mutations of the p53 tumour suppressor gene are the most common genetic lesions in human cancers and have been reported in breast cancer as part of the Li-Fraumeni syndrome. In the present study, we determined frequencies and types of the p53 mutations in breast cancer tissues in women with a history of benign breast disease (BBD) identified in Florence, Italy, with (n = 6) or without (n = 10) a family history of breast cancer. Among the cases with a family history of breast cancer and BBD, 2 out of 6 had p53 gene mutations in cancer samples. 1 patient had a mutation at codon 248 and the other had double mutations at codons 243 and 241. In these cases, the p53 gene was also analysed in the tissue samples from previous BBD lesions; however, no mutations were observed (0 out of 6). These results suggest that the p53 mutations occur during advanced stages of tumour progression. In sporadic breast cancer cases with a history of BBD, p53 point mutations were observed of tumour progression. In sporadic breast cancer cases with a history of BBD, p53 point mutations were observed in four samples (4 out of 10). Two of these mutations turned out to be silent changes and one of the samples showed triple mutations at amino acid positions 267, 277 and 296. No p53 gene mutations were found in the breast tumour tissues of 10 additional women from the same area with a family history of breast cancer, but no previous BBD (0 out of 10). Family history of breast cancer does not appear to affect the frequency of p53 mutations in women with a previous history of BBD.

Base Sequence↗
PubMed1994

Isolation and characterization of simian T-cell leukemia virus type II from New World monkeys.

Since the description of human T-cell leukemia virus type I (HTLV-I) and its simian counterpart, simian T-cell leukemia virus type I (STLV-I), the possible existence of other related simian retroviruses has been raised. Here, we report a new retrovirus, STLV-II, which we have identified in spider monkeys (Ateles fusciceps), a New World primate species. Initially, a recombinant HTLV-II envelope protein (RP-IIB) was used to identify anti-STLV-II antibodies in New World monkeys by Western blot (immunoblot) assays. Subsequently, the virus was characterized by Southern blot hybridization, which showed that STLV-II and HTLV-II have a high degree of nucleotide sequence homology but have different restriction enzyme patterns. Nucleotide sequence analysis of the pX-II region of STLV-II provirus revealed 3% variation with the corresponding region of HTLV-II. Electron micrographic studies revealed HTLV-like, type C retrovirus particles outside the cell membranes of STLV-II-infected cells. This study describes the first link between HTLV-II and a simian reservoir in the New World. Further molecular studies of STLV-II infection in different species of New World monkeys, especially from the wild, may provide valuable information about the origin and intragroup relationships of South American monkeys. Spider monkeys infected with STLV-II may serve as an important animal model for HTLV-II infection in humans.

Animals↗
PubMed1994

The EndoA enhancer contains multiple ETS binding site repeats and is regulated by ETS proteins.

EndoA is a type II keratin and with EndoB (type I keratin), constitutes intermediate filaments in various simple epithelial tissues. EndoA is developmentally regulated and has an enhancer that is located at the 3'- end of the gene. This enhancer contains two single and five dual Ets binding sites. Thus far, no other promoter or enhancer has been shown to contain as many potential clustered Ets binding sites. To study the transcriptional regulation of EndoA by the ETS family proteins, we amplified the EndoA enhancer fragment from mouse genomic DNA by PCR, and cloned it into the pBLCAT2 vector upstream from the CAT reporter gene. Several pBLCAT-ENDOA clones were sequenced to verify the presence of all the ETS binding sites. Clones that did not show any point mutations in the ETS binding sites were chosen to study the transcription regulation by ETS1, ETS2 and ERGB/FLI-1 gene products. EMSA results indicated that the ETS1, ETS2 and ERGB/FLI-1 proteins bind to the enhancer sequence, and DNase I protection data demonstrated that the ETS proteins protect all seven EBS core sequences. Cotransfection of the COS cells with the pBLCAT-ENDOA construct, along with increasing amounts of different ETS expression vectors, resulted in a significant induction of CAT reporter gene expression. Previously, we have shown that the overexpression of the ETS1 gene transforms NIH3T3, and these transformed cells (7AQS2.1) produce high levels of ETS1 protein (Seth & Papas, 1990). In this report, we show that the undifferentiated P19 EC cells do not express detectable levels of ETS1; however, an elevated level of ETS1 is expressed in differentiated derivatives of these cells. We therefore used these two cell lines to examine the activity of the EndoA enhancer with the ETS1 product. Transfection of the pBLCAT-ENDOA construct alone in undifferentiated P19 EC cells results in very low CAT gene expression; however, upon differentiation with retinoic acid the level of CAT gene activity increases dramatically. Similarly, an increase in CAT expression from the same construct (pBLCAT-ENDOA) was also observed in 7AQS2.1 cells. Our results therefore indicate that the EndoA enhancer is regulated by ETS proteins via interaction with multiple ETS-binding site sequences.

