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J A Brockman

Publications and source records attributed to J A Brockman.

At least 19 recordsLinked to original sources
PubMed2001

Target genes of peroxisome proliferator-activated receptor gamma in colorectal cancer cells.

Activation of the nuclear hormone peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and promotes differentiation in a broad spectrum of epithelial derived tumor cell lines. Here we utilized microarray technology to identify PPARgamma gene targets in intestinal epithelial cells. For each gene, the induction or repression was seen with two structurally distinct PPARgamma agonists, and the change in expression could be blocked by co-treatment with a specific PPARgamma antagonist. A majority of the genes could be regulated independently by a retinoid X receptor specific agonist. Genes implicated in lipid transport or storage (adipophilin and liver fatty acid-binding protein) were also activated by agonists of PPAR subtypes alpha and/or delta. In contrast, PPARgamma-selective targets included genes linked to growth regulatory pathways (regenerating gene IA), colon epithelial cell maturation (GOB-4 and keratin 20), and immune modulation (neutrophil-gelatinase-associated lipocalin). Additionally, three different genes of the carcinoembryonic antigen family were induced by PPARgamma. Cultured cells treated with PPARgamma ligands demonstrated an increase in Ca(2+)-independent, carcinoembryonic antigen-dependent homotypic aggregation, suggesting a potential role for PPARgamma in regulating intercellular adhesion. Collectively, these results will help define the mechanisms by which PPARgamma regulates intestinal epithelial cell biology.

Animals↗
PubMed1998

Activation of PPARgamma leads to inhibition of anchorage-independent growth of human colorectal cancer cells.

BACKGROUND & AIMS: Peroxisomal proliferator-activated receptor gamma (PPARgamma) is a nuclear hormone receptor that provides a direct link between fatty acid metabolism and control of gene transcription. The objective of this study was to determine the biological effect(s) of PPARgamma activation in colorectal carcinoma cells. METHODS: PPARgamma expression and activity were measured in 4 human colon cancer cell lines using reverse-transcription polymerase chain reaction, immunoprecipitation and immunoblotting, and transient reporter gene assays. The effects of activated PPARgamma in these cell lines were assessed in cellular proliferation and anchorage-independent growth assays. Flow cytometry was used to determine the effects of PPARgamma activation on progression through the cell cycle. RESULTS: PPARgamma was expressed in all 4 colon cancer cell lines examined and was transcriptionally functional in 3 of the 4. Treatment of these cells with a selective PPARgamma activator (BRL 49653) resulted in inhibition of anchorage-independent growth. The degree of growth inhibition correlated with the level of functional PPARgamma present. Finally, activation of PPARgamma resulted in G1 cell cycle arrest. CONCLUSIONS: Activation of the PPARgamma pathway in colon cancer cells has potent antiproliferative effects, suggesting that this nuclear hormone receptor may provide a novel target for prevention and treatment of colorectal cancer in humans.

Cell Cycle↗
PubMed1997

Antioxidant-induced nuclear translocation of CCAAT/enhancer-binding protein beta. A critical role for protein kinase A-mediated phosphorylation of Ser299.

Alterations in intracellular oxidative status activate several signal transduction pathways resulting in distinct patterns of gene expression. Treatment of colorectal cancer cells with antioxidants can lead to apoptosis by induction of p21 through a mechanism involving CCAAT/enhancer-binding protein beta (C/EBPbeta). Herein, we demonstrate that the antioxidant pyrrolidinedithiocarbamate activates cAMP-dependent protein kinase (PKA) in a colorectal cancer cell line DKO-1. Activation of PKA phosphorylates Ser299 within C/EBPbeta, which is essential for protein translocation to the nucleus. Pharmacological inhibition of PKA and mutation of Ser299 to alanine blocks C/EBPbeta nuclear translocation and induction of p21. Our results indicate that a cAMP-dependent phosphorylation of C/EBPbeta at Ser299 is critical for nuclear translocation of this protein and its subsequent transactivation of genes in response to antioxidant treatment.

Antioxidants↗
PubMed1997

Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-kappaB.

Members of the nuclear factor (NF)-kappaB/Rel family transcription factors are induced during thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor (TCR). Despite these findings, disruption of individual NF-kappaB/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy. To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form of IkappaBalpha that constitutively represses the activity of multiple NF-kappaB/Rel proteins. Transgenic cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes. This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4(+) T cells. Taken together, these in vivo findings indicate that the NF-kappaB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.

