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K T Riabowol

Publications and source records attributed to K T Riabowol.

At least 19 recordsLinked to original sources
PubMed2000

Accelerated replicative senescence of the peripheral immune system induced by HIV infection.

OBJECTIVES: HIV induces rapid turnover of T lymphocytes but whether this leads to replicative senescence of CD4+ and CD8+ cells and contributes to AIDS symptoms is unclear. The aim of this study was to address this question by analyzing telomere length in blood cell populations as a measure of replicative history in a significant number of patients infected with HIV. DESIGN: Total peripheral blood mononuclear cells (PBMCs), CD4+ or CD8+ cells were isolated from blood collected from a total of 73 HIV patients and 27 controls. Samples were isolated to measure telomere length, telomerase activity and proliferative ability, and analyses were carried out in a blind experimental protocol. METHODS: PBMCs isolated on Ficoll-Hypaque gradients were washed and prepared for additional fractionation into CD4+ and CD8+ cells using antibody-bound magnetic beads. Total PBMCs, CD4+ and CD8+ cells were used for cell cycle analysis, for telomerase activity assays and were measured for telomere length using the terminal restriction fragment assay. RESULTS: Telomere analyses in this study show a clear (P < 0.0001) inverse relationship between telomere length and progression of immunosuppression, with HIV infection resulting in a five-fold or greater acceleration of aging of the circulating PBMC component of the immune system. Patients who are 37 years old showed telomere lengths similar to uninfected 75-year-olds. Telomere loss correlated well with progression of AIDS and with reduced proliferative ability of patient PBMCs but was unrelated to telomerase activity. Mean telomere length was shorter in both CD4+ and CD8+ cells, with three-fold higher rates of telomere loss for CD8+ lymphocytes. CONCLUSIONS: These data provide strong support for the occurrence of accelerated replicative aging of the peripheral immune system, possibly resulting in a loss of T cells leading to AIDS symptoms.

Aged↗
PubMed2000

A role for serine proteases in mediating phorbol ester-induced differentiation of HL-60 cells.

Treatment of human HL-60 promyelocytic leukemia cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) results in increases in proteolytic activity and maturation toward the monocytic lineage. To investigate the potential roles that different classes of proteases play in the monocytic differentiation of HL-60 cells, cells were treated with phorbol ester in the presence of various serine and cysteine protease inhibitors. The serine protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), N-alpha-tosyl-phenylalanine chloromethyl ketone (TPCK), and N-alpha-tosyl-lysine chloromethyl ketone (TLCK) repressed a number of phenotypic markers of monocytic differentiation including surface expression of the CD11b integrin, cell aggregate formation, cell cycle exit, and cell death. CD11b was not detected at the cell surface by FACS analysis up to 24 h after induction of differentiation; however, both CD11b mRNA and protein were present. Downregulation of c-myc mRNA and upregulation of c-fos and egr-1 mRNA and protein, which normally occur during TPA-induced differentiation, were not affected by inclusion of the protease inhibitors. These data indicate that serine proteases specifically mediate many of the phenotypic aspects of TPA-induced monocytic differentiation but are not involved with the induction or repression of differentiation-sensitive transcription factors and suggest that serine protease activity is required for intracellular processing of CD11b.

Antigens, CD↗
PubMed1999

Decreased expression and activity of the immediate-early growth response (Egr-1) gene product during cellular senescence.

Human diploid fibroblasts (HDFs) undergo a limited number of population doublings in culture before reaching the end of their proliferative life span, an event termed in vitro cellular senescence. Considerable evidence suggests that altered expression of key genes involved in the mitogenic response may be responsible for the inability of senescent cells to proliferate. Here we examined the expression and activity of the early growth response-1 (egr-1) gene, an "immediate-early" gene that is believed to link extracellular mitogenic signals to cell-cycle progression. We found that egr-1 was strongly downregulated in senescent HDFs at the level of mRNA, protein, and DNA binding activity. Decreased DNA binding activity of Egr-1 in vitro corresponded to decreased transcriptional activation in vivo. To further understand the mechanism of egr-1 downregulation, we examined the potential role of the serum response elements (SREs) present in the egr-1 promoter. Electrophoretic mobility shift studies using young and old cell nuclear extracts showed a marked decrease in serum response factor (SRF) binding activity to the SRE in old compared to young cells. Loss of SRF binding activity has been correlated with the loss of expression of another growth-related immediate-early gene (c-fos). These results suggest a common mechanism for the downregulation of c-fos, egr-1, and other SRE-dependent, mitogen-responsive genes during cellular senescence.

