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M K Offermann

Publications and source records attributed to M K Offermann.

34 records · Page 2Linked to original sources
PubMed1996

Transfection of IFNalpha in human glioblastoma cells and tumorigenicity in association with induction of PKR and OAS gene expression.

Previously these authors and others demonstrated frequent homozygous deletions of the chromosome 9p-localized class I interferon (IFN) gene cluster in glioblastoma tumors and cell lines. To investigate the biological effects of class I IFN gene transfer and constitutive expression in glioblastoma cells devoid of this gene cluster, the authors have developed a stable IFNalpha "transfectant" of the cell line U118. The expression of IFNalpha protein in the U118 transfectant clone is associated with decreased levels of DNA synthesis exhibited by cultures of transfected cells, reduced colony-forming ability in soft agar, and loss of tumorigenicity in athymic nude mice. To address the molecular consequences of constitutive IFNalpha synthesis, they examined the expression of four genes whose transcription has been shown to be responsive to IFN-mediated signal transduction and could be important to the observed antiproliferative and antitumor effects. Northern blot analysis revealed that changes in the levels of messenger (m)RNA for two of these genes, c-myc and mhc class I, are minor. However, mRNAs for oligoadenylate synthetase (OAS) as well as double-stranded RNA-activated protein kinase (PKR), which are not expressed in parental U118 cells, were constitutively expressed in IFNalpha transfectants. These results indicate a differential responsiveness among these four genes to constitutive IFNalpha expression, and suggest that the suppression of U118-transformed phenotypes by IFNalpha transfection may be mediated by the induction of specific IFN response genes thought to have a negative growth-regulatory function.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Activation of the double-stranded-RNA-activated protein kinase and induction of vascular cell adhesion molecule-1 by poly (I).poly (C) in endothelial cells.

Double-stranded RNA (dsRNA) induces the vascular cell adhesion molecule VCAM-1 to high levels of expression in human umbilical vein endothelial (HUVE) cells. Although VCAM-1 is also induced by the cytokine interleukin 1 beta (IL-1 beta), activation of the dsRNA-activated protein kinase (PKR) occurs only in response to incubation with dsRNA but not with IL-1 beta. Incubation of HUVE cells with the synthetic dsRNA, poly (I).poly (C), activates PKR with increased autophosphorylation, increased phosphorylation of the translation factor eIF2 alpha, and increased activation of the transcription factor NF-kappa B. Promoter analysis in HUVE cells using a VCAM-1 promoter linked to CAT reporter gene demonstrates that poly (I).poly (C) responsiveness resides in the minimal VCAM-1 promoter that contains two NF-kappa B sites, and deletion of the NF-kappa B sites eliminates basal and poly (I).poly (C)-induced CAT activity, supporting the importance of NF-kappa B in the poly (I).poly (C)-mediated induction of VCAM-1. In vitro studies using purified reagents demonstrate that PKR is capable of phosphorylating I kappa B alpha (the inhibitory subunit of NF-kappa B) in a dsRNA-dependent manner. This suggests that phosphorylation of I kappa B alpha by PKR could be an initial step in the activation of NF-kappa B by dsRNA. NF-kappa B is also activated by IL-1 beta in HUVE cells, but this activation occurs without increased PKR autophosphorylation or eIF2 alpha phosphorylation. Poly (I).poly (C) induces VCAM-1 mRNA levels that are dramatically higher and sustained longer than levels induced by IL-1 beta. Although phosphorylation of eIF2 alpha interferes with protein translation, sufficient VCAM-1 mRNA translation occurs in response to poly (I).poly (C) to yield VCAM-1 protein levels that are similar to levels that are induced by IL-1 beta. This suggests that the higher, sustained VCAM-1 mRNA levels that occur in response to incubation with poly (I).poly (C) compensate for the partial translational block resulting from increased eIF2 alpha phosphorylation. These studies indicate that transcriptional and translational regulatory events that occur in response to activation of PKR by dsRNA are important in the regulation of VCAM-1 gene expression in HUVE cells.

