Biomedical subjects

O R Colamonici

Publications and source records attributed to O R Colamonici.

At least 37 records · Page 2Linked to original sources

PubMed1995

Cloning and expression of a long form of the beta subunit of the interferon alpha beta receptor that is required for signaling.

The interferon alpha beta receptor (IFN alpha R) or type I IFN-R is formed by a 110-kDa alpha subunit or IFNAR and by a beta subunit, which has short and long forms (molecular masses of 55 and 95-100 kDa, respectively). In this report, we demonstrate that the IFN alpha/beta R cDNA recently cloned corresponds to the 55-kDa or short form of the beta subunit, while the 95-100-kDa species reported here corresponds to a longer form of the IFN alpha/beta R cDNA that is probably produced by alternative splicing of the same gene. Stable transfection of the alpha subunit with either form of the beta subunit results in the expression of low and high affinity receptors, while expression of either form of the beta subunit alone only produces low affinity receptors. More important, only expression of the alpha and long form of the human beta subunits in mouse L-929 cells reconstitutes the activation of the Jak kinases and the Stat factors, as well as the antiviral response to human type I IFNs.

Amino Acid Sequence↗

PubMed1995

Vaccinia virus B18R gene encodes a type I interferon-binding protein that blocks interferon alpha transmembrane signaling.

Poxviruses encode a large number of proteins that attenuate the inflammatory and immune responses to infection. In this report we demonstrate that a number of orthopoxviruses express a type I interferon (IFN)-binding protein, which is encoded by the B18R open reading frame in the WR strain of vaccinia virus. The B18R protein has significant regions of homology with the alpha subunits of the mouse, human, and bovine type I IFN receptors, bound human IFN alpha 2 with high affinity, and inhibited transmembrane signaling as demonstrated by inhibition of Fc receptor factor gamma 1/gamma 2 and interferon-stimulated gene factor-3 formation as well as inhibition of the IFN alpha antiviral response. Among viral host response modifiers, the B18R protein is unique inasmuch as it exists as a soluble extracellular as well as a cell surface protein and thus should effectively block both autocrine and paracrine functions of IFN.

Amino Acid Sequence↗

PubMed1995

Transmembrane signaling by the alpha subunit of the type I interferon receptor is essential for activation of the JAK kinases and the transcriptional factor ISGF3.

The Type I interferon (IFN) receptor has a multisubunit structure. The component of the receptor that has been most thoroughly studied is the alpha subunit. Expression of the alpha subunit in mouse L-929 cells confers antiviral response to human IFN alpha 8, but not to human IFN alpha 2 or IFN beta. This antiviral effect is observed without a significant increase in IFN binding. It has not been determined why mouse cells expressing the human alpha subunit show different response to the antiviral activity of distinct human Type I IFNs. In this report, we demonstrate that the response to human Type I IFNs in mouse cells expressing the alpha subunit is dependent on cross-binding to the mouse receptor. This is supported by the finding that human IFN alpha 8, but not human IFN alpha 2, cross-binds to the mouse receptor even in the absence of expression of the human alpha subunit. We also demonstrate that only mouse cells expressing the human alpha subunit are able to tyrosine-phosphorylate p135tyk2 and JAK-1 and to form the ISGF3 complex in response to human IFN alpha 8. These results demonstrate that the alpha subunit is essential for IFN alpha signaling through the JAK kinases and ISGF3.

Animals↗

PubMed1994

Ligand-independent anti-oncogenic activity of the alpha subunit of the type I interferon receptor.

Two interferon (IFN) alpha-regulated genes, IRF1/ISGF2 and PKR/p68 kinase, may function as tumor suppressor genes suggesting that the IFN system may function as a tumor suppressor system. We report that the expression of the alpha subunit of the type I IFN receptor in human K-562 cells had anti-oncogenic effects that include a marked decrease in: (i) cell proliferation rate, (ii) the cell density at which growth arrest normally occurs, and (iii) the tumorigenicity in nude mice. Furthermore, expression of the alpha subunit in K-562 cells induced erythroid differentiation. While most cytokine receptors become activated after binding their corresponding ligands, the overexpression of the alpha subunit has a physiological effect in the absence of its natural ligand, type I IFNs, suggesting a novel function for this type I IFN receptor subunit. The anti-oncogenic effect of the alpha subunit is mediated by a pathway that does not involve two tumor suppressor genes induced by type I IFNs, the transcriptional regulator IFN response factor-1 and the RNA-dependent protein kinase, or the p135tyk2 tyrosine kinase that directly associates and phosphorylates the alpha subunit.

