New approaches to molecular profiling of tissue samples.
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Alleles↗
Biomedical subjects
E F Petricoin
Publications and source records attributed to E F Petricoin.
Characterization of intracellular prostate-specific antigen from laser capture microdissected benign and malignant prostatic epithelium.
The proportion of unbound serum prostate-specific antigen (PSA; percent-free PSA) is reported to be lower in men with prostate cancer compared to men with benign prostates (U. H. Stenman et al., Cancer Res., 51: 222-226, 1991; H. Lilja et al., Clin. Chem., 37: 1618-1625, 1991; D. L. Woodrum et al., J. Urol., 159: 5-12, 1998; W. J. Catalona et al., J. Am. Med. Assoc., 279: 1542-1547, 1998). The majority of immunoreactive PSA in serum is complexed to alpha-1-antichymotrypsin (ACT). Two major mechanistic questions have previously been unknown: (a) Does PSA in human prostate cancer cells in tissue exist in a free or bound form? and (b) Is PSA produced by malignant cells in the free form because it has lost the ability to form a complex with ACT? Laser capture microdissection (LCM) enables the acquisition of pure populations of defined cell types from tissue (M. R. Emmert-Buck et al., Science, 274: 998-1001, 1996; R. F. Bonner et al., Science, 278: 1481-1483, 1997). This technology provides a unique opportunity to study intracellular protein composition and structure from human cells. In this study, we used LCM to assess the bound versus free form of intracellular PSA in both benign and malignant epithelium procured from prostate tissue. One-dimensional and two-dimensional PAGE were performed on cellular lysates from LCM-procured benign and malignant prostate epithelium from frozen tissue specimens. Western blotting analysis of one-dimensional PAGE gels revealed a strong band at M(r) 30,000 (expected molecular weight of unbound PSA) in all cases demonstrating that the vast majority of intracellular tumor and normal PSA exists within cells in the "free" form. Binding studies showed that PSA recovered from LCM-procured cells retained the full ability to bind ACT, and two-dimensional PAGE Western analysis demonstrated that the PSA/ACT complex was stable under strong reducing conditions. We conclude that intracellular PSA exists in the "free" form and that binding to ACT occurs exclusively outside of the cell.
Blotting, Western↗
An approach to proteomic analysis of human tumors.
A strategy for proteomic analysis of microdissected cells derived from human tumor specimens is described and demonstrated by using esophageal cancer as an example. Normal squamous epithelium and corresponding tumor cells from two patients were procured by laser-capture microdissection and studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Fifty thousand cells resolved approximately 675 distinct proteins (or isoforms) with molecular weights ranging between 10 and 200 kDa and isoelectric points of pH 3-10. Comparison of the microdissected protein profiles showed a high degree of similarity between the matched normal-tumor samples (98% identical). However, 17 proteins showed tumor-specific alterations, including 10 that were uniquely present in the tumors and seven that were observed only in the normal epithelium. Two of the altered proteins were characterized by mass spectrometry and immunoblot analysis and were identified as cytokeratin 1 and annexin I. Acquisition of 2D-PAGE protein profiles, visualization of disregulated proteins, and subsequent determination of the identity of selected proteins through high-sensitivity MS-MS microsequencing are possible from microdissected cell populations. These separation and analytical techniques are uniquely capable of detecting tumor-specific alterations. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms. Mol.
Electrophoresis, Gel, Two-Dimensional↗
Beyond the genome to tissue proteomics.
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Animals↗
Activation of Raf-1 by interferon gamma and oncostatin M requires expression of the Stat1 transcription factor.