Cell Adhesion↗
PubMed1995

Double-stranded RNA induces sickle erythrocyte adherence to endothelium: a potential role for viral infection in vaso-occlusive pain episodes in sickle cell anemia.

Vaso-occlusive pain episodes in sickle cell anemia are hypothesized to be precipitated by adherence of sickle erythrocytes to vascular endothelium in the microcirculation. Febrile episodes, thought to be viral in etiology, are frequently associated with vaso-occlusion; however, a direct link between viral infection and vascular occlusion has not yet been established. Many pathogenic viruses contain double-stranded RNA or replicate through double-stranded RNA intermediates. Double-stranded RNA has been shown to induce vascular cell adhesion molecule-1 (VCAM-1) protein expression on endothelial cells. Recently, a new adhesion pathway has been described between VCAM-1 expressed on cytokine stimulated endothelium and the alpha 4 beta 1 integrin complex expressed on sickle reticulocytes. Based on these observations, the hypothesis was developed that viral infection, through double-stranded RNA intermediates, increases endothelial VCAM-1 expression leading to sickle erythrocyte adhesion to endothelium via an alpha 4 beta 1-VCAM-1--dependent mechanism. In support of this hypothesis, endothelial cells exposed to the synthetic double-stranded RNA poly(I:C) or the RNA virus parainfluenza 1 (Sendai virus) express increased levels of VCAM-1 and support increased sickle erythrocyte adherence under continuous flow at 1.0 dyne/cm2 shear stress as compared with unstimulated endothelium. Blocking antibodies directed against either VCAM-1 on the endothelium or alpha 4 beta 1 on sickle erythrocytes inhibit nearly all of the increased sickle cell adherence caused by poly(I:C) or Sendai virus. These results support the hypothesis that viruses, through double-stranded RNA elements, can induce sickle erythrocyte adherence to endothelium through alpha 4 beta 1-VCAM-1--mediated adhesion and provide a potential link between viral infection and microvascular occlusion precipitating sickle cell pain episodes.

Anemia, Sickle Cell↗
PubMed1994

Induction of IL-6 gene expression in Kaposi's sarcoma cells.

IL-6 is a multifunctional cytokine that functions as an autocrine growth factor for AIDS-derived Kaposi's sarcoma (KS) cells. We report that IL-6 is highly inducible at both the mRNA and protein levels in cultured KS cells by multiple agents, yet the effect of the IL-6 on the proliferation of KS cells is dependent on the agent responsible for its induction. Both IL-1 beta and the synthetic dsRNA, poly (I:C), induced high levels of IL-6 mRNA and protein expression, whereas LPS and TNF-alpha led to only modest increases in IL-6 protein and mRNA. When KS cells were incubated with poly (I:C) in combination with either IL-1 beta or TNF-alpha, there was a synergistic increase in the level of IL-6 production, whereas LPS and TNF-alpha in combination led to only an additive increase in the level of IL-6 production. Exogenous IL-6 was shown to induce proliferation in KS cells, yet there was a dramatic inhibition of proliferation in response to poly (I:C), despite the high levels of IL-6 produced. This inhibition of proliferation by poly (I:C) was unlikely as a result of expression of class I IFN in response to the poly (I:C) because high concentrations of exogenous IFN-alpha had no demonstrable effect on [3H]TdR incorporation under conditions in which poly (I:C) caused a 90% decrease in [3H]TdR incorporation. Pretreatment of KS cells with poly (I:C) for 24 h followed by removal of the poly (I:C) led to high levels of IL-6 secreted into medium that induced proliferation in KS cells. These data suggest that in vivo, multiple agents that occur in response to infection and systemic disease could induce IL-6 production by KS cells, yet the ability of the IL-6 to influence proliferation of KS cells is dependent on the context in which the IL-6 is induced.

Cell Division↗
PubMed1994

Regulation of adhesion molecule expression in Kaposi's sarcoma cells.