Animals↗

PubMed1994

Tyrosine phosphorylation of the alpha and beta subunits of the type I interferon receptor. Interferon-beta selectively induces tyrosine phosphorylation of an alpha subunit-associated protein.

We studied the phosphorylation of the alpha and beta subunits of the Type I interferon (IFN) receptor induced by Type I IFNs in the human U-266 and MOLT-4 cell lines. Both IFN-alpha and IFN-beta induced tyrosine phosphorylation of the beta subunit of the receptor. The Type I IFN-induced tyrosine phosphorylation of the beta subunit was rapid and transient, being detectable within 1 min of Type I IFN treatment and gradually diminishing to almost base-line levels by 60 min. All Type I IFNs studied were found to induce tyrosine phosphorylation of the alpha subunit of the Type I IFN receptor, the p135tyk2 and JAK-1 tyrosine kinases, and the ISGF3 alpha components. Interestingly, IFN-beta, but not IFN-alpha or IFN-omega, induced tyrosine phosphorylation of an alpha subunit-associated protein with an apparent molecular mass of approximately 100 kDa (p100). These data suggest the existence of a common signaling pathway(s) for Type I IFNs involving the alpha and beta subunits of the receptor, the tyrosine kinases p135tyk2 and JAK-1, and the ISGF3 alpha components. However, differences between the signaling pathways of different Type I IFNs exist, as suggested by tyrosine phosphorylation of an alpha subunit-associated protein only in response to IFN-beta.

Humans↗

PubMed1994

Complementation of the interferon alpha response in resistant cells by expression of the cloned subunit of the interferon alpha receptor. A central role of this subunit in interferon alpha signaling.

A subunit of the interferon alpha receptor (IFN alpha R) that confers biologic response to and specific "binding" for IFN alpha 8 has recently been cloned. We have explored the biological consequences of expressing the cloned IFN alpha R subunit in human cells resistant to IFN alpha and in mouse cell lines nonresponsive to human IFN alpha. The expression of the cloned IFN alpha R subunit in the human IFN alpha-resistant K-562 cell line restored sensitivity to the antiviral effect of not only IFN alpha 8 but also IFN alpha 2 and IFN alpha Con1. In mouse L-929 cells the expression of the cloned receptor subunit markedly increased antiviral sensitivity to human type I IFNs. In either human K-562 or mouse L-929 cells these effects were observed without a detectable increase in the binding for any of the subtypes of IFN alpha tested. We propose that the cloned IFN alpha R subunit functions as a transducer subunit for the IFN alpha R. This concept is supported by the finding that the cloned receptor protein, when it is expressed in Cos cells, has an M(r) of 75 kDa, which is different from the main IFN alpha-binding proteins, the alpha and beta subunits of the IFN alpha R. This report also suggests that alterations at the receptor level could be involved in IFN alpha resistance in some cell lines.

Animals↗

PubMed1994

Interferon alpha (IFN alpha) signaling in cells expressing the variant form of the type I IFN receptor.

Two different Type I interferon receptors (IFN-R) have been described: the normal and the variant receptors. The alpha subunit of the Type I IFN-R has a molecular mass of 110 kDa in cells expressing normal and variant receptors. The beta subunit has a molecular mass of approximately 100 kDa in cells that express normal receptors and 55 kDa in cells expressing the variant form of the receptor. The IFN alpha-resistant U-937 cell line expresses variant receptors and fails to down-regulate and phosphorylate the alpha subunit on tyrosine residues. We report that two other myelomonocytic cell lines, YK-M2 and ML-2, also expressing the variant form of the receptor, fail to down-regulate and phosphorylate the alpha subunit on tyrosine residues. However, YK-M2 and ML-2 cells are sensitive to the antiproliferative and antiviral effects of IFN alpha 2, indicating that phosphorylation of the alpha subunit is not necessary to elicit an IFN alpha response and that expression of variant receptors is not a source of IFN alpha resistance. We also determined if other proteins involved in the IFN alpha signal transduction pathway had a different phosphorylation pattern. Treatment of cells expressing variant receptors induced tyrosine phosphorylation of the p135tyk2 tyrosine kinase, and the three interferon-stimulated gene factor 3 alpha (ISGF3 alpha) polypeptides (p113, p91, and p84), albeit at lower levels. These results indicate that cells expressing either form of the Type I IFN-R phosphorylate a similar set of proteins, with the exception of the alpha subunit.

