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Y Chernajovsky

Publications and source records attributed to Y Chernajovsky.

At least 73 records · Page 4Linked to original sources
PubMed1992

Interferon-stimulated genes in interferon-sensitive and -resistant chronic myelogenous leukemia patients.

alpha-Interferon induces hematological and cytogenetic remissions in some individuals with newly diagnosed Philadelphia-positive chronic myelogenous leukemia. However, interferon-resistant disease occurs in a consistent patient subset (primary resistance) and develops during therapy in additional patients (secondary resistance). Several alpha-interferon-inducible genes have been characterized. In interferon-resistant cell line variants, defects in these genes have been implicated in the mechanisms mediating resistance. We have, therefore, evaluated mRNA expression of four interferon-stimulated genes (ISGs) following alpha-interferon therapy. Twenty-seven chronic myelogenous leukemia patients (ten interferon-sensitive patients, 17 interferon-resistant patients) were studied. Peripheral blood samples were collected prior to and 1 to 7 days after starting interferon therapy and analyzed for the expression of 2'-5' oligoadenylate synthetase, ISG-15, ISG-54, and 6-16 transcripts. Following therapy with alpha-interferon, 2'-5' oligoadenylate synthetase, ISG-54, and 6-16 transcripts were discerned in all patients regardless of their response to interferon. The ISG-15 message was detected in eight of nine interferon-sensitive and in 15 of 16 interferon-resistant patients, as well. Overall, no consistent defect in the ISG system could be identified. Therefore, lack of induction of these genes cannot explain resistance to alpha-interferon in chronic myelogenous leukemia patients. Other mechanisms such as posttranslational modification, leading to defects in the ISG corresponding proteins, may play a role in the development of resistance.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

Synergism of glucocorticoids with granulocyte macrophage colony stimulating factor (GM-CSF) but not interferon gamma (IFN-gamma) or interleukin-4 (IL-4) on induction of HLA class II expression on human monocytes.

Peripheral blood monocytes from up to 13 normal donors were stimulated with the cytokines interferon gamma (IFN-gamma), interleukin 4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) in the presence or absence of dexamethasone (Dex), and the effects on HLA class II (HLA-DR, DP and DQ) expression studied. Dex markedly augmented HLA-DR, DP and DQ levels induced by GM-CSF, in all samples tested. Particularly striking were the effects on HLA-DQ expression, since stimulation with a combination of Dex and GM-CSF induced markedly higher levels of HLA-DQ antigen than stimulation with IFN-gamma. Northern blot analysis of samples treated for 40 hours with Dex and GM-CSF indicated that levels of DR alpha, DP alpha and DQ alpha mRNA were also increased. In contrast, despite variation between individual donors, in general Dex weakly inhibited both constitutive and IFN-gamma- or IL-4-induced HLA-DR expression. Variability in the responsiveness of monocytes purified from individual donors to each cytokine was also observed. GM-CSF was less potent than IFN-gamma and IL-4, enhancing HLA class II expression in only seven of 13 donors tested, whereas in the presence of Dex all donors responded to GM-CSF. The differential effects of glucocorticoids in vitro suggest that these cytokines induce HLA class II expression by different mechanisms.

Base Sequence↗
PubMed1991

Predominance of the metastatic phenotype in somatic cell hybrids of the K-1735 murine melanoma.

The purpose of these studies was to determine whether the metastatic phenotype will dominate when metastatic and nonmetastatic clones of the K-1735 mouse melanoma are hybridized by somatic cell fusion. Three nonmetastatic and three metastatic clones were transfected with DNA from plasmids pSV2neo or pSV2hygro, which confer resistance to the drugs neomycin or hygromycin, respectively. The metastatic properties of the six clones were not altered by these transfections. The tumorigenicity and metastatic capacity of cell hybrids formed by somatic cell fusion of nonmetastatic and metastatic clones were examined. To do so, near-tetraploid hybrids containing a nearly complete chromosomal complement from both parental cells were injected i.v. into syngeneic mice, and the number of metastatic nodules in the lung was determined at 45 days or when the mice became moribund. Seven of nine hybrids produced from the fusion of metastatic and nonmetastatic clones exhibited a highly metastatic phenotype, although in most cases the metastatic potential of the hybrids was lower than that of the metastatic parent cells. Very similar results were obtained in athymic nude mice. The metastatic potential of the hybrids was directly correlated with their growth in the subcutis of nude mice. These results indicate that the metastatic capacity of K-1735 cells predominates in somatic cell hybrids between nonmetastatic and metastatic cells. When fusion of nonmetastatic and metastatic cells yields a hybrid with nonmetastatic properties, it may be due to suppression of growth.