A primary signaling cascade responsible for the expression of cytokine-stimulated immediate early genes involves the activation of the Jak/Stat pathway. In addition to being tyrosine-phosphorylated, several signal transducers and activators of transcription (Stats), including Stat1alpha, Stat3, and Stat4, are phosphorylated on a conserved serine residue, which is a consensus phosphorylation site for mitogen-activated protein kinases (MAPKs). Serine phosphorylation of Stat1alpha is required for maximal transcriptional activation of early response genes by interferon gamma (IFNgamma) as well as the antiviral and antigrowth actions of this cytokine. Incubation of cells with either IFNgamma or oncostatin M (OSM) activates Raf-1, a serine/threonine kinase responsible for the ultimate activation of p42 MAPK. To examine whether any of the signaling components that are required for activation of the Jak/Stat pathway are also necessary for activation of Raf-1 by IFNs and OSM, we examined activation of Raf-1 in cell lines that are deficient in either Stat1alpha or Stat2. Unexpectedly, incubation of Stat1-deficient, but not Stat2-deficient cells with IFNgamma or OSM for 5 min displayed no increase in Raf-1 activity. In peripheral blood lymphocytes Raf-1 was associated with Stat1, and this interaction was disrupted after incubation of cells with IFNgamma. Stat1-negative cells reconstituted with either Stat1alpha or Stat1alpha with a point mutation in the site where it is serine-phosphorylated displayed normal activation of Raf-1 by IFNgamma and OSM. However, activation of Raf-1 was not observed in lines that expressed Stat1alpha containing a mutation in its tyrosine phosphorylation site or in its SH2 domain. These results provide the first example of a novel role of Stat1alpha not as a transcription factor, but as a protein which may function to scaffold signaling components required for activation of the distinct Raf/MEK/MAPK signaling cascade.
Animals↗
Prolonged STAT1 activation related to the growth arrest of malignant lymphoma cells by interferon-alpha.
Multiple biologic effects of interferon-alpha (IFN-alpha), including cell growth inhibition and antiviral protection, are initiated by tyrosine phosphorylation of STAT proteins. Although this signal pathway has been intensively investigated, the relevance of STAT signal persistence has received scant attention. Using paired isogenic lymphoma cells (Daudi), which either are sensitive or resistant to growth inhibition by IFN-alpha, we found comparable initial tyrosine phosphorylation of multiple STAT proteins; however, the phosphorylation durations and associated DNA-binding activities diverged. Phosphorylation and DNA-binding capacity of STAT1 decreased after 4 to 8 hours in resistant cells, as compared with 24 to 32 hours in sensitive cells, whereas phosphorylation of STAT3 and STAT5b was briefer in both lines. Functional significance of the prolonged STAT1 signal, therefore, was explored by experimental interruption of tyrosine phosphorylation, either by premature withdrawal of the IFN-alpha or deferred addition of pharmacologically diverse antagonists: staurosporine (protein kinase inhibitor), phorbol 12-myristate 13-acetate (growth promoter), or aurintricarboxylic acid (ligand competitor). Results indicated that an approximately 18-hour period of continued STAT1 phosphorylation was associated with growth arrest, but that antiviral protection developed earlier. These differences provide novel evidence of a temporal dimension to IFN-alpha signal specificity and show that duration of STAT1 activation may be a critical variable in malignant cell responsiveness to antiproliferative therapy.
Antineoplastic Agents↗
Antiproliferative action of interferon-alpha requires components of T-cell-receptor signalling.
Signal transduction through both cytokine and lymphocyte antigen receptors shares some common pathways by which they initiate cellular responses, such as activation of mitogen-activated protein kinase(s). However, other signalling components appear to be uniquely coupled to each receptor. For example, the interferon receptors transduce regulatory signals through the JAK/STAT pathway, resulting in an inhibition of growth and of antiviral effects, whereas this pathway apparently plays no role in T-cell-receptor (TCR)-dependent gene expression. Conversely, signal transduction through the TCR requires the tyrosine kinases Lck and ZAP-70 and the tyrosine phosphatase CD45. Here we show that, unexpectedly, transmission of growth-inhibitory signals by interferon-alpha (IFN-alpha) in T cells requires the expression and association of CD45, Lck and ZAP-70 with the IFN-alpha-receptor signalling complex.
Animals↗
Phosphorylation events modulate the ability of interferon consensus sequence binding protein to interact with interferon regulatory factors and to bind DNA.
Two families of transcription factors mediate interferon (IFN) signaling. The first family, signal transducers and activators of transcription (STATs), is activated within minutes of IFN treatment. Specific phosphorylation events lead to their translocation to the nucleus, formation of transcriptional complexes, and the induction of the second family of transcription factors termed interferon regulatory factors (IRFs). Interferon consensus sequence binding protein (ICSBP) is a member of IRF family that is expressed only in cells of the immune system and acts as a transcriptional repressor. ICSBP binds DNA through the association with other transcription factors such as IRF-1 or IRF-2. In this communication, the domain that is involved in protein-protein interactions was mapped to the carboxyl terminus of ICSBP. This domain is also important for mediating ICSBP-repressing activity. In vitro studies demonstrated that direct binding of ICSBP to DNA is prevented by tyrosine (Tyr) phosphorylation. Yet, Tyr-phosphorylated ICSBP can bind target DNA only through the association with IRF-2 and IRF-1. This type of phosphorylation is essential for the formation of heterocomplexes. Tyr-phosphorylated ICSBP and IRF-2 are detected in expressing cells constitutively, and Tyr-phosphorylated IRF-1 is induced by IFN-gamma. These results strongly suggest that like the STATs, the IRFs are also modulated by Tyr phosphorylation that affects their biological activities.