Animals↗
PubMed1997

Antioxidants enhance the cytotoxicity of chemotherapeutic agents in colorectal cancer: a p53-independent induction of p21WAF1/CIP1 via C/EBPbeta.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Five-fluorouracil (5FU) remains the single most effective treatment for advanced disease, despite a response rate of only 20%. Herein, we show that the antioxidants pyrrolidinedithiocarbamate and vitamin E induce apoptosis in CRC cells. This effect is mediated by induction of p21WAF1/CIP1, a powerful inhibitor of the cell cycle, through a mechanism involving C/EBPbeta (a member of the CCAAT/enhancer binding protein family of transcription factors), independent of p53. Antioxidants significantly enhance CRC tumor growth inhibition by cytotoxic chemotherapy in vitro (5FU and doxorubicin) and in vivo (5FU). Thus, chemotherapeutic agents administered in the presence of antioxidants may provide a novel therapy for colorectal cancer.

Acetylcysteine↗
PubMed1996

Corepression of RelA and c-rel inhibits immunoglobulin kappa gene transcription and rearrangement in precursor B lymphocytes.

Multiple members of the NF-kappa B/Rel protein family are induced during B cell differentiation and have been implicated in transcriptional activation of the immunoglobulin kappa (Ig kappa) locus. Despite these findings, normal numbers of Ig kappa + B lymphocytes are produced by mice bearing targeted mutations in individual NF-kappa B/Rel genes. In the present study, precursor B lymphocytes were engineered to express a trans-dominant form of I kappa B alpha that simultaneously impairs the c-Rel and RelA transactivating subunits of NF-kappa B. This dual block in NF-kappa B/Rel signaling led to potent inhibition of germline Ig kappa transcription and rearrangement, whereas recombinase activity was unaffected. These findings suggest that c-Rel and RelA serve compensatory functional roles in the developmental mechanisms that govern Ig kappa gene assembly.

Animals↗
PubMed1996

Inactivation of IkappaBbeta by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-kappaB.

In resting T lymphocytes, the transcription factor NF-kappaB is sequestered in the cytoplasm via interactions with members of the I kappa B family of inhibitors, including IkappaBalpha and IkappaBbeta. During normal T-cell activation, IkappaBalpha is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional NF-kappaB. In contrast to its transient pattern of nuclear induction during an immune response, NF-kappaB is constitutively activated in cells expressing the Tax transforming protein of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that HTLV-1 Tax targets IkappaBalpha to the ubiquitin-proteasome pathway. However, it remains unclear how this viral protein induces a persistent rather than transient NF-kappaB response. In this report, we provide evidence that in addition to acting on IkappaBalpha, Tax stimulates the turnover Of IkappaBbeta via a related targeting mechanism. Like IkappaBalpha, Tax-mediated breakdown of IkappaBbeta in transfected T lymphocytes is blocked either by cell-permeable proteasome inhibitors or by mutation Of IkappaBbeta at two serine residues present within its N-terminal region. Despite the dual specificity of HTLV-1 Tax for IkappaBalpha and IkappaBbeta at the protein level, Tax selectively stimulates NF-kappaB-directed transcription of the IkappaBalpha gene. Consequently, IkappaBbeta protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with IkappaBbeta provide a potential mechanism for the constitutive activation of NF-kappaB in Tax-expressing cells.

Amino Acid Sequence↗
PubMed1995

Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination.

The inhibitor protein I kappa B alpha controls the nuclear import of the transcription factor NF-kappa B. The inhibitory activity of I kappa B alpha is regulated from the cytoplasmic compartment by signal-induced proteolysis. Previous studies have shown that signal-dependent phosphorylation of serine residues 32 and 36 targets I kappa B alpha to the ubiquitin-proteasome pathway. Here we provide evidence that lysine residues 21 and 22 serve as the primary sites for signal-induced ubiquitination of I kappa B alpha. Conservative Lys-->Arg substitutions at both Lys-21 and Lys-22 produce dominant-negative mutants of I kappa B alpha in vivo. These constitutive inhibitors are appropriately phosphorylated but fail to release NF-kappa B in response to multiple inducers, including viral proteins, cytokines, and agents that mimic antigenic stimulation through the T-cell receptor. Moreover, these Lys-->Arg mutations prevent signal-dependent degradation of I kappa B alpha in vivo and ubiquitin conjugation in vitro. We conclude that site-specific ubiquitination of phosphorylated I kappa B alpha at Lys-21 and/or Lys-22 is an obligatory step in the activation of NF-kappa B.

Arginine↗
PubMed1995

Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation.