Animals↗
PubMed1991

The role of receptor-ligand endocytosis and degradation in interleukin-2 signaling and T-lymphocyte proliferation.

The specific intracellular signaling pathways for interleukin-2 (IL-2) that lead to delivery of the proliferative stimulus are currently unknown. We and others have excluded signaling pathways used by other growth factors and by the antigen-specific T-cell receptor, such as increased intracellular Ca2+ concentrations, activation of protein kinase C, or ion transport across the plasma membrane. One feature of IL-2 signaling that may be important in delivery of the proliferative stimulus is endocytosis and processing of the lymphokine receptor-ligand complex. In this study we examined these steps in receptor signaling by mouse CTLL-2 cells and human OKT3-activated T-cells using monoclonal antibodies specific for the 55 kDa alpha-subunit of the IL-2R that allow IL-2 binding but block endocytosis, and with lysosomotrophic amines that selectively inhibit receptor mediated endocytosis and/or processing of IL-2. Our results demonstrate that these inhibitors block receptor endocytosis, ligand degradation, c-fos protooncogene activation, and ultimately proliferation of the IL-2-dependent T-cell line, CTLL-2. In heterogeneous populations of activated human T cells the lysosomotrophic amines demonstrated a greater inhibition of degradation than of endocytosis. These observations support the hypothesis that IL-2/IL-2R endocytosis and ligand/receptor processing or degradation may be important steps in lymphokine signal transduction.

Blotting, Northern↗
PubMed1990

Transformation injury and the unicellular phenotype of malignant cells.

In 1984, Philippe Shubik wrote in an editorial in the Journal of the National Cancer Institute that: 'In terms of general biology, the multicellular organisms have evolved a complex series of defensive responses to extracellular injury involving various inflammatory reactions and their systemic concomitants. Needless to say, these reactions are by no means always successful from the standpoint of the host and indeed may well be the immediate cause of the obvious ill effects noted. The unicellular organisms, in contrast, react to injury by dividing and moving. In the search for the features that may link chemical, physical, and viral carcinogens apart from their ability to induce neoplasia, only one characteristic in common is obvious, namely, their ability to produce intracellular change or injury while leaving the cell viable. Perhaps the initial and fundamental characteristics of neoplasia is a reversion of the cell to unicellular behavior. Division and invasiveness are the characteristics of the neoplastic cell, and increased motility certainly seems to be the most likely mechanism for invasion'. Dr. Shubik presented these views as a basis for 'further discussion' regarding the nature of the neoplastic response. We hope that this presentation will augment Shubik's plea by reviewing his idea in the context of our current knowledge of tumor development. In addition, we will attempt to integrate the concept of the unicellular behavior of tumor cells with Foulds' and subsequently Nowell's insightful hypothesis concerning tumor progression.

Cell Transformation, Neoplastic↗
PubMed1990

Regulation of the human interferon-inducible 6-16 promoter in tumor necrosis factor-sensitive and resistant mouse cells: role of cAMP as a mediator of signal transduction.

The human intgerferon (IFN)-inducible 6-16 gene is also induced by tumor necrosis factor (TNF) in human and mouse cells. A reporter gene carrying the IFN regulatory sequences of the human IFN-inducible 6-16 promoter linked to the bacterial chloramphenicol acetyl-transferase gene was transfected into the adipocyte-differentiating fibroblast cell line TA1 and a TNF-resistant derivative (TA1 R-6). Both IFN and TNF induced the 6-16 reporter gene in TA1 cells. However, the induction of 6-16 by TNF was abolished by antibodies to IFNs-alpha/beta. In the TNF-resistant derivative (TA1 R6) TNF failed to induce the 6-16 plasmid construct. IFN, on the other hand, effectively induced the 6-16 promoter in TA1-R6. The block in activation of the 6-16 promoter in TNF-resistant cells can be reversed by the addition of compounds that increased the intracellular concentration of cAMP. These cAMP analogs induced IFN secretion in parental cells but not in the TA1-R6 cells.

Animals↗
PubMed1990

A cis-acting sequence, located at -450 in the promoter of the human interferon-inducible gene 6-16, binds constitutively to a nuclear protein and decreases the expression of a reporter interferon-inducible promoter.

An interferon-inducible synthetic promoter was constructed with oligonucleotides which correspond to two regions of the promoter of the human interferon-inducible gene 6-16 that interact in-vitro with nuclear proteins. Firstly, we cloned a direct repeat sequence that was necessary for interferon regulation and that interacted with a 55 kilodalton nuclear protein. 5' to the direct repeat we introduced a 45 bp long oligonucleotide located at -450 in the native 6-16 promoter that interacted in-vitro with an 80 kilodalton nuclear protein. Expression after transfection of these plasmids showed that the direct repeat is necessary for interferon-inducible control and the -450 oligonucleotide by itself has no effect on transcriptional activity while in conjunction with the direct repeat it decreased the basal and interferon-inducible transcriptional activity of the reporter promoter in human cells.