Amino Acid Sequence↗
Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway.
Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways.
Animals↗
Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes.
Interleukin 12 (IL-12) is an important immunoregulatory cytokine whose receptor is a member of the hematopoietin receptor superfamily. We have recently demonstrated that stimulation of human T and natural killer cells with IL-12 induces tyrosine phosphorylation of the Janus family tyrosine kinase JAK2 and Tyk2, implicating these kinases in the immediate biochemical response to IL-12. Recently, transcription factors known as STATs (signal transducers and activators of transcription) have been shown to be tyrosine phosphorylated and activated in response to a number of cytokines that bind hematopoietin receptors and activate JAK kinases. In this report we demonstrate that IL-12 induces tyrosine phosphorylation of a recently identified STAT family member, STAT4, and show that STAT4 expression is regulated by T-cell activation. Furthermore, we show that IL-12 stimulates formation of a DNA-binding complex that recognizes a DNA sequence previously shown to bind STAT proteins and that this complex contains STAT4. These data, and the recent demonstration of JAK phosphorylation by IL-12, identify a rapid signal-transduction pathway likely to mediate IL-12-induced gene expression.
Base Sequence↗
Prolactin activates the interferon-regulated p91 transcription factor and the Jak2 kinase by tyrosine phosphorylation.
The prolactin (PRL) receptor is a member of the family of cytokine receptors that lack intrinsic tyrosine kinase activity but contain two conserved cysteines in their N-terminal regions and a WSXWS motif adjacent to their transmembrane domains. In a manner similar to the interferons (IFNs), exposure of cells to PRL results in tyrosine phosphorylation of several cellular proteins and the rapid transcriptional induction of the IFN regulatory factor 1 gene. In this communication, we demonstrate that treatment of rat Nb2 lymphoma cells with PRL activates a latent protein factor so that it binds to an enhancer in the IFN regulatory factor 1 gene. This enhancer has been shown to be required for IFN-gamma-activated expression of this gene. PRL-induced assembly of the DNA binding complex, PRL-stimulated factor, required tyrosine phosphorylation. PRL-stimulated factor contained at least one protein that was antigenically similar to the p91 transcription factor, a component of several transcription complexes required for cytokine-activated gene expression. PRL not only induced the tyrosine phosphorylation of p91 but also induced tyrosine phosphorylation of Jak2, a tyrosine kinase required for IFN-gamma-activated gene expression. These results provide evidence for a signaling mechanism, some of whose components are shared by both PRL and IFN-gamma receptors, that results in the expression of early response genes.
Animals↗
Cytokines that associate with the signal transducer gp130 activate the interferon-induced transcription factor p91 by tyrosine phosphorylation.
Interleukin-6, leukemia inhibitory factor, and oncostatin M exert a broad range of similar biological activities through association of their receptors with the signal-transducing component gp130. Although it is known that these cytokines trigger rapid tyrosine phosphorylation of a common set of cellular proteins as well as induction of several of the same early response genes, the mechanisms by which these genes are activated is not well understood. In this report, we show that interleukin-6, leukemia inhibitory factor, and oncostatin M stimulate the assembly of protein complexes that recognize conserved sequences within the enhancers of two genes (interferon regulatory factor 1 and Fc gamma receptor type I) that are rapidly activated by these cytokines. These enhancers are known to be required for transcriptional induction of these genes by interferon-gamma. Assembly of the DNA-binding protein complexes occurs within minutes after ligand addition and depends upon tyrosine phosphorylation. These complexes contain the p91 transcription factor, which is tyrosine-phosphorylated in response to these cytokines. An additional tyrosine-phosphorylated protein of 93 kDa can be coimmunoprecipitated with antibodies against p91. These findings further expand the network of cytokines known to activate p91 and, in addition, support the concept that sets of tyrosine-phosphorylated proteins may be responsible for the cytokine-regulated expression of early response genes.