Cells, Cultured↗
PubMed1998

Increased expression of cyclin D2 during multiple states of growth arrest in primary and established cells.

Cyclin D2 is a member of the family of D-type cyclins that is implicated in cell cycle regulation, differentiation, and oncogenic transformation. To better understand the role of this cyclin in the control of cell proliferation, cyclin D2 expression was monitored under various growth conditions in primary human and established murine fibroblasts. In different states of cellular growth arrest initiated by contact inhibition, serum starvation, or cellular senescence, marked increases (5- to 20-fold) were seen in the expression levels of cyclin D2 mRNA and protein. Indirect immunofluorescence studies showed that cyclin D2 protein localized to the nucleus in G0, suggesting a nuclear function for cyclin D2 in quiescent cells. Cyclin D2 was also found to be associated with the cyclin-dependent kinases CDK2 and CDK4 but not CDK6 during growth arrest. Cyclin D2-CDK2 complexes increased in amounts but were inactive as histone H1 kinases in quiescent cells. Transient transfection and needle microinjection of cyclin D2 expression constructs demonstrated that overexpression of cyclin D2 protein efficiently inhibited cell cycle progression and DNA synthesis. These data suggest that in addition to a role in promoting cell cycle progression through phosphorylation of retinoblastoma family proteins in some cell systems, cyclin D2 may contribute to the induction and/or maintenance of a nonproliferative state, possibly through sequestration of the CDK2 catalytic subunit.

3T3 Cells↗
PubMed1997

Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix.

Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes.

Animals↗
PubMed1997

Immunoreactivity for the Fas ligand in the mammalian enteric nervous system.

The Fas ligand induces apoptosis in activated immunocytes that express the Fas receptor. Fas-ligand transcripts have been found previously in murine intestine but the intestinal tissues that express Fas ligand have not been identified. We used immunohistochemistry to examine the expression of the Fas ligand in the enteric nervous system of rats, mice, guinea-pigs, ferrets and humans. Fas-ligand immunoreactivity was detectable in enteric nerve fibres and neurons in all species tested, representing 25%-50% of the neurons in rats, mice and guinea-pigs. An antigen of approximately 48 kDa was detected by Western blot analysis with Fas-ligand antiserum in the dissected enteric plexuses of duodenum from a C3H/HeJ mouse. In gld mice that harbour a Fas-ligand mutation, Fas-ligand immunoreactivity was slightly more intense in neurons and fibres and was also apparent in submucosal lymphocytes. In the myenteric plexuses of guinea-pig ileum and human colon, Fas-ligand immunoreactivity was not contained in neurons exhibiting nicotinamide-adenine dinucleotide phosphate-diaphorase activity. In the submucosal plexus of guinea-pig ileum, labelled neurons included some neuropeptide-Y-containing neurons but none with vasoactive intestinal polypeptide. We conclude that the Fas ligand is expressed by a large subset of enteric neurons and may provide the basis for cytotoxic neuroimmune interactions in the intestines.

Animals↗
PubMed1996

Selective inhibition of telomerase activity during terminal differentiation of immortal cell lines.

Telomerase, the enzyme that maintains the ends of linear eukaryotic chromosomes, is active in human germ cells and in a majority of tumor tissues and immortalized cell lines. In contrast, most mature somatic cells and tissues contain low or undetectable telomerase activity, implying a stringent negative regulatory control mechanism. We report here that telomerase activity is dramatically inhibited during the terminal differentiation of HL-60 human promyelocytic leukemia cells to monocytic and granulocytic lineages. A loss of telomerase activity was seen in response to three different inducers of differentiation, was independent of differentiation-induced apoptosis, and occurred in the presence of unaltered expression of the RNA component of telomerase. Reduction in telomerase activity was also observed during the differentiation of murine F9 teratocarcinoma and C2C12 myoblast cells. In contrast, induced differentiation of murine p19 embryonal carcinoma and Neuro 2a neuroblastoma cells did not result in a loss of telomerase activity. These results are therefore consistent with the absence of telomerase activity in human somatic cells and the presence of telomerase activity in many somatic murine cells and tissues.