The eukaryotic transcription factor NF-kappa B plays a central role in the induced expression of human immunodeficiency virus type 1 and in many aspects of the genetic program mediating normal T-cell activation and growth. The nuclear activity of NF-kappa B is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called I kappa B alpha. This cytoplasmic inhibitor is rapidly phosphorylated and degraded in response to a diverse set of NF-kappa B-inducing agents, including T-cell mitogens, proinflammatory cytokines, and viral transactivators such as the Tax protein of human T-cell leukemia virus type 1. To explore these I kappa B alpha-dependent mechanisms for NF-kappa B induction, we identified novel mutants of I kappa B alpha that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of I kappa B alpha failed to disrupt its ability to form latent complexes with NF-kappa B in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of I kappa B alpha from NF-kappa B in Tax-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of I kappa B alpha also yielded constitutive repressors that escaped from Tax-induced turnover and that potently inhibited immune activation pathways for NF-kappa B induction, including those initiated from antigen and cytokine receptors. In contrast, introduction of a phosphoserine mimetic at these sites rectified this functional defect, a finding consistent with a causal linkage between the phosphorylation status and proteolytic stability of this cytoplasmic inhibitor. Together, these in vivo studies define a critical signal response domain in I kappa B alpha that coordinately controls the biologic activities of I kappa B alpha and NF-kappa B in response to viral and immune stimuli.

Base Sequence↗
PubMed1994

Transcription of the murine class II Eb gene is regulated primarily at the level of transcriptional initiation.

The constitutive and inducible expression of MHC class II genes is known to be regulated by multiple upstream promoter elements. Transient transfection assays implicate the proximal promoter region as both necessary and sufficient for appropriate tissue-specific and inducible expression. However, transgenic mouse experiments suggest that additional control regions are important. In this study we tested the hypothesis that additional regulation of class II expression occurred at the level of RNA polymerase II elongation. Nuclear run-on analysis using single-stranded probes spanning the entire Eb gene was performed on a variety of class II-positive, class II-negative, and class II-inducible cell lines. The results demonstrate that, while there is not an even distribution of RNA polymerase along the gene, there is no evidence for a regulated block in elongation. These data further support the idea that the primary mechanism of class II gene regulation is at the level of transcriptional initiation.

Animals↗
PubMed1994

Cellular mechanisms mediating the stimulation of ovine endometrial secretion of prostaglandin F2 alpha in response to oxytocin: role of phospholipase C and diacylglycerol.

The first objective was to describe and evaluate the relationship between the ability of oxytocin to stimulate the activity of phospholipase (PL) C and its ability to stimulate the release of prostaglandin (PG) F2 alpha in ovine endometrial tissue. Caruncular endometrial tissue was collected from ovariectomized ewes after completion of an 11-day steroid replacement protocol. In experiment 1, explants were incubated either in the presence (10(-6) M) or absence of oxytocin for 0, 1, 3, 10, 30 or 100 min to examine the time-course for activation of PLC and release of PGF2 alpha in response to oxytocin. An increase in the activity of PLC was detected at 3 min while an increase in the release of PGF2 alpha was not detected until 10 min (P < 0.05). In experiment 2, explants were incubated in the presence of various oxytocin analogues (10(-6) M) to compare their abilities to activate PLC and release PGF2 alpha. Oxytocin and three receptor agonists stimulated the activity of PLC and the release of PGF2 alpha (P < 0.05) while two oxytocin receptor antagonists had no effect on either response. In experiment 3, explants were incubated in the presence of oxytocin or arginine vasopressin at 10(-9) to 10(-6) M to establish dose-response curves for the activation of PLC and release of PGF2 alpha. For both hormones, significant increases (P < 0.05) in the release of PGF2 alpha were observed at 10(-8) M while increases in PLC activity were not detected until 10(-7) M was used. In experiment 4, explants were pretreated with either U-73122 (an inhibitor of PLC activity) or U-73343 (an inactive analogue of U-73122). Explants were then treated with control medium, oxytocin or AlF4-. Both oxytocin and AlF4-stimulated the activity of PLC and the release of PGF2 alpha (P < 0.05). U-73122 blocked the ability of oxytocin to stimulate the release of PGF2 alpha (P < 0.05) but had no effect on its ability to stimulate the activity of PLC (P > 0.1). Based on the results from these experiments, the role of PLC in mediating the stimulatory effect of oxytocin on the release of PGF2 alpha remains unclear. The second objective was to evaluate the role of diacylglycerol (DAG) in mediating the stimulatory effect of oxytocin on endometrial secretion of PGF2 alpha. In experiment 5, explants were incubated in vitro with varying doses of two DAG analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Preparation of triazolo[1,5-c]pyrimidines as potential antiasthma agents.