Animals↗
Use of xylE fusions to demonstrate that lsi-1, a Neisseria gonorrhoeae lipooligosaccharide biosynthetic gene, and lsi-3 are not transcriptionally linked.
The Neisseria gonorrhoeae lipooligosaccharide biosynthetic gene lsi-1 is preceded by a 281-bp non-protein-encoding sequence, lsi-3, that contains two pairs of inverted repeats. Gonococcal chromosomal lsi-xylE gene fusions were generated to measure the effect of the secondary structure on transcriptional attenuation. The data obtained indicate that lsi-3 and lsi-1 are not transcriptionally linked and therefore that lsi-3 is not involved in the regulation of lsi-1.
Base Sequence↗
Growth hormone and erythropoietin differentially activate DNA-binding proteins by tyrosine phosphorylation.
Binding of growth hormone (GH) and erythropoietin (EPO) to their respective receptors results in receptor clustering and activation of tyrosine kinases that initiate a cascade of events resulting not only in the rapid tyrosine phosphorylation of several proteins but also in the induction of early-response genes. In this report, we show that GH and EPO induce the tyrosine phosphorylation of cellular proteins with molecular masses of 93 kDa and of 91 and 84 kDa, respectively, and that these proteins form DNA-binding complexes which recognize an enhancer that has features in common with several rapidly induced genes such as c-fos. Assembly of the protein complexes required tyrosine phosphorylation, which occurred within minutes after addition of ligand. The activated complexes translocated from the cytoplasm to the nucleus. The protein activated by GH is antigenically similar to p91, a protein common to several transcription complexes that are activated by interferons and other cytokines. In contrast, the proteins activated by EPO are distinct from p91. These findings establish the outlines for a cytokine-induced intracellular signaling pathway, which begins with ligand-induced receptor clustering that activates one or more tyrosine kinases. These data are the first to demonstrate that GH- and EPO-activated tyrosine-phosphorylated proteins can specifically recognize a well-defined enhancer and therefore provide a mechanism for rapidly transducing signals from the membrane to the nucleus.
Base Sequence↗
Tyrosine phosphorylation of DNA binding proteins by multiple cytokines.
Interferon-alpha (IFN-alpha) and IFN-gamma regulate gene expression by tyrosine phosphorylation of several transcription factors that have the 91-kilodalton (p91) protein of interferon-stimulated gene factor-3 (ISGF-3) as a common component. Interferon-activated protein complexes bind enhancers present in the promoters of early response genes such as the high-affinity Fc gamma receptor gene (Fc gamma RI). Treatment of human peripheral blood monocytes or basophils with interleukin-3 (IL-3), IL-5, IL-10, or granulocyte-macrophage colony-stimulating factor (GM-CSF) activated DNA binding proteins that recognized the IFN-gamma response region (GRR) located in the promoter of the Fc gamma RI gene. Although tyrosine phosphorylation was required for the assembly of each of these GRR binding complexes, only those formed as a result of treatment with IFN-gamma or IL-10 contained p91. Instead, complexes activated by IL-3 or GM-CSF contained a tyrosine-phosphorylated protein of 80 kilodaltons. Induction of Fc gamma RI RNA occurred only with IFN-gamma and IL-10, whereas pretreatment of cells with GM-CSF or IL-3 inhibited IFN-gamma induction of Fc gamma RI RNA. Thus, several cytokines other than interferons can activate putative transcription factors by tyrosine phosphorylation.
Base Sequence↗
IL-4 attenuates the transcriptional activation of both IFN-alpha and IFN-gamma-induced cellular gene expression in monocytes and monocytic cell lines.
