Biomedical subjects

M B Prystowsky

Publications and source records attributed to M B Prystowsky.

At least 19 recordsLinked to original sources

PubMed1996

A1, a Bcl-2 family member, prolongs cell survival and permits myeloid differentiation.

A1, a bcl-2 family member, has been identified as a hematopoietic-specific, early inducible gene. In this study it is shown that stable transfection of A1 into an interleukin-3 (IL-3)-dependent myeloid precursor cell line, 32D c13, leads to a retardation of IL-3 withdrawal-induced cell death similar to that observed with transfection of bcl-2. However, unlike bcl-2. A1 expression permits the accumulation of differentiated myeloid cells both before and after IL-3 withdrawal. Total cell accumulation, on the other hand, is considerably greater after IL-3 deprivation in the bcl-2 transfectant than in A1-expressing cells. Cells cotransfected with the two genes behave similarly to cells singly transfected with bcl-2, except that viability following IL-3 withdrawal is somewhat further enhanced. These results suggest that these two proteins have distinct roles that may be related to the divergent regulation of their expression during myeloid differentiation.

Animals↗

PubMed1996

Identification of an essential cis-element near the transcription start site for transcriptional activation of the proliferating cell nuclear antigen gene.

Interleukin 2 (IL-2) stimulates T lymphocyte proliferation and induces the expression of proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerase delta. Previously, deletion analysis suggested cis-element(s) in the proximal region of the PCNA promoter (-40 to +143) are required for IL-2 induction in cloned T lymphocytes. The sequence 5'-TTGCGGGC-3' located at +10 to +17 is similar to the E2F consensus binding site and is required for optimal PCNA promoter activity. In IL-2-stimulated T cells, nuclear proteins are induced to bind to this sequence as demonstrated using electrophoretic mobility shift assay (EMSA), competition EMSA, and methylation interference analysis. A 180-kDa polypeptide was detected by UV cross-linking to bind specifically to the PCNA E2F-like sequence. Our data indicate that the protein bound to the PCNA E2F-like site is not one of the transcription factor E2F proteins. Our results demonstrate that the E2F-like sequence and the protein(s) binding to it are required for optimal PCNA promoter activity and IL-2 induction of PCNA expression.

Animals↗

PubMed1995

Effect of non-H-2-linked genes on anti-virus immune responses and long-term survival in mice persistently infected with E-55+ murine leukemia virus.

We have previously demonstrated that BALB/c-H-2k (BALB.K) mice are susceptible to the development of thymic lymphoma induced by E-55+ murine leukemia virus (MuLV). In the present studies, C57BL/10-H-2k (B10.BR) mice were found to be resistant to E-55+ MuLV-induced lymphoma despite the fact that these mice become persistently infected. This resistance to lymphomagensis is mediated by the anti-virus immune response since immunosuppressed mice progress to develop disease. The protective immune response in B10.BR mice is bimodal with respect to time after virus infection. The early immune response results in a dramatic decrease in the number of virus-infected cells within 4-8 weeks after infection. This decrease in virus-infected cells occurs in immunocompetent mice from strains that are either resistant (B10.BR) or susceptible (BALB.K) to E-55+ MuLV-induced disease. Subsequently, susceptible mice develop an increase in infected cells, whereas no increase in infected cells occurs in resistant mice despite the fact that they are persistently infected. This later phase of resistance in B10.BR appears to be mediated by T cells. Since B10.BR and BALB.K both express the H-2k haplotype, resistance appears to be mediated by a non-H-2-linked gene(s). (BALB.K x B10.BR)F1 mice are resistant to disease development, indicating resistance is a dominant trait.

Animals↗

PubMed1995

Rapamycin selectively blocks interleukin-2-induced proliferating cell nuclear antigen gene expression in T lymphocyte. Evidence for inhibition of CREB/ATF binding activities.