Animals↗
PubMed1994

Differential expression of ets-1 and ets-2 proto-oncogenes during murine embryogenesis.

ets-1 and ets-2 genes have previously been identified by their sequence homology to the v-ets oncogene of the avian erythroblastosis virus, E26. These cellular genes have been shown to function as transcription factors important in lymphoid differentiation and activation and cellular proliferation. In this study, we have broadly analysed the differential expression of ets-1 and ets-2 during murine development using in situ hybridization. Our results indicate that these transcription factors are expressed in multiple tissues during critical stages of embryo formation and organogenesis, suggesting that these genes may serve multiple functions during mouse development. The patterns of expression of both genes are quite different as early as day 8.0 of gestation. ets-1 expression is clearly observed during a narrow developmental stage in the developing nervous system, including the presumptive hindbrain regions, the neural tube, as well as neural crest and the first and second branchial arches, ets-2 expression is limited to the developing limb buds and distal tail. At later times, ets-1 expression is observed in developing vascular structures, including the heart, arteries, capillaries and meninges, whereas ets-2 is highly expressed in developing bone, tooth buds, epithelial layers of the gut, nasal sinus and uterus, and several regions of the developing brain. Both ets-1 and ets-2 are expressed in developing lung, gut and skin. High levels of expression in both genes is observed in adult lymphoid tissues, but in different tissue subsets. ets-1 is expressed in the adult lung, gut mesenchyme and bone marrow. ets-2 continues to be expressed at low levels in several adult tissues, except in the differentiated brain, where substantial levels of expression are found in particular regions of the mature brain. These results demonstrate that ets-1 and ets-2 are differentially regulated, are widely expressed in many tissues during murine embryogenesis and may play important roles in cellular proliferation and differentiation during mouse development.

Animals↗
PubMed1994

Structural inferences of the ETS1 DNA-binding domain determined by mutational analysis.

The ets family of transcription factors is characterized by a conserved region that harbors the DNA-binding activity. We performed extensive deletion and mutational analyses, as well as DNA-peptide interaction studies necessary to identify the determinants of the DNA-binding activity of the ETS1 oncoprotein. We found that amino acids beyond the 85 amino acid conserved region are required in order to afford maximum DNA-binding activity in a heterologous system. Mutation throughout the binding domain can have a detrimental effect on binding activity, indicating that proper folding of the entire domain is necessary for DNA binding. A peptide, as small as 37 residues (K37N), derived from the basic region of the ETS1 binding domain, is sufficient to exhibit sequence-specific DNA recognition. Total randomization of Lysine 379, Lysine 381 and Arginine 391 within this region fails to provide functional substitutions, indicating that these specific amino acids within the basic region are required for binding. Transactivation activity of the ETS1 proteins bearing mutations was consistent with their DNA-binding activity, indicating that the primary (if not only) function of this domain is to provide sequence-specific DNA recognition activity. Our mutational analysis, as well as modeling predictions, lead us to propose a helix-turn-helix structure for the basic region of the ETS1 binding domain that is able to interact directly with DNA. We also propose that the hydrophobic alpha-helical region, surrounding tryptophan 338, is fundamental for proper protein folding and functioning of the ets binding domain.

Amino Acid Sequence↗
PubMed1993

The Ets1 transcription factor is widely expressed during murine embryo development and is associated with mesodermal cells involved in morphogenetic processes such as organ formation.

