Phorbol ester induces c-sis gene transcription in stem cell line K-562.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced megakaryoblastic differentiation and c-sis expression in the human hematopoietic stem cell line K-562. This induction occurred at the transcriptional level, as determined by a nuclear runoff transcriptional assay, and was not a generalized effect of TPA, since the treatment of other hematopoietic cell lines and normal peripheral blood lymphocytes with TPA did not result in the appearance of c-sis mRNA.

Transcriptional inactivation of c-myc and the transferrin receptor in dibutyryl cyclic AMP-treated HL-60 cells.

Treatment of HL-60 cells with dibutyryl cyclic AMP induced rapid transcriptional inactivation of c-myc and the transferrin receptor. Transcriptional inactivation was followed by loss of c-myc and transferrin receptor mRNA and protein. Treated cells completed one round of proliferation, followed by growth arrest, G1 synchronization, and monocytic differentiation. These data suggest that cyclic AMP-mediated control of growth and differentiation may be achieved, at least in part, by transcriptional regulation of certain growth-associated proto-oncogenes and growth factor receptor genes.

The type-I interferon receptor. The long and short of it.

The type-I interferon receptor is a multisubunit receptor of the cytokine receptor superfamily. The production of specific monoclonal antibodies against the receptor and the cloning of different receptor subunits have contributed to understanding the type-I interferon receptor structure and function. The present article analyzes these new advances and the role of the different receptor subunits in type-I interferon signaling.

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.

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.

Homodimerization and intermolecular tyrosine phosphorylation of the Tyk-2 tyrosine kinase.

The Jak kinases and Stat transcription factors play a major role in signaling of various cytokines including IFN alpha. In this report we show a ligand-independent interaction between Tyk-2 and Jak-1 kinases. We also demonstrate that the Tyk-2 kinase forms a homodimer that has the ability to undergo intermolecular tyrosine phosphorylation. The formation of the Tyk-2 homodimer is independent of both tyrosine phosphorylation and the presence of the tyrosine kinase domain.

The vav proto-oncogene product (p95vav) interacts with the Tyk-2 protein tyrosine kinase.

The vav proto-oncogene product participates in the signaling pathways activated by various cell-surface receptors, including the type I IFN receptor. During engagement of the type I IFN receptor, p95vav is phosphorylated on tyrosine residues, but the kinase regulating its phosphorylation has not been identified to date. Our studies demonstrate that p95vav forms a stable complex with the IFN-receptor-associated Tyk-2 kinase in vivo, and strongly suggest that this kinase regulates its phosphorylation on tyrosine. Thus, p95vav is engaged in IFN-signaling by a direct interaction with the functional type I IFN receptor complex to transduce downstream signals.

Structure-function study of the extracellular domain of the human type I interferon receptor (IFNAR)-1 subunit.

Despite accumulating information about the different effector molecules and signaling cascades that are invoked on interferon-alpha (IFN-alpha) binding to the type 1 IFN receptor, little is known about the specifics of the binding interactions between the ligand and the receptor complex. The IFN-alpha/beta receptor (IFNAR)-2 subunit of the IFN receptor is considered the primary binding chain of the receptor, yet it is clear that both receptor subunits, IFNAR-1 and IFNAR-2, cooperate in the high-affinity binding of IFN to the receptor complex. Earlier results from our laboratory suggested that an association of IFNAR-1 with membrane Galalpha1-4Gal-containing glycolipids facilitates receptor-mediated signaling. The data implicated amino acid residues in the SD100 domain of IFNAR-1 in the glycosphingolipid (GSL) modification of the type 1 IFN receptor. Interestingly, the human and murine counterparts of IFNAR-1 exhibit remarkable species specificity despite their considerable amino acid sequence identity. Certainly, those amino acid residues that effect GSL modification of IFNAR-1 are conserved between species, yet specific regions of IFNAR-1 that confer species specificity have not been defined. To delineate further the role of the IFNAR-1 SD100A domain in receptor function, a chimeric cDNA was assembled, in which the SD100A domain of the murine IFNAR-1 chain was replaced with the human sequence. This construct was expressed in IFNAR-1-/- mouse embryonic fibroblasts, and stable transfectants were established. Transfectants are fully sensitive to murine IFN-alpha4 treatment with respect to the induction of IFN-stimulated gene factor 3 (ISGF3) and sis-inducing factor (SIF) signal transducer and activator of transcription factor (Stat) complexes, exhibiting comparable levels of Stat activation to those observed in IFNAR-1-/- cells reconstituted with intact MuIFNAR-1. Similar results were obtained with IFN-induced antiviral and growth inhibitory responses. Viewed together, these data suggest that the SD100A domain of IFNAR-1 does not contribute to species-specific IFN binding.