The interaction of IFN-alpha and IFN-gamma with monocytes results in several actions that significantly influence the course of an immune response. Many of these effects are proinflammatory and can contribute to the degree of tissue injury at a site of inflammation. Whereas recent investigations target IL-4 as a T cell product that can antagonize some of the responses induced by IFN, little is known regarding the mechanisms involved. We have taken advantage of two well defined systems: the transcriptional activation of the cellular genes ISG-54 by IFN-alpha and IP-10 by IFN-gamma. IL-4 treatment of both the monocytic leukemia cell line, THP-1, and normal peripheral blood monocytes resulted in inhibition of IFN-induced RNA levels for both genes. Nuclear run-on assays in THP-1 cells indicated that the effects of IL-4 were due to the inhibition of the transcriptional activation of these genes by both IFN-alpha and IFN-gamma. This inhibition was not due to alteration in the binding characteristics of IFN-alpha or IFN-gamma to the cell. In the IFN-alpha system, we were able to show that IL-4 treatment resulted in reduced formation of the transcriptional activator, IFN-stimulated gene factor 3. This reduction appears to be the result of a defect in the ability of IFN alpha to activate the IFN-stimulated gene factor 3 alpha component of IFN-stimulated gene factor 3.
Base Sequence↗
Interferon-alpha-induced gene expression: evidence for a selective effect of ouabain on activation of the ISGF3 transcription complex.
Binding of interferons (IFNs) to their cell surface receptors stimulates rapid translocation of cytoplasmic proteins to the nucleus and the expression of a variety of cellular genes within minutes. Translocated proteins subsequently bind to the interferon-stimulated response element (ISRE) located in the promoters of all IFN-activated cellular genes. We report here that ouabain, a specific inhibitor of the Na/K ATPase, selectively inhibited transcription of several IFN-alpha-induced cellular RNAs under conditions in which some other well-described signal transduction pathways remained intact. The latter included induction of human metallothionein 2A (HMT2A) by phorbol ester and induction of IP-10 RNA by IFN-gamma. Ouabain itself induced RNA of the protooncogene c-fos which conversely was inhibited by IFN-alpha. Specificity of the ouabain effects on IFN alpha-induced RNAs with respect to a direct action on the Na/K ATPase was shown with a transfected monkey CV-1 cell line which expresses the ouabain-insensitive rat alpha 1 subunit. Electrophoretic mobility shift assays (EMSAs) using nuclear extracts from ouabain-treated cells demonstrated that ouabain decreased IFN alpha-induced binding of the ISGF3 complex to the ISRE. Reconstitution experiments showed that this effect of ouabain is not due to the inhibition of IFN alpha activation of the ISGF3 alpha subcomponent, which occurs in the cytoplasm, but a selective depletion of the ISGF3 gamma factor which in concert with activated ISGF3 alpha induces interferon-stimulated gene (54 kDa) transcription. These findings imply that intracellular ion balance can selectively regulate the half-life of the ISGF3 gamma protein or the ability of this protein to complex with ISGF3 alpha to activate IFN alpha-regulated cellular genes.
Base Sequence↗
Modulation of interferon signaling in human fibroblasts by phorbol esters.
Phorbol esters activate the expression of a variety of early-response genes through protein kinase C-dependent pathways. In addition, phorbol esters may promote cell growth by the inhibition of expression of cellular gene products regulated by antiproliferative agents such as interferons (IFN)s. In human diploid fibroblasts, phorbol 12-myristate 13-acetate (PMA) selectively inhibits the IFN-alpha-induced cellular gene ISG54. Using transient transfection assays, we have delineated two elements in the promoter of this gene that are necessary for the inhibitory actions of PMA. These elements include (i) the IFN-stimulated response element (ISRE) which is necessary for IFN-alpha-induced cellular gene expression, and (ii) an element located near the site of transcription initiation. IFN-alpha treatment resulted in the rapid induction of ISGF3, a multisubunit transcription factor which binds to the ISRE. PMA caused a substantial reduction in IFN alpha-induced ISGF3 in both nuclear and cytoplasmic extracts, as determined by electrophoretic mobility shift assays with the ISRE as a probe. In vitro reconstitution experiments revealed that IFN-alpha activation of the ISGF3 alpha component of ISGF3 was not affected by PMA. Further experiments were consistent with the possibility that PMA regulated the activity of a cellular factor which competed with ISGF3 gamma for binding of the activated ISGF3 alpha polypeptides. Electrophoretic mobility shift assays using the cap site of ISG54 as a probe demonstrated the formation of a specific complex whose DNA binding activity was not affected by treatment of cells with PMA or IFN-alpha. Competitive inhibition studies were consistent with the DNA-protein complex at the cap site of ISG54 containing proteins with DNA binding sites in common with those which also interact with the ISRE. These data suggest a unique regulatory mechanism by which phorbol esters can modulate IFN signaling.
Base Sequence↗