Animals↗
PubMed1989

Constitutive in vitro binding of nuclear proteins to the 5'-flanking region of 6-16, a human gene inducible by alpha, beta-interferons.

Proteins in nuclear extracts of HeLa cells that constitutively bound in vitro to three regions upstream of the interferon-inducible gene 6-16 were separated partially by chromatography on DEAE-Sepharose. Region one, a CCAAT box in the non-coding strand at position --63 to --67, was protected from DNase digestion by the bound protein(s) and was required for transcription in vitro. Region two, a tandem duplication sequence at position --89 to --168 contains two copies of a sequence essential for strong induction of the 6-16 gene by interferon in vitro. Region three, a palindromic sequence at position --449 to --465, not necessary for induction of 6-16 by interferon, was also protected from DNase digestion by nuclear protein(s). Templates with or without regions of two and three were transcribed equally well in extracts from interferon-treated or untreated cells.

Base Sequence↗
PubMed1988

Interferon response element of the human gene 6-16.

1046 base-pairs (bp) of genomic DNA spanning the first exon of the human alpha/beta-interferon (IFN)-inducible gene 6-16 have been analysed for their role in induction. The whole gene or 5'-flanking deletion derivatives of it were assayed for inducibility in populations of stably transfected mouse cells. 5'-Flanking DNA fragments were assayed for their ability to confer inducibility on a reporter gene in stably and transiently transfected mouse and human cells. The data suggest that a 39 bp sequence is sufficient to confer transcriptional inducibility and can account in large part for the response of 6-16. Two copies of this sequence, one of which contains a dinucleotide insert, are located in tandem 88 bp upstream of the 6-16 transcriptional initiation site. For at least one of the repeat units the 5' limit of a subregion required for induction lies in the sequence GGGAAAAT. The motif GGAAA occurs in several well characterized enhancers. Furthermore, one residue 3' of the GGAAA there is a second motif, TGAAACT, which is conserved in the regulatory regions of other IFN-induced genes. In gel retardation assays the oligonucleotide GGGAAAATGAAACT competes with the repeat element for binding to IFN-modulated protein(s) but a mutated oligonucleotide, GGGAAAATGACACT does not. These results identify an alpha/beta IFN response element partially homologous to those described previously for the genes of the MHC complexes.

Animals↗
PubMed1986

Characterization of a human gene inducible by alpha- and beta-interferons and its expression in mouse cells.

An intact interferon-inducible gene has been isolated from a cosmid library of human genomic DNA. The gene (designated 6-16) encodes a mRNA of approximately 1 kb which is induced well by alpha- and beta- but poorly by gamma-interferons. Genomic and cDNA sequences indicate that the gene contains five exons, and that the mRNA encodes a hydrophobic polypeptide of 130 amino acids with a putative NH2-terminal signal sequence. The 5' end has been identified by primer extension. The corresponding genomic DNA contains a TATA box 20 nucleotides upstream of the putative transcription initiation site. After transfection of the human genomic cosmid into mouse Ltk- cells, human 6-16 mRNA is expressed in response to mouse alpha- and beta- but not gamma-interferons with the same kinetics and dose-response as in the human cells. No such expression is observed in response to human interferons. It can be concluded that the human cosmid DNA contains all of the sequences necessary for alpha- and beta-interferon-induced gene expression and that the mechanisms governing such expression are conserved between murine and human cells.

Amino Acid Sequence↗
PubMed1985

Family of human alpha-interferon-like sequences.

An interferon-alpha-like sequence was isolated from a human genomic library by hybridization with a 15-base oligonucleotide. The sequence also showed homology to alpha-interferon and was most closely related to the leukocyte interferon-M gene fragment. The original isolate cross-hybridized to a family of sequences, 10 of which were isolated as clones. Some of these sequences were located within a few kilobases of alpha-interferon genes, consistent with our assignment of several members of the family to human chromosome 9 which also has the beta 1- and alpha-interferon genes.

Amino Acid Sequence↗
PubMed1984

Efficient constitutive production of human fibroblast interferon by hamster cells transformed with the IFN-beta 1 gene fused to an SV40 early promoter.