Kaposi's sarcoma (KS) is a neoplasm with multifocal vascular lesions that is often seen in homosexual HIV-infected individuals. Infiltrates of leukocytes are characteristic components of KS lesions, and the products of leukocytes have been shown to enhance the proliferation of KS cells in vitro and most likely are crucial for the development of KS lesions in vivo. It is therefore likely that the expression of cellular adhesion molecules (CAM) is a critical determinant in the pathogenesis of KS by dictating the numbers and types of leukocytes that accumulate in areas predisposed to KS. We report that in the absence of inducers, KS cells in culture expressed low levels of ICAM-1 and undetectable VCAM-1 and E-selectin. ICAM-1, VCAM-1, and E-selectin were all induced by dsRNA (poly (I:C)), IL-1 beta, TNF-alpha, and LPS in KS cells. All of these agents increased NF-kappa B binding activity in nuclear extracts from KS cells. Neither human dermal fibroblasts nor human aortic smooth muscle cells had detectable VCAM-1 protein expression in response to conditions that led to high levels of VCAM-1 expression in KS cells. Although E-selectin expression was induced in KS cells, the peak cell surface protein levels were less than 25% the levels achieved on HUVEC or human dermal microvascular endothelial cells (HMEC). These low levels resembled the levels that were induced in HMEC immortalized with SV 40 large T Ag. These data indicate that multiple proinflammatory agents can induce NF-kappa B binding activity and can enhance ICAM-1, VCAM-1, and E-selectin expression in KS cells. The increased CAM expression enhances leukocyte binding to KS cells. Thus, the induction of CAM expression could be an early event in the development of KS by recruiting leukocytes into KS lesions, thereby providing factors that could potentiate the development of KS.

Base Sequence↗
PubMed1993

Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells.

Oxidative stress and expression of the vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells are early features in the pathogenesis of atherosclerosis and other inflammatory diseases. Regulation of VCAM-1 gene expression may be coupled to oxidative stress through specific reduction-oxidation (redox) sensitive transcriptional or posttranscriptional regulatory factors. In cultured human umbilical vein endothelial (HUVE) cells, the cytokine interleukin 1 beta (IL-1 beta) activated VCAM-1 gene expression through a mechanism that was repressed approximately 90% by the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC). Furthermore, PDTC selectively inhibited the induction of VCAM-1, but not intercellular adhesion molecule-1 (ICAM-1), mRNA and protein accumulation by the cytokine tumor necrosis factor-alpha (TNF alpha) as well as the noncytokines bacterial endotoxin lipopolysaccharide (LPS) and double-stranded RNA, poly(I:C) (PIC). PDTC also markedly attenuated TNF alpha induction of VCAM-1-mediated cellular adhesion. In a distinct pattern, PDTC partially inhibited E-selectin gene expression in response to TNF alpha but not to LPS, IL-1 beta, or PIC. TNF alpha and LPS-mediated transcriptional activation of the human VCAM-1 promoter through NF-kappa B-like DNA enhancer elements and associated NF-kappa B-like DNA binding proteins was inhibited by PDTC. These studies suggest a molecular linkage between an antioxidant sensitive transcriptional regulatory mechanism and VCAM-1 gene expression that expands on the notion of oxidative stress as an important regulatory signal in the pathogenesis of atherosclerosis.

Antioxidants↗
PubMed1990

Effect of cellular density and viral oncogenes on the major histocompatibility complex class I antigen response to gamma-interferon in BALB-c/3T3 cells.