The macrolide rapamycin arrests T lymphocytes stimulated by interleukin-2 (IL-2) at G1/S. We have recently found that IL-2 induced an increase in the binding of discrete transcription factors of the ATF/cAMP-responsive element binding factor (CREB) family at G1/S, and that this effect was inhibited by rapamycin (Feuerstein, N., Huang, D., Hinrichs, S. H., Orten, D. J., Aiyar, N., and Prystowsky, M. B. (1995) J. Immunol. 154, 68-79). We now show, by using high resolution two-dimensional gel electrophoresis, that rapamycin inhibited selectively the synthesis of three discrete IL-2-induced soluble proteins (35 kDa/pI approximately 5, 68 kDa/pI approximately 4, 110 kDa/pI approximately 4.3). Analysis of nuclear proteins demonstrated that rapamycin selectively blocked the expression of proliferating cell nuclear antigen (PCNA), an obligate cofactor of DNA polymerase-delta, an important component for DNA replication. Rapamycin inhibited the IL-2-induced PCNA mRNA, and the murine PCNA promoter activity in IL-2-stimulated cells. Inducible CRE-binding proteins were shown previously to be required for PCNA promoter activity in IL-2-stimulated T lymphocytes. Using DNA binding gel mobility shift assay we demonstrated that rapamycin potently inhibited the binding of CREB/ATF transcription factors to CRE elements in the murine proximal PCNA promoter. These results suggest that PCNA is a preferred target in a rapamycin-sensitive transduction pathway, and that the mechanism by which rampamycin inhibits PCNA gene expression may involve the inhibition of the interaction of CREB/ATF transcription factors with CRE elements in the proximal PCNA promoter.

Activating Transcription Factors↗

PubMed1995

Genomic organization and genetic mapping of the neuroimmune gene I2rf5 to mouse chromosome 4.

The nervous and immune systems share many functional and molecular similarities, including shared surface antigens, secretions of soluble factors, and cross-modulatory effects. We have identified previously a novel mRNA termed F5, which is expressed only in activated T lymphocytes and mature, postmitotic neurons. Tissue specificity and sequence conservation suggest an important function for F5 in T-lymphocyte proliferation and neuronal maturation. The F5 gene product is an evolutionarily conserved, cytoskeletal-associated phosphoprotein. A full-length mouse genomic clone has been isolated. The protein coding region of the F5 gene is approximately 16 kb in length and is composed of 13 coding exons. The gene encoding F5, termed I2rf5, was mapped using interspecies mouse crosses in close proximity to a number of genes associated with neuronal defects on distal chromosome 4.

Animals↗

PubMed1995

Regulation of cAMP-responsive enhancer binding proteins during cell cycle progression in T lymphocytes stimulated by IL-2.

IL-2 stimulates the proliferative response of various lymphoid cells. Previous studies showed an increase in intracellular levels of cAMP concomitant with an increase in phosphorylation of discrete proteins by protein kinase A at late G1 phase in mitogen-stimulated lymphocytes. Thus, experiments were undertaken to study nuclear proteins that bind to the cAMP-responsive enhancer (CRE) in cloned T lymphocytes stimulated with IL-2. With the use of a 32P-labeled CRE consensus sequence in a DNA binding gel mobility shift assay, we showed that IL-2 stimulation resulted in the induction of two major DNA-protein complexes at late G1/S during the cell cycle. This binding was competed in a dose-dependent manner by a nonlabeled CRE oligonucleotide but was not competed by a nonlabeled AP-1 oligonucleotide. Rapamycin, a potent immunosuppressant, which arrests IL-2-stimulated T lymphocytes at G1/S, inhibited the IL-2-induced CRE binding activities concomitantly with inhibition of DNA synthesis. By using specific Abs in a gel mobility shift assay, we identified two known CREB/ATF transcription factors in the IL-2-induced CRE complexes: the CRE binding factor (CREB), and ATF1. The induction of CREB binding by IL-2 was not associated with an increase in its abundance but was associated with a major increase in CREB phosphorylation that was particularly prominent at late G1/S. However, we found that G1/S progression induced by IL-2 was not associated with an increase in the intracellular levels of cAMP. These results suggest that 1) the transcription factors CREB and ATF1 and possibly other CRE binding proteins may have an important role in the modulation of specific gene expression at G1/S during cell cycle progression induced by IL-2. 2) The involvement of these CRE binding transcription factors in IL-2-stimulated cells is regulated via a mechanism that is not cAMP dependent.