The Ets family of genes encodes a class of transcription factors. Ets1 is predominantly expressed in the lymphoid organs of neonatal and adult mice, whereas Ets2 is expressed in every organ examined. In this study, we investigate the expression of Ets1 and Ets2 during murine embryonic development. Our data show that Ets1 expression increases in embryos after implantation and during organogenesis such that it is expressed in all the organs of day-15 embryos studied. In later fetal stages, Ets1 expression is predominant in the lymphoid tissues, brain, and organs that are undergoing branching morphogenesis (e.g., lung) but is dramatically reduced in other organs such as the stomach and intestine. In neonatal development, Ets1 is expressed only in the lymphoid organs and brain. In situ hybridization analysis demonstrates that expression of Ets1 occurs in mesenchymal cells of developing organs, in the nervous system, and in forming bone. Furthermore, expression of Ets1 is upregulated in P19 cells induced to differentiate into mesoderm-like cells. Ets2, on the other hand, is expressed in differentiated and undifferentiated P19 and F9 cells and in all organs of embryonic, neonatal, and adult mice studied. These data suggest that Ets1 plays an important role in mesodermal cells associated with morphogenetic processes such as organ formation and tissue modeling, whereas Ets2 plays a more fundamental role in cells.

Animals↗
PubMed1993

Identification of a colon mucosa gene that is down-regulated in colon adenomas and adenocarcinomas.

A cDNA, which we call DRA (for down-regulated in adenoma) has been isolated. Its mRNA is expressed exclusively in normal colon tissue, probably only in the mucosal epithelia. Expression of the DRA gene is significantly decreased in adenomas (polyps) and adenocarcinomas of the colon. The DRA gene appears to be a single-copy gene present on chromosome 7, a chromosome associated with colorectal tumorigenesis. The predicted DRA polypeptide is an 84,500-Da protein that contains charged clusters of amino acids, primarily at the NH2 and COOH termini. Together with potential nuclear targeting motifs, an acidic transcriptional activation domain, and a homeobox domain, these elements suggest a transcription factor or a protein that may interact with transcription factors. Such a function may be consistent with a role in tissue-specific gene expression and/or as a candidate tumor-suppressor gene.

Adenocarcinoma↗
PubMed1993

Restriction endonuclease cleavage of 5-methyl-deoxycytosine hemimethylated DNA at high enzyme-to-substrate ratios.

We have investigated the ability of a large number of restriction enzymes to digest non-canonically hemimethylated DNA at high enzyme-to-substrate ratios. A single-stranded unmethylated phagemid was used as a template to complete synthesis of the second strand using 5-methyl-dCTP to substitute for all the deoxycytosine residues. A fragment of this double-stranded hemimethylated DNA which contains the multiple cloning site region was used as a substrate. For all the enzymes tested, at least some degree of protection from digestion is observed. Sites completely protected from digestion by their cognate enzymes are SalI, BstXI, SacI, SacII, SmaI, SstI, XhoI, PstI, HinfI, BamHI and AccI. Sites partially protected from digestion by their cognate enzymes are XbaI, HindIII, KpnI, SpeI, ClaI, EcoRI and PvuII. Knowledge of the sensitivity of commonly used restriction enzymes to hemimethylated substrates is useful for several applications, which will be discussed.

5-Methylcytosine↗
PubMed1993

ETS family proteins activate transcription from HIV-1 long terminal repeat.

ets is a multigene family and its members share a common ETS DNA-binding domain. ETS proteins activate transcription via binding to a purine-rich GGAA core sequence located in promoters/enhancers of various genes, including several that are transcriptionally active in T cells. The ETS1, ETS2, and ERBG/Hu-FLI-1 gene expression pattern also suggests a role for these genes in cells of hematopoietic lineage. The HIV-1 LTR core enhancer contains two 10-base pair direct repeat sequences (left and right) that are required for regulation of HIV-1 mRNA expression by host transcription factors, including NF kappa B. Two ETS-binding sites are present in the core enhancer of all the HIV-1 isolates reported so far. In our studies, we utilized HIV-1 HXB2 and HIV-1 Z2Z6 core enhancers because the Z2Z6 strain has a single point mutation flanking the right ETS-binding site. We demonstrate that the ETS1, ETS2, and ERGB/Hu-FLI-1 proteins can trans-activate transcription from both the HXB2 and Z2Z6 core enhancer when linked to a reporter (cat) gene. In addition, we show that the DNA binding and trans-activation with the Z2Z6 core enhancer is at least 40-fold higher than that observed with the HXB2 core enhancer. Further, we provide evidence that the marked increase in binding and trans-activation with Z2Z6 core enhancer sequences is due to the substitution of a flanking T residue in HXB2 TGGAA) by a C residue in Z2Z6 (CGGAA) isolate, thus generating an optimal ETS-binding core (CGGAA) sequence.