The coding sequence of the human interferon (IFN)-beta 1 gene, fused 60 bp downstream from the RNA start site of the SV40 early gene, was transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells together with a selectable DHFR gene. Most transformants continuously secreted IFN-beta 1 into the medium. Induction did not stimulate expression of the fused SV40-IFN-beta 1 gene. The role of the SV40 promoter was verified by transforming cells with the unmodified human IFN-beta 1 gene, or by the IFN-beta 1 coding region fused to another poly(rI):(rC)-inducible gene. In these cases, the transformants showed strictly inducible (not constitutive) IFN secretion. By selection for methotrexate resistance, CHO clones with a 10-20-fold amplification of the SV40-IFN-beta 1 DNA were obtained. Such clones constitutively produce up to 350,000 units IFN/ml per 10(6) cells/24 hr, i.e., over 10 times more than fully induced human fibroblasts. In continuous culture with daily changes of medium, accumulation of IFN-beta 1 is constant at a rate of 300,000 molecules per cell/hr. Batches of up to 16 mg of IFN-beta 1 produced by the transformed CHO cells were purified to homogeneity by affinity chromatography on monoclonal antibodies. This IFN appears identical in size, activity, and immunospecificity to the native human IFN-beta 1 glycoprotein.

Animals↗
PubMed1983

Expression of human interferon genes using the recA promoter of Escherichia coli.

Interferon beta 1 and three alpha-interferon genes were cloned on Eco RI fragments isolated from a human genomic library into the Eco RI site of a plasmid containing the recA promoter of E. coli. Expression of interferon activity from cells carrying these plasmids was nalidixic acid inducible. The alpha-interferon genes were expressed only when in the same transcriptional orientation as the recA promoter while the beta 1 interferon gene was expressed in either orientation. Interferon activity was also inducibly expressed from the recA promoter in cells containing a plasmid carrying a fusion of the recA gene with the beta 1 interferon gene. This interferon activity was thirty-fold less sensitive to neutralization by polyclonal antibodies than authentic interferon, implying that the change near the amino terminus affects either antibody recognition or specific activity or both.

Bacterial Proteins↗
PubMed1981

Synthesis of human interferon beta 1 in Escherichia coli infected by a lambda phage recombinant containing a human genomic fragment.

DNA from a human adult was fragmented by partial digestion with restriction endonuclease EcoRI and cloned in lambda Charon 4A. Clone C15, with a human DNA insert of 17 X 10(3) bases, was identified as containing a gene for the fibroblast interferon, interferon beta 1. Restriction mapping shows that this gene, located on a 1840-base EcoRI fragment, is not interrupted by introns. Moreover, we show that this human genomic DNA fragment is able to direct the synthesis of active human interferon beta 1 in Escherichia coli. Interferon activity of up to 7 X 10(6) U/l was recovered from phage lysates by chromatography on Cibacron blue--Sepharose, and had the same immunological properties and species specificity as interferon produced by human fibroblasts.

Adult↗
PubMed1980

Two interferon mRNAs in human fibroblasts: in vitro translation and Escherichia coli cloning studies.

Two mRNA species that produce biologically active interferon were isolated from human fibroblasts and studied by size fractionation and cloning in Escherichia coli plasmid pBR322. The major fibroblast interferon (Hu IFN-beta 1) is coded for by the smaller of the two mRNAs, an 11S species, 900 nucleotides long, which in cell-free systems yields a 20,000 Mr protein. The second interferon mRNA species (Hu IFN-beta 2) is 14S, about 1300 nucleotides long, and codes for another protein of 23,000-26,000 Mr. The two interferon mRNAs do not cross-hybridize. Both are induced by poly(rI.rC), but IFN-beta 2 mRNA is induced to about 10% in cells by cycloheximide treatment alone whereas under these conditions IFN-beta 1 is not induced.

Biological Assay↗
PubMed1979

Differential effects of two interferon-induced translational inhibitors on initiation of protein synthesis.

At least two different mechanisms for the inhibition of mRNA translation operate in extracts of interferon-treated L cells. One is mediated by an interferon-induced protein kinase which, when activated by double-stranded RNA and ATP, phosphorylates the small subunit of initiation factor eIF-2. Addition of the purified interferon-induced protein kinase to L cell extracts, strongly reduces the amount of methionyl-tRNA bound to 40-S ribosomal subunits. The second translational inhibition is due to the synthesis of (2'-5')oligo(adenylate) by interferon-induced enzyme E. The oligonucleotide in turn activates a ribonuclease F constitutively present in L cells. Addition of the purified nuclease with its oligonucleotide activator to L cell extracts produces a strong decrease in polyribosome formation and an accumulation of initiation complex. These experiments differentiate the effects of the two interferon-induced inhibitors on mRNA translation.

Animals↗