Activating Transcription Factor 1↗

PubMed1995

CCAAT-box contributions to human thymidine kinase mRNA expression.

In order to examine the role of two inverted CCAAT boxes near the start of transcription of the human thymidine kinase (TK) gene, a series of constructs were prepared in which one or both CCAAT boxes were deleted or mutated. These altered promoters (1.2 kb of 5'-flanking sequence) were used to express a TK minigene containing the first two exons and introns followed by the remainder of the cDNA. RNA blots were prepared from stable cell lines of ts13 cells containing these constructs under three conditions: 1) serum deprived cells, 2) serum stimulated cells, and 3) cells that had been stimulated with serum, but were arrested in the G1 phase of the cell cycle by the temperature sensitive mutation carried by these cells. TK mRNA expression from each construct was suppressed by the temperature sensitive block to cell cycle progression. Measurement of protein expression from the various altered TK promoters indicated that both CCAAT boxes contribute to promoter strength. These experiments also suggested that the two CCAAT boxes were not equivalent and that the distal CCAAT could substitute for the proximal CCAAT, but the converse was not true.

Animals↗

PubMed1995

Protein that binds to the distal, but not to the proximal, CCAAT of the human thymidine kinase gene promoter.

Mobility shift assays were used to examine protein binding to the human TK gene CCAAT boxes. Similar protein binding patterns were observed with probes containing either the proximal or distal CCAAT. However, probes containing both CCAAT boxes in which one of the CCAAT boxes was inactivated by mutation did not demonstrate identical binding patterns. One of the complexes formed with the longer probes was only observed when the distal CCAAT was intact. This species was not formed with probes that only contained an intact proximal CCAAT, and its formation could only be competed by oligonucleotides containing the distal CCAAT motif. This observation reveals the existence of a protein that can bind to the distal, but not to the proximal, CCAAT of the human TK promoter. This protein may account for the previous observation that the two CCAAT motifs are not functionally equivalent. The protein that binds to the distal, but not to the proximal, CCAAT (DTK-CBP) was also present in two human cell lines. Significantly more DTK-CBP was present in nuclear extracts of HepG2 and WI38 cells than in TK-ts13 cells. However, this protein was not observed in three different murine cell lines and one primary culture. Its abundance in some human cell lines suggests it might modulate the expression of human TK mRNA in cells that express this protein.

3T3 Cells↗

PubMed1995

Environmental lighting alters the infection process in an animal model of AIDS.

In this study, we examined the effects of altered environmental lighting on the infection process of a murine leukemia virus, E-55(+), which induces a thymic lymphoma/leukemia in 100% of BALB.K mice inoculated as adults. One to two weeks after inoculation, high levels of proviral DNA are usually found. This is followed by an asymptomatic period of many weeks during which proviral DNA becomes essentially undetectable. Leukemia develops approximately 28 weeks postinoculation. In this experiment, one group of mice was exposed a consistent 10L: 14D cycle while a second was maintained in constant light (LL). A third group was exposed to a rotating cycle characterized by phase shifting a 10L: 14D cycle every three 24-h days (rLD). All cycles began 2 weeks prior to inoculation and were maintained thereafter. Animals were sacrificed at 1, 5, 10, and 15 weeks, and hematopoietic tissue was examined for proviral DNA content. At 1 week, LL- and rLD-exposed animals showed considerably less proviral DNA in bone marrow and spleen compared with controls. At 15 weeks, thymuses from controls were showing signs of infection whereas tissue from LL and rLD mice remained at background levels. We conclude that environmental lighting does alter the infective pattern displayed by this retrovirus, although whether this effect is mediated by changes in the target stem cells or through immunoenhancement has not yet been determined.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Differential effects of monoclonal antibodies on activating transcription factor-1 and cAMP response element binding protein interactions with DNA.