Activation of the Jak-Stat pathway in cells that exhibit selective sensitivity to the antiviral effects of IFN-beta compared with IFN-alpha.

We determined whether selective activation of components of the Jak-Stat pathway by different type I interferons (IFN) occurs in human myocardial fibroblasts that exhibit much higher sensitivity to the antiviral effects of IFN-beta than of IFN-alpha. Similar levels of activation of the Tyk2 kinase and the Stat3 transcription factor were induced in response to either IFN-beta or IFN-alpha treatment. However, activation of the Jak1 tyrosine kinase was detectable only in IFN-beta-treated but not IFN-alpha-treated cells. Consistent with this, tyrosine phosphorylation of Stat1 and Stat2 and formation of the IFN-stimulated gene factor 3 (ISGF3) complex occurred to a much higher degree in response to IFN-beta stimulation. These findings demonstrate that differential activation of distinct components of the Jak-Stat pathway by different type I IFN can occur. Furthermore, they strongly suggest that such selective activation accounts for the occurrence of differences in the antiviral properties of distinct type I IFN in certain cell types.

Detection of functional interferon alpha receptors in human neuroendocrine tumor cell lines using a new monoclonal antibody.

While first described as antiviral agents, interferons (IFNs) exhibit significant antiproliferative and antitumor effects as well. IFN alpha has been successfully used in clinical trials to treat several malignancies, including leukemias and certain solid tumors. While many cell types have been studied for IFN alpha receptor expression, very little is known about receptor expression on human neuroendocrine cells. Using a novel anti-IFN alpha receptor monoclonal antibody, we examined IFN alpha receptor expression in 10 human cell lines derived from tumors of neuroendocrine origin, including neuroblastoma, neuroepithelioma and small cell lung carcinoma. All cell lines studied displayed a similar pattern of IFN alpha receptor expression and 5 of 8 cell lines demonstrated reduced thymidine incorporation following IFN alpha treatment. Addition of exogenous IFN alpha caused a decrease in IFN alpha receptor expression, while differentiating agents, such as phorbol esters and retinoic acid, induced an increase in receptor number without altering receptor affinity.

Expression of type I interferon receptor and its relation with other prognostic factors in human neuroblastoma.

Expression of type I interferon receptor (IFN-R) has been found in several normal tissues and in malignant neoplasms, mainly those with epithelial differentiation. In order to analyze the immunohistochemical expression of type I IFN-R we studied 79 cases of neuroblastoma. Results of expression of type I IFN-R were statistically correlated with histopathology, stage, bcl-2 and PCNA expression, N-myc amplification and apoptosis. We found expression of type I IFN-R in 54/79 cases showing statistical correlation with bcl-2 expression (P=0.017) and favourable histopathology (P=0.015). The overexpression found in ganglion cells suggests that IFN-R could be involved in the pathway of neuroblastoma differentiation. Moreover, the expression of type I IFN-R in stage 4 cases (12/20), even with N-myc amplification (6/8), opens new possibilities for therapeutic management in advanced cases that do not respond to any chemotherapeutic protocol.

ARF-induced downregulation of Mip130/LIN-9 protein levels mediates a positive feedback that leads to increased expression of p16Ink4a and p19Arf.

The ARF-MDM2-p53 pathway constitutes one of the most important mechanisms of surveillance against oncogenic transformation, and its inactivation occurs in a large proportion of cancers. Here, we show that ARF regulates Mip130/LIN-9 by inducing its translocation to the nucleolus and decreasing the expression of the Mip130/LIN-9 protein through a post-transcriptional mechanism. The knockdown of Mip130/LIN-9 in p53(-/-) and Arf(-/-) mouse embryonic fibroblasts (MEFs) mimics some effects of ARF, such as the downregulation of B-Myb, impaired induction of G2/M genes, and a decrease in cell proliferation. Importantly, although the knockdown of Mip130/LIN-9 reduced the proliferation of p53 or Arf-null MEFs, only p53(-/-) MEFs showed a senescence-like state and an increase in the expression of Arf and p16. Interestingly, the increase in p16 and ARF is indirect because the Mip130/LIN-9 knockdown decreased the transcription of negative regulators of the Ink4a/Arf locus, such as BUBR1 and CDC6. Chromatin immunoprecipitation assays also reveal that Mip130/LIN-9 occupies the promoters of the BubR1 and cdc6 genes, suggesting that Mip130/LIN-9 is necessary for the expression of these genes. Altogether, these results indicate that there is a feedback mechanism between ARF and Mip130/LIN-9 in which either the increase of ARF or the decrease in Mip130/LIN-9 causes a further increase in the expression of Arf and p16.