Base Sequence↗

PubMed1994

p135tyk2, an interferon-alpha-activated tyrosine kinase, is physically associated with an interferon-alpha receptor.

Recent genetic studies have linked the tyk2 gene, which encodes a novel type of non-receptor tyrosine kinase, to the interferon-alpha intracellular signaling pathway. In this report, biochemical evidence is presented which supports this proposed function for the tyk2 tyrosine kinase and further defines its role in the interferon-alpha signaling cascade. Specifically, the tyk2 gene is shown to encode a 135-kDa protein which is rapidly phosphorylated on tyrosine in response to interferon-alpha treatment. Indirect evidence suggests that the tyrosine phosphorylation of p135tyk2 is the result of autokinase activity, implying that the Tyk2 tyrosine kinase is activated by interferon-alpha treatment. Two complementary methods demonstrate a physical association between p135tyk2 and the alpha-subunit of the interferon-alpha receptor. First, immunoblots show that monoclonal antibodies against the alpha-subunit of the interferon-alpha receptor can co-immunoprecipitate p135tyk2. Second, interferon-alpha receptor proteins which have been labeled by affinity cross-linking with 125I-interferon-alpha 2 can be co-immunoprecipitated using anti-tyk2 antisera. Taken together, these data suggest that an interferon-alpha receptor-p135tyk2 complex functions, in a manner analogous to the CD4-lck tyrosine kinase complex, to initiate the interferon-alpha signaling cascade.

Animals↗

PubMed1993

Identification of a novel subunit of the type I interferon receptor localized to human chromosome 21.

Expression in mouse cells of the cloned human IFN alpha receptor (IFN alpha R) subunit selectively confers response and binding to human IFN alpha 8, indicating that other subunits are involved in IFN alpha binding. We report here that a new monoclonal antibody (mAb), termed IFNaR beta 1, recognizes a novel IFN alpha R subunit different from the one recently cloned and distinct from the alpha subunit recognized by the IFN alpha R3 mAb. The IFNaR beta 1 mAb blocks the biological effect of seven different Type I IFNs. Immunoprecipitations after cell surface iodination demonstrate that the IFNaR beta 1 mAb recognizes a protein with a molecular mass of 100 kDa in Daudi and U-266 cells that express normal IFN alpha R. However, a 55-kDa protein instead of the 100-kDa product was immunoprecipitated in the IFN alpha-resistant U-937 cell line that express the variant form of the receptor. We also demonstrate that the gene that codes for this novel IFN alpha R subunit maps to human chromosome 21, as do the cloned IFN alpha R subunit and the alpha subunit, indicating the existence of a locus on this chromosome that regulates binding for Type I IFNs.

Animals↗

PubMed1993

Characterization of the alpha subunit of the IFN-alpha receptor. Evidence of N- and O-linked glycosylation and association with other surface proteins.

We studied the association of the alpha subunit of the (IFN-alpha-receptor) to other receptor components in the human H-929 and U-266 myeloma cell lines. Immunoprecipitation performed with the IFNaR3 mAb showed that two proteins with molecular masses of 205 and 145 kDa are co-precipitated with the alpha subunit. These complexes may not bind IFN-alpha as shown by studies using the heterobifunctional cross-linking reagent Denny-Jaffe and by partial cleavage of the homobifunctional cross-linker dithio succinimidyl propionate. These studies also provided evidence that at least two subunits with molecular mass of 130 kDa (alpha subunit) and 110 kDa (including 20 kDa corresponding to IFN-alpha) contribute to the formation of the IFN-alpha-receptor complex. To further characterize the alpha subunit of the IFN-alpha-receptor, immunoprecipitates using the mAb IFNaR3 were sequentially treated with N-glycanase, neuraminidase and O-glycanase. These studies showed that the alpha subunit is heavily glycosylated and has a protein precursor with a molecular mass of 68 kDa. Binding studies provided evidence for high and low affinity binding sites for IFN-alpha 2. Affinity cross-linking experiments under low and high affinity conditions suggest that the high affinity binding site of the IFN-alpha-receptor is formed by a complex containing the alpha subunit, whereas the 110-kDa subunit may bind IFN-alpha 2 under low affinity conditions.

Binding Sites↗

PubMed1993

Direct association of interleukin-6 with a 130-kDa component of the interleukin-6 receptor system.