Activating transcription factor-1 (ATF1) and cAMP response element binding protein (CREB) have been implicated in cAMP-, calcium-, and virus-induced transcriptional alterations. Although CREB and ATF1 share extensive homology, they appear to mediate distinct cellular functions. We investigated the effect on DNA binding and in vitro transcription of four monoclonal antibodies (mAb) that bound to domains in either the regulatory region (mAb 1 and 3) or unique regions near the DNA-binding domains (mAb 4 and 5) of ATF1.mAb 1 and 3 supershifted both ATF1 and CREB in a DNA binding assay but did not affect in vitro transcription. mAb 4 prevented ATF1-DNA binding while supershifting CREB.DNA complexes and inhibited in vitro transcription by 95% from the CRE-containing murine proliferating cell nuclear antigen promoter. mAb 5 reacted specifically with ATF1 and did not prevent DNA binding or affect in vitro transcription. The mAb 4 epitope was located within ATF1 amino acid residues 205-219, including the first 3 basic residues in the putative DNA-binding domain. Secondary structural analysis predicted that this region comprises a transition site from alpha-helix to a turn-like conformation in ATF1. The transition to turn-like motifs is predicted to occur in CREB after 5 additional residues, with a correspondingly longer alpha-helical domain. Although regulatory domains distinct from DNA binding regions are thought to account for most of the differences in activity of members of the CREB subfamily, our results suggest that small structural variations adjacent to DNA binding regions may also contribute to the distinct functional activities of ATF1 and CREB.

Activating Transcription Factor 1↗

PubMed1994

Selective induction of the beta chemokine C10 by IL-4 in mouse macrophages.

The beta chemokines are a family of 8- to 12-kDa leukocyte chemoattractants that are typically produced by activated macrophages or lymphocytes. We examined the expression in primary macrophages of a recently described, and as yet functionally uncharacterized, murine beta chemokine, C10, and contrasted its regulation with that of several other beta chemokines. Although three other beta chemokines, macrophage inflammatory protein-1 alpha (MIP-1 alpha), JE, and RANTES, were all induced by LPS treatment of bone marrow-derived macrophages (BMM) and/or resident peritoneal macrophages (RPM), LPS stimulation of C10 was never observed. Conversely, IL-3 and granulocyte macrophage-CSF (GM-CSF) strongly induced C10 in both macrophage populations, whereas MIP-1 alpha and RANTES showed a weaker induction restricted to BMM. JE was strongly induced but only in BMM. Finally, IL-4 strongly induced C10 in a dose-dependent manner in both BMM and RPM but failed to stimulate any of the other three beta chemokines. The accumulation of C10 protein in culture supernatants paralleled the induction of mRNA, and the combination of IL-4 and GM-CSF led to enhanced protein levels. The expression of the C10 message in response to cytokines was completely blocked by cycloheximide, whereas the other three chemokines were all overexpressed in the presence of this inhibitor. These results demonstrate a sharp divergence between the regulation of C10 expression and that of other chemokines and suggest that this molecule may have distinct functions in host defense.

Amino Acid Sequence↗

PubMed1994

Prolactin as a second messenger for interleukin 2.

The generation of an effective immune response requires the selection and proliferation of antigen-specific lymphocytes. Activation of at least some T lymphocytes involves the sequential stimulation of cell surface receptors, including the T-cell receptor for antigen, the interleukin 2 receptor, and the prolactin receptor. Prolactin signaling in T lymphocytes appears to utilize a classical receptor-mediated kinase cascade and a novel peptide hormone activation pathway involving nuclear translocation.