Cellular density in culture has profound effects on major histocompatibility complex (MHC) class I antigen expression in BALB/c-3T3 cells. Cells which have been confluent for greater than 24 h demonstrate a 2- to 6-fold increase in MHC class I antigen expression compared to subconfluent cells. These density-associated changes in MHC class I antigen expression occur both in untransformed and in v-mos or v-rasKi-transformed cells. The density-associated increases are specific for MHC class I antigens and do not occur with the cytoskeletal antigen actin. Transformation of the BALB/c-3T3 cells by either v-rasKi or v-mos has little or no direct effect on MHC class I expression under standard culture conditions. However, both oncogenes can indirectly alter the enhancement of MHC class I antigen expression in response to gamma-interferon. Incubation of untransformed BALB/c-3T3 cells with gamma-interferon leads to greater relative and absolute increases in MHC class I antigen expression in confluent cells than it does in subconfluent cells. In contrast, in v-rasKi- and v-mos-transformed cells, the subconfluent cells have a greater increase in MHC class I antigen expression in response to gamma-interferon than the cells which have exceeded monolayer confluence. The dense v-rasKi- and v-mos-transformed BALB/c-3T3 cell cultures are able to deplete their medium of the exogenous gamma-interferon, and this depletion of gamma-interferon causes the increase in the MHC class I antigen expression to be less sustained with lower peak expressions than the expression found in subconfluent cells. Supplementation with additional gamma-interferon can restore the full enhancement of MHC class I antigen in the transformed BALB/c-3T3 cells. The v-mos- and v-rasKi-transformed cells are more likely to deplete their medium of exogenous gamma-interferon because these cells can exceed monolayer confluence and thus achieve 10-fold higher densities than the untransformed BALB/c-3T3 cells. At high cellular densities, untransformed cells can partially deplete their medium of exogenous gamma-interferon, but this phenomenon is generally less pronounced than in the transformed cells.

Animals↗
PubMed1989

Autocrine induction of major histocompatibility complex class I antigen expression results from induction of beta interferon in oncogene-transformed BALB/c-3T3 cells.

By varying growth conditions, we identified a novel mechanism of autocrine regulation of major histocompatibility complex (MHC) class I gene expression by induction of beta interferon gene expression in transformed BALB/c-3T3 cells. Low-serum conditions enhanced MHC class I antigen expression in v-rasKi- and v-mos-transformed BALB/c-3T3 cells but not in untransformed BALB/c-3T3 cells. Transformed and untransformed cells grown under standard serum conditions (10% bovine calf serum) expressed similar cell surface levels of MHC class I antigens. However, low-serum conditions (0.5% bovine calf serum) induced four- to ninefold increases in cell surface levels of MHC class I antigens in both v-rasKi- and v-mos-transformed cells but not in untransformed cells. These increases in MHC class I gene expression were seen at both the mRNA and cell surface protein levels and involved not only the heavy-chain component of the class I antigens but also beta 2 microglobulin. Beta 1 interferon mRNA and beta interferon-inducible 2',5'-oligoadenylate synthetase mRNA were induced by growth under low-serum conditions in transformed BALB/c-3T3 cells, and antibodies to beta interferon blocked the induction of MHC class I antigen expression by serum deprivation in these cells. These results demonstrate that growth under low-serum conditions leads to induction of beta interferon expression in oncogene-transformed cells which then directly mediates autocrine enhancement of MHC class I gene expression.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Glutathione disulfide inactivates, destabilizes, and enhances proteolytic susceptibility of fructose-1,6-bisphosphate aldolase.

Disulfides (glutathione disulfide, cystine, cystamine) caused a first-order inactivation of rabbit-muscle fructose-1,6-bisphosphate aldolase at pH values of 7.4 and above. Inactivation by glutathione disulfide was partially reversed by reducing agents, but the enzyme became irreversibly inactivated with time. The disulfide-inactivated aldolase had a lower transition temperature and enthalpy of denaturation than the native enzyme. In addition, the disulfide-inactivated enzyme was extensively degraded by proteinases, whereas the native enzyme was resistant. Mixed disulfides were formed; a maximum ratio of 4-5 mol of glutathione/mol of the aldolase tetramer was found. The number of titratable--SH groups on aldolase decreased by 16 (out of 32 total on the control enzyme) after inactivation by glutathione disulfide, indicating that other oxidation reactions in addition to those resulting in mixed disulfides occurred. The substrate, fructose 1,6-bisphosphate, prevented inactivation of aldolase by glutathione disulfide, the formation of glutathione-enzyme mixed disulfides, thermodynamic destabilization of the enzyme, and a decrease of--SH groups on the enzyme. These data indicate that covalent modification of aldolase by biological disulfides is important in modulating enzyme stability and vulnerability to proteinases as well as enzyme activity and that the substrate protects against modification by disulfides.

Animals↗
PubMed1984

Hairy cell leukemia.