Affinity cross-linking of membrane bound 125I-interleukin-6 (IL-6) on several cell lines revealed a three-band pattern of IL-6-containing cross-linked complexes with molecular masses of 100, 120, and 150 kDa. To identify the membrane components that were associated with IL-6 in the three complexes, we employed the Denny-Jaffe reagent, a heterobifunctional, cleavable cross-linker that allows the transfer of 125I from the ligand to its receptor. Samples cross-linked with Denny-Jaffe reagent were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis in which the cross-linker was cleaved prior to the second dimension. This analysis revealed that IL-6 directly associates with a 130-kDa membrane protein thus allowing the formation of the 150-kDa complex. In addition, both the 100- and 120-kDa cross-linked complexes were shown to include an 80-kDa membrane glycoprotein associated with one and two IL-6 molecules, respectively.

Animals↗

PubMed1992

Interferon alpha induces rapid tyrosine phosphorylation of the alpha subunit of its receptor.

The mechanisms of generation of second messengers after binding of interferon alpha (IFN alpha) to its receptor remain unknown. We have studied the phosphorylation of the alpha subunit of the IFN alpha receptor, which is recognized by the monoclonal antibody IFNa receptor 3. Immunoblotting experiments showed that IFN alpha induced rapid tyrosine phosphorylation of the alpha subunit in the IFN alpha-sensitive H-929, U-266, and Daudi cell lines. Immunoprecipitation experiments performed with 32P-labeled cells showed that the alpha subunit is phosphorylated before IFN alpha treatment and that the level of phosphorylation increases after IFN alpha stimulation. Phosphoamino acid analysis confirmed the IFN alpha-induced tyrosine phosphorylation and demonstrated that the base-line phosphorylation corresponded to serine phosphorylation that increased 50% upon IFN alpha treatment. Tyrosine phosphorylation of the alpha subunit was time- and dose-dependent, further demonstrating the specificity of the process. Phosphorylation of the alpha subunit of the receptor occurred rapidly after IFN alpha binding, both at 37 and 4 degrees C. Exposure of the cells to the tyrosine kinase inhibitor genistein blocked the IFN alpha-induced tyrosine phosphorylation of this subunit of the IFN alpha receptor. In contrast H7, a specific protein kinase C inhibitor, and acute and chronic exposure to phorbol esters had no effect on tyrosine phosphorylation, suggesting that protein kinase C does not regulate the tyrosine phosphorylation of the alpha subunit of the IFN alpha receptor. No IFN alpha-induced tyrosine phosphorylation was observed in the IFN alpha-resistant U-937 cell line that expresses a variant IFN alpha receptor. Altogether these data suggest that tyrosine phosphorylation of the alpha subunit may play a role in the signal transduction pathway of IFN alpha.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Correlation between interferon (IFN) alpha resistance and deletion of the IFN alpha/beta genes in acute leukemia cell lines suggests selection against the IFN system.

Homozygous and hemizygous deletions of the interferon A (IFNA) and IFNB genes have been frequently observed in acute leukemia cell lines, primary acute leukemia cases, and gliomas. Because IFNs have an antiproliferative effect, selection against the IFN alpha/beta system could play a role or accompany the development of the malignant phenotype. Although the deletion of the IFNA/B genes could interrupt an autocrine loop that controls cell proliferation, cells would still respond to exogenous IFN alpha/beta and, thus, lesions at the receptor or signal transduction level should also be present to render cells resistant to exogenous IFN alpha/beta. To test if selection against the IFN system was operating in acute leukemias, the sensitivity to the antiproliferative effect of IFN alpha 2 was studied in acute leukemia cell lines with and without alterations of the IFNA/B genes. We found that 10 of 11 acute leukemia cell lines with alterations of the IFNA/B genes were resistant to the antiproliferative effect of IFN alpha 2, whereas only two of eight cell lines with normal IFNA/B genes were IFN-resistant. We then examined the possibility that an alteration of the receptor expression could account for the lack of response to IFN alpha 2. No significant alteration in the expression or structure of the IFN alpha receptor was observed. We also studied the downmodulation of the alpha subunit of the IFN alpha receptor upon IFN alpha 2 binding. One cell line with deletion of the IFNA/B genes showed impaired downmodulation of the IFN alpha receptor. The data presented here suggest that selection against the IFN alpha/beta system could play a role or accompany the development of the malignant phenotype.

Acute Disease↗

PubMed1992

Multichain structure of the IFN-alpha receptor on hematopoietic cells.

