Biomedical subjects

R E Shackelford

Publications and source records attributed to R E Shackelford.

10 recordsLinked to original sources

PubMed2001

The Ataxia telangiectasia gene product is required for oxidative stress-induced G1 and G2 checkpoint function in human fibroblasts.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond to t-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G(1) and G(2) phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage.

Adolescent↗

PubMed2000

Oxidative stress and cell cycle checkpoint function.

Oxidative stress and the damage that results from it have been implicated in a wide number of disease processes including atherosclerosis, autoimmune disorders, neuronal degeneration, and cancer. Reactive oxygen species (ROS) are ubiquitous and occur naturally in all aerobic species, coming from both exogenous and endogenous sources. ROS are quite reactive and readily damage biological molecules, including DNA. While the damaging effects of ROS on DNA have been intensively studied, the effects of oxidative damage on cell cycle checkpoint function have not. Here will we review several biologically important ROS and their sources, the cell cycle, checkpoints, and current knowledge about the effects of ROS on initiating checkpoint responses.

Aerobiosis↗

PubMed1999

Lack of involvement of ataxia telangiectasia mutated (ATM) in regulation of nuclear factor-kappaB (NF-kappaB) in human diploid fibroblasts.

It has been suggested that the cellular response to exposure to ionizing radiation involves activation of the transcription factor nuclear factor-kappaB (NF-kappaB) and that this response is defective in cells from individuals with ataxia telangiectasia (AT). In one study, it was found that SV40 large T-transformed cells derived from a patient null for the AT mutated (ATM) gene exhibited constitutive activation of NF-kappaB and that in those cells, inhibition of NF-kappaB by expression of a modified form of IkappaBalpha led to correction of the radiosensitivity associated with the AT phenotype [M. Jung et al., Science (Washington DC), 268: 1691-1621, 1995]. From those data, it was suggested that NF-kappaB played a role in the AT phenotype. We show here that normal diploid cells derived from AT patients do not exhibit constitutive activation of NF-kappaB. Furthermore, we provide data that the transformation process associated with SV40 large T antigen expression in AT-/- cells leads to aberrant cellular responses. Our studies highlight the importance of using diploid, nontransformed AT-/- cells for in vitro studies relevant to the AT phenotype whenever possible.

Ataxia Telangiectasia↗

PubMed1999

Interferon-gamma, bacterial lipopolysaccharide, and tumor necrosis factor-alpha induce CD11a mRNA and protein via Na+/H+ exchange and protein kinase C-dependent mechanisms in tissue macrophages.

Previously CD11a or leukocyte function-associated antigen alpha-1 was found to be induced at the surface protein level in thioglycolate-elicited peritoneal macrophages by bacterial lipopolysaccharide and interferon-gamma. To investigate this induction further, Northern blotting and enzyme-linked immunosorbent assays were used to examine the role of second messengers in CD11a gene product induction by these agents. Here I report that CD11a RNA and cell surface protein induced by bacterial lipopolysaccharide and tumor necrosis factor-alpha are sensitive to inhibition of protein kinase C, while insensitive to inhibition of Na+/H+ exchange. CD11a induction by interferon-gamma conversely is sensitive to inhibition of Na+/H+ exchange and insensitive to inhibition of protein kinase C. These observations indicate that CD11a may be induced by multiple and separate second messenger systems in primary macrophages.

Amiloride↗

PubMed1999

Protein kinase C activation increases binding of transcription factor PU.1 in murine tissue macrophages.

PU.1 is a transcription factor found in macrophages, B cells, neutrophils, and hemopoietic stem cells. In macrophages PU.1 regulates a number of genes, including c-fms, CD11b, CD18, and FcgammaR1b. Previously, in primary macrophages PU.1 binding to the sequence GAGGAA was found to be induced by treatment with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Here we investigated the role of protein kinase C (pKC) in the induction of PU.1 binding in macrophages. We report that pharmacological activation of pKC increases PU.1 binding, while inactivation of pKC inhibits the increases in PU.1 binding by agents which activate pKC in macrophages (LPS and tumor necrosis factor-alpha), but not by an agent which does not activate pKC (IFN-gamma). pKC activation may therefore be one pathway by which PU.1 binding may be increased in primary macrophages.

Animals↗

PubMed1999

Cell cycle control, checkpoint mechanisms, and genotoxic stress.

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling.

Animals↗

PubMed1998

Maleylated-BSA enhances production of nitric oxide from macrophages.