Animals↗
PubMed1994

Monitoring mRNA expression by polymerase chain reaction: the "primer-dropping" method.

We have developed a method to monitor mRNA expression that is based upon the reverse transcriptase-polymerase chain reaction (RT-PCR) and includes multiple sets of primer pairs in coamplification reactions. To observe relative changes in mRNA steady-state levels, each target in a multiplex reaction was amplified to within a predetermined range by using PCR cycle numbers specific for each target. Optimal PCR cycle numbers for target templates were determined by preliminary titration experiments performed using the "primer-dropping" method. By employing this method, the overall amplification reaction was limited, permitting the PCR products to remain within the exponential range of the amplification curve and yet be detectable on ethidium bromide-stained gels. We demonstrated the utility of this method by monitoring the expression kinetics of cyclins A, B1, D1, and E, and of the immediate-early genes c-fos, c-myc, and beta-actin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included in the multiplex reactions as an endogenous internal standard to control for variations in product abundances due to differences in individual RT and PCR reaction efficiencies. Changes in gene expression of less than twofold to greater than 75-fold were readily distinguished.

Actins↗
PubMed1994

Loss of serum response element-binding activity and hyperphosphorylation of serum response factor during cellular aging.

Human diploid fibroblasts undergo a limited number of population doublings in vitro and are used widely as a model of cellular aging. Despite growing evidence that cellular aging occurs as a consequence of altered gene expression, little is known about the activity of transcription factors in aging cells. Here, we report a dramatic reduction in the ability of proteins extracted from the nuclei of near-senescent fibroblasts to bind the serum response element which is necessary for serum-induced transcription of the c-fos gene. In contrast, the activities of proteins binding to the RNA polymerase core element, TATA, as well as to the cyclic AMP response element were maintained during cellular aging. While no major differences in the expression of the serum response factor (SRF) that binds the serum response element were seen between early-passage and late-passage cells, hyperphosphorylation of SRF was observed in near-senescent cells. Furthermore, removal of phosphatase inhibitors during the isolation of endogenous nuclear proteins restored the ability of SRF isolated from old cells to bind the SRE. These data, therefore, indicate that hyperphosphorylation of SRF plays a role in altering the ability of this protein to bind to DNA and regulate gene expression in senescent cells.

Base Sequence↗
PubMed1992

Testing the in vivo role of protein kinase C and c-fos in neurite outgrowth by microinjection of antibodies into PC12 cells.

To define the molecular bases of growth factor-induced signal transduction pathways, antibodies known to block the activity of either protein kinase C (PKC) or the fos protein were introduced into PC12 cells by microinjection. The antibody against PKC significantly inhibited neurite outgrowth when scored 24 h after microinjection and exposure to nerve growth factor (NGF). Microinjection of antibodies to fos significantly increased the percentage of neurite-bearing cells after exposure to either NGF or basic fibroblast growth factor (bFGF) but inhibited the stimulation of DNA synthesis by serum, suggesting that in PC12 cells, fos is involved in cellular proliferation. Thus, activation of PKC is involved in the induction of neurite outgrowth by NGF, but expression of the fos protein, which is induced by both NGF and bFGF, is not necessary and inhibits neurite outgrowth.

Animals↗
PubMed1989

Multiple sequence elements of a single functional class are required for cyclic AMP responsiveness of the mouse c-fos promoter.