The structure of IFN-alpha receptor was studied by 1) developing antibodies against the receptor, and 2) screening a number of cell lines by affinity cross-linking to identify cells that express different IFN-alpha 2 receptor structures. We report that two different patterns of IFN-alpha 2 receptor are observed in human cells of hematopoietic origin. The predominant form of the IFN-alpha receptor is a multichain structure in which IFN-alpha 2 forms complexes of 110 and 130 kDa (alpha-subunit). A high Mr complex of 210 kDa results from the association of alpha-subunit and other receptor components. In contrast, another form of the receptor has been identified in the IFN-alpha-resistant U-937 cell line and in some cases of acute leukemia. This form of the IFN-alpha receptor is characterized by the presence of the alpha- subunit, and the absence of the 110- and 210-kDa bands. Also a novel 180-kDa complex and a more prominent 75-kDa band are observed. Functional studies performed in U-937 cells showed that this cell line is not only partially resistant to the antiproliferative and antiviral effects of IFN-alpha, but also fails to down-regulate the alpha-subunit of the IFN-alpha receptor upon IFN-alpha binding.

Cell Line↗

PubMed1992

Expression of the IFN alpha receptor in hairy cell leukaemia.

The expression of IFN alpha receptors on the surface of hairy cell leukaemia cells was studied using affinity crosslinking methods, flow cytometry with a recently produced anti-IFN alpha receptor monoclonal antibody (IFNaR3), and binding techniques. IFN alpha receptors were detected by flow cytometry and affinity crosslinking in eight cases showing a normal receptor structure with 125I-IFN alpha 2-receptor complexes with molecular weights of 210, 130 and 110 kD. The hairy cell leukaemia cells from one patient did not express IFN alpha receptors as determined by flow cytometry, affinity crosslinking and binding studies. Northern blot analysis showed expression of mRNA for the gene coding for the putative IFN alpha receptor in all cases studied, including the patient with lack of IFN alpha receptor expression on hairy cells. This patient was refractory to IFN alpha 2 treatment. Our data suggest that lack of expression of IFN alpha receptors in rare cases may be associated with refractoriness to IFN alpha 2 therapy.

Adult↗

PubMed1992

A phase I study of interleukin-2 in children with cancer.

Recombinant interleukin-2 (IL-2) produces clinical responses in approximately 20% of adult patients with renal cell carcinoma and melanoma, with both high-dose bolus and continuous infusion regimens. Because of the lower toxicity of continuous infusion, we elected to investigate in a Phase I trial a 5-day continuous infusion repeated for three weeks in children with malignancies refractory to standard therapy. Nineteen children with solid tumors and eight children with hematologic malignancies were entered into the study. The maximum tolerated dose was 3 x 10(6) U/m2/day, with dose-limiting toxicities occurring in five of seven patients treated at the 5 x 10(6) U/m2/day dose level. Dose-limiting toxicities included hypotension, hyperbilirubinemia, thrombocytopenia, pulmonary/pleural effusion, and nephrotoxicity. Serum IL-2 levels were detectable at the higher dose levels and were comparable to those observed in adult patients. Hematologic changes at the higher dose levels included rebound lymphocytosis occurring within 48 h of discontinuation of IL-2, eosinophilia, and decreased platelet counts. No objective responses to therapy were seen. We have identified a dose and schedule of administration for IL-2 in pediatric patients that can be given without intensive care unit support. Pediatric Phase II trials examining the anti-tumor activity of IL-2 given by this schedule are in progress.

Adolescent↗

PubMed1991

Transmembrane signalling by interferon-alpha.

Human leukocyte interferon (IFN-alpha) binds to discrete cell surface receptors on target cells, and thereby alters gene expression. Transmembrane signaling by IFN-alpha involves the production of DAG without an increased intracellular free calcium concentration, and the subsequent activation of calcium-independent isoforms of PKC (beta and epsilon). Selective PKC inhibitors (H-7 and staurosporine) can block the ability of IFN-alpha to activate the transcription of a distinct set of genes, called the IFN-stimulated genes (ISG), and to protect cells against viral infection. IFN-alpha also induces the rapid changes in protein phosphorylation, which may include latent transcription factors for ISGs.

Animals↗

PubMed1991

Structure of the human interferon alpha receptor.

The structure of the IFN alpha receptor has been studied by methods such as affinity crosslinking and gel chromatography over the last 8 years. The recent development of monoclonal antibodies against the receptor, and the cloning of an IFN alpha receptor cDNA has provided new important tools to understand the IFN alpha receptor structure. Thus, it has become obvious that the IFN alpha receptor has a more complex structure than first anticipated, probably involving more than one subunit. This review analyzes the present knowledge about the structure of the IFN alpha receptor, as well as many unresolved issues concerning this topic.

Animals↗