With the use of the human basophil histamine release assay, 5-aryl-2-amino[1,2,4]triazolo[1,5-c]pyrimidines were found to be active as mediator release inhibitors. These compounds were prepared by reacting arylamidines with sodium ethyl formylacetate or with ethyl propiolate to give pyrimidinones. Treatment with phosphorus oxychloride gave a chloropyrimidine, which was converted to a hydrazinopyrimidine with hydrazine. Cyclization, using cyanogen bromide, gave the triazolo[1,5-c]pyrimidines, after a Dimroth rearrangement. Following a structure-activity evaluation, the 5-[3-(trifluoromethyl)phenyl]-2-amino (8-10), 5-(3-bromophenyl)-2-amino (8-13), 5-[3-(difluoromethoxy)-phenyl]-2-amino (8-11), and 5-(4-pyridinyl)-2-amino (6-7) compounds were found to have the best activity. They were chosen for further pharmacological and toxicological study.

Animals↗
PubMed1985

Imidazo[1,5-d][1,2,4]triazines as potential antiasthma agents.

By using inhibition of histamine release from antigen-challenged, sensitized human basophils as a means of identifying a potentially prophylactic drug for the treatment of asthma, a series of substituted imidazo[1,5-d][1,2,4]triazines were found, which were active. These compounds were prepared by treating imidazolecarboxaldehydes with excess Grignard agent and then oxidizing the resulting alcohols to ketones with Jones reagent. Pyrolysis of a mixture of ketone and methyl carbazate at 200 degrees C in diphenyl ether produced the desired imidazo[1,5-d][1,2,4]triazines. Those compounds with the greatest basophil activity were tested for in vivo activity in the mouse passive cutaneous anaphylaxis (PCA) and the guinea pig passive anaphylaxis tests. The best compounds, 1-ethyl-8-methyl-6-propylimidazo[1,5-d][1,2,4]triazin-4(3H)- one (4-17) and 1,8-dimethyl-6-propylimidazo[1,5-d][1,2,4]triazin-4-(3H)-one (4-16) were chosen for further study.

Anaphylaxis↗
PubMed1982

Lymphokine-induced uptake of [14C]glucosamine, [14C]glucose, and [3H]leucine by macrophages.

Lymphokine-activated (LK+) and control (LK-) macrophages were cultured for 66 h and then pulsed with [14C]glucosamine. Uptake of [14C]glucosamine was greater in LK+ than in LK- cultures. If, after 66 h, the medium was replaced with fresh medium and then pulsed with either [14C]glucose or [14C]glucosamine, the uptake of isotope was greatly reduced compared to cultures with no change of medium. However, uptake of both radiolabeled substances was still found to be greater in LK+ cultures than in LK- cultures. Although uptake of both substances was enhanced by lymphokines, the uptake kinetics of each isotope was different. Under similar conditions the uptake of [3H]leucine was not enhanced by lymphokine activation. These data are interpreted to mean that LK+ macrophages are metabolically stimulated and utilize more glucose and glucosamine. The difference in kinetics implies a different utilization by macrophages for each substance.

Animals↗
PubMed1982

Effects of iodipamide on human C3 and factor B in vitro.

The effects of iodipamide on C3 and factor B in normal human serum and in purified form have been examined by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Temperature-dependent changes in immunoelectrophoretic profiles have been observed; however, these are not the same as those obtained after treatment of normal human serum (NHS) with cobra venom factor Naja naja. Analyses of iodipamide-treated NHS and purified C3 and factor B by reducing SDS-PAGE indicate that no macromolecular changes have occurred in C3 and factor B that can be ascribed to proteolysis (i.e., activation). The changes observed in C3 and factor B, including loss of hemolytic activity, appear to be due to direct interactions between iodipamide and C3 and factor B. In the case of factor B, iodipamide treatment at 37 degrees C induces aggregation, which is reversible upon reduction with beta-mercaptoethanol.

Complement Activation↗
PubMed1981

Systematic discovery and evaluation of complement inhibitors.

Methods are presented for an orderly search of a chemical file for complement inhibitors. Compounds are initially examined for intrinsic activity against dilute human components in vitro, using hemolytic assays to detect inhibitors of fluid phase C1, of late components lysis of EAC142, and of CVF-induced passive lysis of AET-treated human erythrocytes. Active compounds are then examined for activity against undiluted serum in vitro. Compounds passing this test are examined for activity in vivo against serum complement and complement-dependent lesions, viz. Forssman vasculitis, the reverse passive Arthus phenomenon, and Forssman shock. Methods are given for quantitation of these lesions.

Animals↗