Hairy cell leukemia is a malignancy with a variable course that can be relatively indolent or rapidly fatal. Alterations in the immune system are responsible for much of the morbidity and mortality from hairy cell leukemia. More than 60% of patients die from infection, and infections are both pyogenic and nonpyogenic. Many patients have transfusion requirements, and bleeding complications can also occur. Treatment strategies for hairy cell leukemia have evolved and are being modified as more is learned about the disease. Splenectomy is the initial treatment when patients become symptomatic, and if the disease progresses after splenectomy, chlorambucil offers good control in many patients. Radiation can be used for local palliation, as when complications such as bulky adenopathy or bone lesions occur. Initial studies using interferon in the treatment of hairy cell leukemia look encouraging, but more investigation is necessary before the role of interferon in treatment of hairy cell leukemia is determined.

Androgens↗
PubMed1983

Action of human liver cathepsin B on the oxidized insulin B chain.

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.

Binding Sites↗
PubMed1983

Action of cathepsin D on fructose-1,6-bisphosphate aldolase.

Cathepsin D inactivated aldolase at pH values between 4.2 and 5.2; the chloride, sulphate or iodide, but not citrate or acetate, salts of sodium or potassium accelerated the rate of inactivation. Cathepsin D cleaved numerous peptide bonds in the C-terminus of aldolase, but the major site of cleavage in this region was Leu354-Phe355. The most prominent peptide products of hydrolysis were Phe-Ile-Ser-Asn-His-Ala-Tyr and Phe-Ile-Ser-Asn-His. Up to 20 amino acids were removed from the C-terminus of aldolase, but no further degradation of native aldolase was observed. By contrast, extensive degradation of the 40 000-Mr subunit was observed after aldolase was denatured. The cathepsin D-inactivated aldolase cross-reacted with antibodies prepared against native aldolase and had the same thermodynamic stability as native aldolase, demonstrated by differential scanning calorimetry and fluorescence quenching of tryptophan residues. Furthermore, the cathepsin-modified and native forms of aldolase were both resistant to extensive proteolysis by other purified cellular proteinases and lysosomal extracts at pH values of 4.8-8.0.

Amino Acids↗
PubMed1981

Initial events in the degradation of soluble cellular enzymes: factors affecting the stability and proteolytic susceptibility of fructose-1,6-bisphosphate aldolase.

The effects of disulfides (oxidized glutathione or cystine) and of cellular proteinases on rabbit muscle aldolase activity, thermal stability and susceptibility to proteolysis were determined. Native aldolase was reversibly inactivated by cystine and oxidized glutathione. Disulfide-inactivated aldolase had a lower transition temperature and enthalpy for denaturation than the native enzyme and was extensively degraded by lysosomal enzymes or a metallo-proteinase, meprin. Native aldolase was also inactivated by lysosomal enzymes or meprin; this inactivation was due to limited proteolysis in the C-terminus. However, aldolase inactivated by limited proteolysis had the same thermal stability as native aldolase and was resistant to extensive proteolysis by lysosomal enzymes or meprin. These data provide insight into the molecular basis whereby formation of mixed disulfides between proteins and glutathione or cysteine may result in unstable protein conformations and may be an initial event in the process of degradation of soluble cellular enzymes to amino acids and small peptides.

Cathepsins↗
PubMedDate not supplied

Antioxidants and atherosclerosis: a molecular perspective.

Current models of atherogenesis link abnormalities in the oxidative state of the vascular wall with interactions with the immune system, leading to a cycle of localized inflammatory and growth responses that result in the characteristics of the mature atherosclerotic lesion. The oxidative modification of LDL may be an important manifestation and mediator of this process, although the degree to which this contributes to atherogenesis has not been directly assessed. Another important mechanism may involve the linkage of the oxidative state of the vascular endothelial cell, through specific transcriptional regulatory factors, to control the expression of a gene involved in this disease process. This further expands the idea of oxidative stress as an important regulatory signal in the pathogenesis of atherosclerosis and provides important paradigms for the development of novel therapeutic treatment regimens, drug design, and diagnostic assessments of disease state.

Animals↗