Antigens↗

PubMed1994

Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes.

The proliferating-cell nuclear antigen (PCNA) gene encodes an auxiliary factor of DNA polymerase delta and functions in DNA replication during S phase. It is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the regulatory role of the 5'-flanking sequence of the murine PCNA gene in interleukin 2 (IL-2)-responsive cloned T cells (L2). Analysis of a set of deletion constructs in transient transfection assays measuring heterologous reporter gene (luciferase) activity demonstrated that the 182-bp 5'-flanking region provides full promoter activity in IL-2-stimulated L2 cells. While many elements contribute to PCNA promoter strength in IL-2-stimulated cells, the largest decrease in activity occurred with deletion of the tandem CRE (cyclic AMP response element) binding sites located at nucleotides -37 to -52. With a gel mobility shift assay, several IL-2-inducible DNA-protein complexes were detected, including CREB (CRE-binding) and ATF1 (activating transcription factor) proteins that are specific for the PCNA-CRE sequence. Methylation interference analysis confirmed specific binding of these proteins to the CRE sites. Mutation at the PCNA-CRE motif abolishes IL-2-inducible binding and reduces substantially PCNA promoter activity. These results indicate that IL-2-stimulated PCNA transcription may be partially mediated by these CRE-binding proteins.

Animals↗

PubMed1993

Characterization of A1, a novel hemopoietic-specific early-response gene with sequence similarity to bcl-2.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates hemopoietic cell proliferation, differentiation, and functional activation by inducing the expression of specific genes. As part of an investigation of the regulation of gene expression by GM-CSF, we have previously identified a novel murine GM-CSF-inducible gene, A1. In this report, we present the complete nucleotide sequence of the A1 mRNA as well as a portion of the 5' flanking region, and describe the expression pattern of the gene. The results demonstrate that A1 is a hemopoietic tissue-specific gene that is expressed in several hemopoietic cell lineages, including T-helper lymphocytes, macrophages, and neutrophils. In murine bone marrow-derived macrophages, A1 gene expression is rapidly and transiently induced by GM-CSF, and the induction was independent of de novo protein synthesis. In addition to GM-CSF, a transient induction of A1 mRNA accumulation was observed in response to LPS in macrophages. This induction is not mediated by IL-1 alpha or IL-6, neither of which stimulate A1. In the myeloid precursor cell line, 32D cl3, A1 gene expression is stably induced during granulocyte colony-stimulating factor-stimulated myeloid cell differentiation. The A1 message encodes a predicted polypeptide with an M(r) of 20,024 and no signal peptide. The peptide sequence contains a region of 80 amino acids that shows similarity to bcl-2 and to the recently described bcl-2-related gene, MCL1. These data demonstrate that A1 is a novel early-response gene whose expression is associated with a variety of stimuli and occurs in several hemopoietic cell types.

Amino Acid Sequence↗

PubMed1993

Corticotropin-releasing factor mRNA in rat thymus and spleen.

Corticotropin-releasing factor (CRF) initiates stress-induced immunosuppression via the hypothalamic-pituitary-adrenal axis. CRF has also been shown to have direct stimulatory and suppressive effects on immune cells. We have previously detected immunoreactive and bioactive CRF in the rat spleen and thymus. To determine if CRF is synthesized in these tissues, we analyzed rat spleen and thymus for the presence of CRF mRNA. RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction with CRF gene-specific oligonucleotide primers. After Southern blotting and hybridization with an internal CRF gene probe, a product of the expected size was detected in the spleen, thymus, and hypothalamus (positive control) but not in liver or kidney (negative controls), indicating that CRF is synthesized in the spleen and thymus. Furthermore, CRF could be secreted from splenic and thymic adherent cells in culture. Secretion increased severalfold in response to nordihydroguaiaretic acid (NDGA), a lipoxygenase pathway inhibitor, whereas interleukin 1 had no effect, suggesting that regulation of CRF secretion may differ from that in the hypothalamus. CRF mRNA was detected in NDGA-stimulated thymic adherent cells and in both control and NDGA-stimulated splenic nonadherent cells. The finding that CRF is synthesized in the spleen and thymus suggests that locally synthesized "immune" CRF, acting as an autocrine or paracrine cytokine, may have direct regulatory effects on immune function.