Maleylated-bovine serum albumin (maleyl-BSA) elicits transcription and secretion of a number of proinflammatory genes via ligation of the low-affinity scavenger receptor (SR) on macrophages. We now demonstrate that while neither maleyl-BSA, nor interferon-gamma (INF-gamma) alone induce nitric oxide (NO) production, when combined they promote release of NO from murine peritoneal macrophages. This effect was blocked by treatment with oxidized-low density lipoprotein. Maleyl-BSA activated NF-kappaB dimers capable of binding the NF-kappaBd sequence unique to the iNOS promoter, but this failed to induce significant new transcription or accumulation of iNOS mRNA. The combination of maleyl-BSA and IFN-gamma failed to demonstrate synergy at the transcriptional or mRNA levels, as these levels were comparable to those elicited by IFN-gamma alone. These studies suggest that the synergy in NO production between maleyl-BSA and IFN-gamma occurs after the accumulation of iNOS-specific mRNA, possibly at the translational or post-translational level.

Animals↗

PubMed1997

Tumor necrosis factor-alpha induces c-fms RNA expression in murine tissue macrophages.

The macrophage-colony stimulating factor receptor, c-fms, is induced by IL-2 in monocytes and is suppressed by LPS, LPS plus IFN-gamma, phorbol esters, and colony stimulating factor in monocytes/macrophages. Here we show that c-fms RNA is induced by treatment of murine tissue macrophages with TNF-alpha. This induction is suppressed by co-treatment with LPS, IFN-gamma, LPS plus IFN-gamma, aspirin, and ox LDL. c-fms expression is required for the proliferation, differentiation, and survival of monocytes/macrophages. Our findings indicate a way by which TNF-alpha, as well as LPS, IFN-gamma, aspirin, and ox LDL impinge upon macrophage biology.

Animals↗

PubMed1997

Aspirin inhibits tumor necrosis factoralpha gene expression in murine tissue macrophages.

Aspirin has been reported to inhibit the activation of nuclear factor-kappaB (NF-kappaB) through stabilization of inhibitor kappaB (IkappaB). This observation led us to investigate the role of aspirin in suppressing the activation of the NF-kappaB-regulated tumor necrosis factor-alpha (TNF-alpha) gene expression in primary macrophages. We now report that therapeutic doses of aspirin suppress lipopolysaccharide-inducible NF-kappaB binding to an NF-kappaB binding site in the TNF-alpha promoter, lipopolysaccharide-induced TNF-alpha mRNA accumulation, and protein secretion. IkappaB is also stabilized under these conditions. The aspirin-initiated stabilization of IkappaB, suppression of induced TNF-alpha mRNA, and NF-kappaB binding to the TNF-alpha promoter are blocked by pretreatment with pertussis toxin. These studies suggest that aspirin may exert significant anti-inflammatory effects by suppressing the production of macrophage-derived inflammatory mediators.

Animals↗

PubMed1996

Maleylated-BSA induces hydrolysis of PIP2, fluxes of Ca2+, NF-kappaB binding, and transcription of the TNF-alpha gene in murine macrophages.

The interaction of altered lipids or proteins with the several scavenger receptors (SR) on macrophages can lead to disparate results in both gene expression and cell function. However, the molecular bases of signaling induced by SR ligation have remained obscure. Here we report that maleylated-bovine serum albumin (maleyl-BSA) binds a low-affinity SR, initiating PIP2 hydrolysis, [Ca2+]i spikes, phospholipase A2 (PLA2) activation, nuclear factor-kappa(B) (NF-kappa(B)) binding to its cognate nucleotide and tumor necrosis factor alpha (TNF-alpha) gene transcription. We recently reported that oxidized low-density lipoprotein (ox-LDL), which binds another macrophage SR, induced pertussis-toxin-sensitive hydrolysis of PIP2 and elevations in [Ca2+]i [J. Biol. Chem. 270, 3475-3478, 1995]. By contrast, maleyl-BSA-initiated events were not pertussis toxin-sensitive and produced less [Ca2+]i spiking than ox-LDL. Furthermore, maleyl-BSA led to binding of NF-kappa(B) to its cognate nucleotide and TNF-alpha gene transcription, whereas ox-LDL suppressed these events. Collectively, this data suggests that maleyl-BSA and ox-LDL bind to distinct SR on murine macrophages, initiate distinct signal transduction pathways, and produce different functional effects.

Albumins↗