Mammalian Mip/LIN-9 interacts with either the p107, p130/E2F4 repressor complex or B-Myb in a cell cycle-phase-dependent context distinct from the Drosophila dREAM complex.

Mammalian Mip/LIN-9 is a cell cycle regulatory protein that is negatively regulated by CDK4/cyclin D. It has been demonstrated that Mip/LIN-9 collaborates with B-Myb during S and G(2)/M in the induction of cyclins A and B, and CDK1. The ortholog of Mip/LIN-9 in Drosophila, Mip130, is part of a large multisubunit protein complex that includes RBF, repressor E2Fs and Myb, in what was termed the dREAM complex. A similar complex, although lacking B-Myb, was also described in Caenorhabditis elegans. Here, we demonstrate that unlike Drosophila, Mip/LIN-9 has mutually exclusive and cell cycle-phase-specific interactions with the mammalian orthologs of the dREAM complex. In G(0)/early G(1), Mip/LIN-9 forms a complex with E2F4 and p107 or p130, while in late G(1)/S phase, it associates with B-Myb. The separation of Mip/LIN-9 from p107,p130/E2F4 is likely driven by phosphorylation of the pocket proteins by CDK4 since Mip/LIN-9 fails to interact with phosphorylated forms of p107,p130. Importantly, the repressor complex that Mip/LIN-9 forms with p107 takes functional precedence over the transcriptional activation linked to the Mip/LIN-9 and B-Myb interaction since expression of p107 blocks the activation of the cyclin B promoter triggered by B-Myb and Mip/LIN-9.

Role of the cytoplasmic domains of the type I interferon receptor subunits in signaling.

Type I interferons are imperative in maintaining a defense against viral infection. These cytokines also play an important role in the control of cell proliferation. These effects are triggered by ligand binding to a specific cell surface receptor. In the present article, we attempt to analyze the advances made in the last four years on type I interferon signaling. This review will focus on the contribution of the cytoplasmic domain of the alpha and betaL chains of the receptor to the activation of the Jak-Stat pathway. We also analyze the possible role of other pathways in interferon signaling.

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.

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.

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.

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.

Phorbol ester enhances deoxynucleoside incorporation while inhibiting proliferation of K-562 cells.

Recently a new technique has been developed to study bromodeoxyuridine incorporation into DNA as a function of the cell cycle phase distribution. We have made the observation that treatment of the hematopoietic cell line K-562 results in a complete and immediate cessation of cell growth, although tritiated thymidine incorporation is markedly elevated for as long as 5 days. We have made use of that newly described technique to study this phenomenon.

Immunohistochemical detection of the type I interferon receptor in human fetal, adult, and neoplastic tissues.

We have used the monoclonal antibody IFNaR3 that recognizes the alpha subunit of the type I interferon (IFN) receptor to study the expression of this receptor in a large series of normal human adult and fetal tissues, as well as in a large number of tumors of diverse origin. Among fetal tissues (8-20 weeks) the type I IFN receptor was expressed in liver, striated muscle, epidermis, renal tubules, choroid plexus of the CNS, and epithelia of different origins (bronchial, gastrointestinal, and pancreatic). Adult tissues showed a similar pattern that includes epithelia from salivary ducts, genital tract, bladder, breast, as well as germinal centers of lymph nodes, tonsils, and spleen. The study of a large series of tumors revealed that the type I IFN receptor is expressed in most, but not all, melanomas, bladder, kidney, small bowel, lung, and breast adenocarcinomas. The majority of lymphomas, sarcomas, and endocrine tumors proved negative. These results support the concept that the type I IFN receptor is rather ubiquitously expressed in normal and malignant epithelial tissues. More interestingly, the expression of the type I IFN receptor was not detected in all tumors, raising the question of whether some cases may fail IFN alpha therapy due to the lack of receptor expression. This report demonstrates that the IFNaR3 monoclonal antibody can be used for receptor detection in paraffin-embedded sections and it could represent a useful tool in the search for correlations between IFN alpha response and receptor expression in different diseases.