Animals↗

PubMed1993

Murine leukemia virus infection in immunocompetent adult mice.

The course of infection of a chronic murine leukemia virus (MuLV), E-55+, which induces thymic lymphoma/leukemia in 100% of mice infected as adults, was followed after inoculation of virus into adult mice which generate both cytotoxic T cell and neutralizing antibody responses (Blank 1976). DNA from the tissues of these infected mice was analyzed using a polymerase chain reaction assay to detect integrated provirus. Infection in adult immunocompetent BALB.K mice results in an acute phase characterized by a high number of cells containing provirus DNA followed by a period in which provirus DNA in most tissues reaches background levels until the development of thymic lymphoma/leukemia at 4-7 months. This pattern of infection is different from what has been observed in immunosuppressed mice in which levels of virus-infected cells remain high throughout the preleukemic and leukemic phases.

Animals↗

PubMed1993

Loss of antigenic epitopes as the result of env gene recombination in retrovirus-induced leukemia in immunocompetent mice.

The murine leukemia virus, E-55+ virus, induces a thymic lymphoma/leukemia in 100% of BALB.K mice infected as adults after a latent period of 4 months or more (Pozsgay et al., Virology 173, 330-334, 1989). Two molecular clones of virus designated E-55+ and E-55- based on their ability to encode the E-55 epitope detected by the monoclonal antibody 55 (mAb 55) were isolated from a leukemic BALB.K mouse inoculated with a biologically cloned E-55+ virus (Chesebro et al., Virology 112, 131-144, 1981). Env gene sequence analysis of E-55+ and E-55- clones showed that the E-55- virus was generated from the E-55+ virus as the result of a recombination between E-55+ virus and the endogenous ecotropic virus, emv-1, carried in the genome of the BALB.K mouse strain. The recombinant E-55- virus is replication competent. This recombination event and the consequential expression of E-55- virus consistently occur in immunocompetent BALB.K mice inoculated with the E-55+ virus and appear to play a role in the loss of epitopes recognized by virus neutralizing antibodies. The loss of these epitopes apparently allows the virus to evade the host immune response.

Animals↗

PubMed1993

The gene for C10, a member of the beta-chemokine family, is located on mouse chromosome 11 and contains a novel second exon not found in other chemokines.

C10 is a recently described member of the beta-chemokine subfamily of the chemokine superfamily of cytokine proteins. Genomic clones encoding murine C10 were isolated and sequenced. The other members of the beta-chemokine family have a three-exon genomic structure containing, among other sequence similarities, four cysteines spaced in a highly conserved manner. In each of these genes, the second exon contains the first three of the four conserved cysteines, and the third exon contains the last. In contrast to this genomic structure, the C10 gene has four exons, with a novel second exon of 48 nucleotides. Exons 3 and 4 of C10 contain four cysteines distributed in the same manner as in exons 2 and 3 of other beta-chemokine family members. The novel second exon codes for a large number of charged amino acids, and this exon shows no homology to any previously described sequences in computer databases. Linkage studies showed that the C10 gene (Scya6) is closely linked to the Scya2 locus on mouse chromosome 11, indicating that the C10 gene is located in the same region of mouse chromosome 11 as other members of the beta-chemokine family. Thus, although the C10 gene contains a novel exon not found in any other members of the chemokine superfamily, its chromosomal location and conservation of cysteine residues and other structural features suggest that it evolved from the same ancestral gene as other members of the beta-chemokine family.

Amino Acid Sequence↗