Animals↗
PubMed1993

Purification of an Escherichia coli-expressed Nef protein from the human immunodeficiency virus-type 2.

The entire nef gene sequence of HIV-2, NIH-Z strain, has been cloned into the pJL6 expression vector and used for the synthesis of a 23-kDa protein in E. coli. The expressed protein is a fusion between the N-terminal 13 amino acids of the cII gene, 8 amino acids resulting from the ligation procedure, and the 180 amino acids that comprise the HIV-2 Nef sequence from the NIH-Z strain. The bacterially expressed Nef protein has been purified to apparent homogeneity on analytical scale (10-20 micrograms) by a combination of sequential detergent extraction, gel filtration, and reversed-phase high-performance chromatography. The expressed Nef protein is highly susceptible to proteolysis (chymotryptic-like activity) and this property accounts for the low yield obtained by gel filtration and RP-HPLC. Larger amounts (> 100 micrograms) of the purified Nef protein have been produced by a purification procedure that employs sequential detergent extraction, chromatography on Q-Sepharose in the presence of 7 M urea, and chromatography on hydroxylapatite, also in 7 M urea. The purified HIV-2 Nef protein has been used for the production of polyclonal and monoclonal antibodies. The milder method of purification should facilitate structure-function studies of the Nef protein and its role in the life cycle of HIV.

Amino Acid Sequence↗
PubMed1993

Cost-effective diagnosis of HIV-1 and HIV-2 by recombinant-expressed env peptide (566/996) dot-blot analysis.

OBJECTIVE: To characterize the recombinant env peptides, 566 (HIV-1) and 996 (HIV-2), for their ability to serodiagnose HIV-1 and HIV-2 infection. To develop a cost-effective dot-blot format for these peptides, and to evaluate its performance in a developing country laboratory. DESIGN: The recombinant env peptides were evaluated using a select panel of sera (n = 327) with known serostatus from geographically diverse areas. A dot-blot assay was developed and tested on a second set of immunoblotted sera (n = 331) and further evaluated in the field on a third set of sera (n = 2718) from study populations. METHODS: All sera were evaluated by immunoblot with both HIV-1 and HIV-2 viral lysates. The recombinant env peptides were characterized in immunoblot assay before development of the dot-blot assay. RESULTS: The 566 (HIV-1) peptide showed 100% sensitivity and specificity. The 996 (HIV-2) peptide performed similarly, but showed the presence of HIV-1 cross-reactive epitopes. When the two env peptides were used together, there was high specificity and sensitivity for detecting HIV-positive sera in both immunoblot and dot-blot formats. The dot-blot assay performed in the field showed slightly lower specificity and sensitivity for HIV diagnosis. The relative cost of this assay combined with non-commercial immunoblot confirmation was 10-fold lower than conventional commercial assays. CONCLUSIONS: The 566 and 996 env peptides are appropriate antigens for HIV serotype diagnosis. A dot-blot assay using these peptides may be a useful cost-effective method for HIV diagnosis applicable in developing country laboratories.

AIDS Serodiagnosis↗
PubMed1993

Inversion of a chicken ets-1 proto-oncogene segment in avian leukemia virus E26.

The segment of the avian leukemia virus E26 genome near the termination of the p135gag-myb-ets open reading frame contains an inversion of the chicken ets-1 sequence. The inversion contains at least 41 bp and may be as large as 46 bp. This results in the replacement of 13 amino acids of chicken ets-1, with 16 amino acids derived from reverse complement of the normal ets-1 coding strand or read-through into E26 env sequences. At least 13 of these codons are specified by the inverted ets sequences. This represents the first reported occurrence of inverted oncogene sequences in a natural retrovirus. The inverted ets sequences are immediately followed by sequences homologous to the Rous sarcoma virus Prague B env gene. Since the E26 env sequence is more closely related to subgroup B avian retroviruses than to avian retroviruses from subgroups A, C, D, or E, the progenitor of E26 was a virus belonging to avian retrovirus subgroup B.

Avian Leukosis Virus↗