Agents that elevate the intracellular concentration of cyclic AMP (cAMP) rapidly and transiently induce expression of the c-fos proto-oncogene in BALB/c 3T3 cells. We show that the mouse c-fos promoter-enhancer region contains multiple elements that contribute to cAMP responsiveness of the promoter in transient expression assays. The most potent element was found to correspond to a previously mapped basal promoter element and protein-binding site located 65 base pairs upstream of the transcriptional initiation site. This element and two less potent sites contained a match to the cAMP response element (CRE) core sequence defined in several mammalian genes. The relative potencies of these elements corresponded with their relative affinities for cellular factors that bound to the CRE in vitro. Mutation of all three elements failed to abolish completely cAMP responsiveness of the c-fos promoter in the transient expression assay. However, we present evidence that this residual responsiveness may have been due to sequences present in vector DNA. Finally, we show, by using a new microinjection competition assay, that a double-stranded oligonucleotide carrying the major c-fos CRE is sufficient to block induction of the endogenous c-fos gene by cAMP. Therefore, induction of the endogenous gene requires positively acting cellular factors that interact with a single functional class of regulatory sites in the c-fos gene. Unrelated regulatory elements, such as the serum response element and putative AP-2 sites, are not by themselves sufficient to mediate the cAMP response.

Animals↗
PubMed1988

The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements.

Transcriptional regulation of eukaryotic genes by cyclic AMP requires a cAMP-dependent protein kinase (A kinase). Two hypotheses have been proposed to explain how the holoenzyme of the A kinase induces transcription. The regulatory subunits of the A kinase, which bind cAMP and DNA, and have amino-acid homology with the Escherichia coli catabolite activator protein could directly stimulate gene expression. Alternatively, phosphorylation by the catalytic subunits could induce transcription by activating proteins involved in gene transcription. To distinguish between these models, we microinjected purified preparations of the catalytic and regulatory subunits of A kinase into tissue culture cells and monitored expression of a stably integrated fusion gene containing a cAMP-responsive human promoter fused to a bacterial reporter gene, or of the endogenous c-fos gene. The catalytic subunit stimulated expression of these genes, whereas the regulatory subunit did not. These results indicate that the catalytic subunit of A kinase is sufficient to induce expression of two cAMP-responsive genes, without increasing levels of cAMP.

Animals↗
PubMed1988

Identification of microinjected cells using biotinylated antibodies and Strep-avidin-conjugated horseradish peroxidase.

Results from experiments using needle microinjection of cells are often compromised by an inability to readily demonstrate which cells within a population have been injected. The technique described here allows the unambiguous identification of cells that have been successfully microinjected. Sequential incubation of fixed cells with biotinylated anti-immunoglobulin antibodies, followed by horseradish peroxidase (HRP)-conjugated Strep-avidin and HRP substrate, provides a sensitive assay for identification of cells containing trace amounts of immunoglobulins. This allows direct correlation to the presence of injected molecules of effects on cell morphology, the ability to enter into DNA synthesis, or expression of specific genes. By a variety of criteria, nonspecific immunoglobulins do not adversely affect cellular processes when injected by themselves or in the presence of other proteins known to have biological effects when injected, such as cAMP-dependent protein kinase and the ras oncogene protein.

Animals↗
PubMed1988

Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70.

Synthesis of a small group of highly conserved proteins in response to elevated temperature and other agents that induce stress is a universal feature of prokaryotic and eukaryotic cells. Although correlative evidence suggests that these proteins play a role in enhancing survival during and after stress, there is no direct evidence to support this in mammalian cells. To assess the role of the most highly conserved heat shock protein (hsp) family during heat shock, affinity-purified monoclonal antibodies to hsp70 were introduced into fibroblasts by needle microinjection. In addition to impairing the heat-induced translocation of hsp70 proteins into the nucleus after mild heat shock treatment, injected cells were unable to survive a brief incubation at 45 degrees C. Cells injected with control antibodies survived a similar heat shock. These results indicate that functional hsp70 is required for survival of these cells during and after thermal stress.

Animals↗
PubMed1988

Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells.

Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis.

Animals↗
PubMed1985

Extrachromosomal circular DNA and aging cells.

A DNA sequence situated in the human genome between Alu-repeat clusters ("Inter-Alu" DNA) is progressively amplified in extrachromosomal DNA, including covalently closed DNA circles, during serial passage of diploid fibroblasts. A single size-class of Inter-Alu circles is also amplified in lymphocytes from 16 of 24 old donors and yet is not detected in cells from 18 young donors.

Cell Survival↗