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• W2007662857 abstract "Certain mutations in the mammalianras gene are oncogenic and are often detected in human cancers. Oncogenic Ras induces the transcription activity of NF-κB that confers cell survival. Oncogenic Ras also down-modulates the expression of Par-4, a transcriptional repressor protein, that is essential but not sufficient on its own to induce apoptosis. Here we show that reintroduction of Par-4 by transient transfection leads to apoptosis in cells expressing oncogenic Ras but not in those that lack oncogenic Ras expression. Par-4 abrogates oncogenic Ras-inducible NF-κB transcription activity but does not interfere with cytoplasmic activation, or the DNA binding activity, of NF-κB. Because abrogation of NF-κB transcription activity is sufficient to cause apoptosis in cells expressing oncogenic Ras, our findings identify Par-4 as a novel example of a pro-apoptotic protein that selectively inhibits oncogenic Ras-dependent NF-κB function at the transcription level and suggest a mechanism by which Par-4 expression may selectively induce apoptosis in oncogenic Ras-expressing cells. Certain mutations in the mammalianras gene are oncogenic and are often detected in human cancers. Oncogenic Ras induces the transcription activity of NF-κB that confers cell survival. Oncogenic Ras also down-modulates the expression of Par-4, a transcriptional repressor protein, that is essential but not sufficient on its own to induce apoptosis. Here we show that reintroduction of Par-4 by transient transfection leads to apoptosis in cells expressing oncogenic Ras but not in those that lack oncogenic Ras expression. Par-4 abrogates oncogenic Ras-inducible NF-κB transcription activity but does not interfere with cytoplasmic activation, or the DNA binding activity, of NF-κB. Because abrogation of NF-κB transcription activity is sufficient to cause apoptosis in cells expressing oncogenic Ras, our findings identify Par-4 as a novel example of a pro-apoptotic protein that selectively inhibits oncogenic Ras-dependent NF-κB function at the transcription level and suggest a mechanism by which Par-4 expression may selectively induce apoptosis in oncogenic Ras-expressing cells. The cellular Ras protein is a central point of convergence for a number of signaling pathways that originate at the cell surface and lead to phenotypic alteration in the cell (1Der C.J. Krontiris T.G. Cooper G.M. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3637-3640Crossref PubMed Scopus (506) Google Scholar, 2Denhardt D.T. Biochem. J. 1996; 318: 724-729Crossref Scopus (452) Google Scholar, 3White M.A. Nicolette C. Minden A. Polverino A. Van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (628) Google Scholar, 4Meier P. Evan G. Cell. 1998; 95: 295-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The Ras family of GTPases, which includes Ha-Ras, K-Ras, R-Ras, and N-Ras, is conserved during evolution and is important in the regulation of cellular growth, survival, and differentiation (3White M.A. Nicolette C. Minden A. Polverino A. Van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (628) Google Scholar, 4Meier P. Evan G. Cell. 1998; 95: 295-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 5Tamanoi F. Biochim. Biophys. Acta. 1988; 948: 1-15PubMed Google Scholar, 6Marshall M.S. FASEB J. 1995; 9: 1311-1318Crossref PubMed Scopus (271) Google Scholar). Certain mutations in theras gene occur at high frequency in mammalian cells resulting in transformation and malignant progression to cancer (7Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar, 8Park M. Vogelstein B. Kinzler K.W. Oncogenes. McGraw-Hill Inc., New York1998: 205-228Google Scholar). In fact, ras is the most commonly occurring oncogene in about 30% of human cancers (7Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar, 8Park M. Vogelstein B. Kinzler K.W. Oncogenes. McGraw-Hill Inc., New York1998: 205-228Google Scholar). The oncogenic effects of Ras are mediated by activation of the downstream serine/threonine kinase Raf. Mutant forms of Ras that are unable to bind to Raf but that can bind to other Ras targets are incompetent for transformation indicating that the Raf-mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase pathway is critical for transformation and malignant progression (9Vojtek A.B. Holienberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1662) Google Scholar). Both extracellular signal-regulated protein kinase mitogen-activated protein kinase-dependent and -independent pathways are induced by oncogenic Ras and involve the activation of transcription factors Ets, c-Jun, c-Myc, and NF-κB (10Marte B.M. Downward J. Trends Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (647) Google Scholar, 11Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar, 12Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 13Mayo M.W. Wang C.-Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (507) Google Scholar). NF-κB, a key regulator of cytokine-inducible gene expression (14Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5578) Google Scholar, 15Maniatis T. Science. 1997; 278: 818-819Crossref PubMed Scopus (233) Google Scholar, 16Stancovski I. Baltimore D. Cell. 1997; 91: 299-302Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 17Tartaglia L.A. Weber R.F. Figari I.S. Reynolds C. Palladino M.A. Goddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9292-9296Crossref PubMed Scopus (770) Google Scholar), serves to block the process of apoptosis (18Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2935) Google Scholar, 19Wang C.-Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2512) Google Scholar, 20Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2449) Google Scholar). NF-κB is also essential for focus formation, a hallmark of transformation, by oncogenic Ras (12Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). The most common form of NF-κB is a heterodimer consisting of p50 and RelA/p65 protein subunits (14Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5578) Google Scholar, 15Maniatis T. Science. 1997; 278: 818-819Crossref PubMed Scopus (233) Google Scholar, 16Stancovski I. Baltimore D. Cell. 1997; 91: 299-302Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 17Tartaglia L.A. Weber R.F. Figari I.S. Reynolds C. Palladino M.A. Goddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9292-9296Crossref PubMed Scopus (770) Google Scholar). When in an inactive state, this heterodimer is bound to an inhibitory molecule IκBα and restricted to the cytoplasm (14Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5578) Google Scholar). Treatment with cytokines, chemotherapeutic agents, or ionizing radiation activates a second messenger cascade that causes phosphorylation of IκBα (14Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5578) Google Scholar). This modification event is required for the dissociation, and subsequent ubiquitination and degradation, of IκBα (14Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5578) Google Scholar). RelA contains a nuclear localization signaling sequence that is exposed upon dissociation of IκBα, thereby allowing translocation of the heterodimer to the nucleus, where it executes its transcription regulatory functions (14Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5578) Google Scholar). Interestingly, NF-κB transcription activity can be induced by oncogenic Ras or -Raf, and this induction of activity is not preceded by cytoplasmic activation of NF-κB or an increase in the amount of nuclear NF-κB bound to its target response site in the DNA (12Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 13Mayo M.W. Wang C.-Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (507) Google Scholar). Most importantly, inhibition of oncogenic Ras-inducible NF-κB activation by a super-repressor form of IκBα (IκBα-SR) is sufficient to induce apoptosis (13Mayo M.W. Wang C.-Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (507) Google Scholar). Par-4 is the product of the prostate apoptosis response-4 (par-4) gene that shows widespread expression in human and rodent tissues (21Sells S.F. Wood D.P. Joshi-Barve S.S. Muthukkumar S. Jacob R.J. Crist S.A. Humphreys S. Rangnekar V.M. Cell Growth Differ. 1994; 5: 457-466PubMed Google Scholar, 22Sells S.F. Han S.-S. Muthukkumar S. Maddiwar N. Johnstone R. Boghaert E. Gillis D. Liu G. Nair P. Monnig S. Collini P. Mattson M.P. Sukhatme V.P. Zimmer S.G. Wood D.P. McRoberts J.W. Shi Y. Rangnekar V.M. Mol. Cell. Biol. 1997; 17: 3823-3832Crossref PubMed Scopus (180) Google Scholar, 23Boghaert E.R. Sells S.F. Walid A.-J. Malone P. Williams N.M. Weinstein M.H. Strange R. Rangnekar V.M. Cell Growth Differ. 1997; 8: 881-890PubMed Google Scholar, 24Rangnekar V.M. Apoptosis. 1998; 3: 61-66Crossref PubMed Scopus (33) Google Scholar). The deduced amino acid sequence ofpar-4 predicts a protein with a leucine zipper domain and nuclear localization sequences (21Sells S.F. Wood D.P. Joshi-Barve S.S. Muthukkumar S. Jacob R.J. Crist S.A. Humphreys S. Rangnekar V.M. Cell Growth Differ. 1994; 5: 457-466PubMed Google Scholar, 22Sells S.F. Han S.-S. Muthukkumar S. Maddiwar N. Johnstone R. Boghaert E. Gillis D. Liu G. Nair P. Monnig S. Collini P. Mattson M.P. Sukhatme V.P. Zimmer S.G. Wood D.P. McRoberts J.W. Shi Y. Rangnekar V.M. Mol. Cell. Biol. 1997; 17: 3823-3832Crossref PubMed Scopus (180) Google Scholar, 23Boghaert E.R. Sells S.F. Walid A.-J. Malone P. Williams N.M. Weinstein M.H. Strange R. Rangnekar V.M. Cell Growth Differ. 1997; 8: 881-890PubMed Google Scholar, 24Rangnekar V.M. Apoptosis. 1998; 3: 61-66Crossref PubMed Scopus (33) Google Scholar, 25Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 26Berra E. Diaz-Meco M.T. Moscat J. J. Biol. Chem. 1998; 273: 10792-10797Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 27Johnstone R.W. See R.H. Sells S.F. Wang J. Muthukkumar S. Englert C. Haber D.A. Licht J.D. Sugrue S.P. Roberts T. Rangnekar V.M. Shi Y. Mol. Cell. Biol. 1996; 16: 6945-6956Crossref PubMed Scopus (210) Google Scholar). When brought to the DNA as a Gal4-Par-4 fusion protein or by protein-protein interaction with the Wilms' tumor protein WT1, Par-4 represses transcription of reporter constructs with Gal4- or WT1-binding sites, respectively (27Johnstone R.W. See R.H. Sells S.F. Wang J. Muthukkumar S. Englert C. Haber D.A. Licht J.D. Sugrue S.P. Roberts T. Rangnekar V.M. Shi Y. Mol. Cell. Biol. 1996; 16: 6945-6956Crossref PubMed Scopus (210) Google Scholar). Functional studies suggest that Par-4 is not sufficient on its own to cause apoptosis but can sensitize cells to the action of apoptotic agents (22Sells S.F. Han S.-S. Muthukkumar S. Maddiwar N. Johnstone R. Boghaert E. Gillis D. Liu G. Nair P. Monnig S. Collini P. Mattson M.P. Sukhatme V.P. Zimmer S.G. Wood D.P. McRoberts J.W. Shi Y. Rangnekar V.M. Mol. Cell. Biol. 1997; 17: 3823-3832Crossref PubMed Scopus (180) Google Scholar, 23Boghaert E.R. Sells S.F. Walid A.-J. Malone P. Williams N.M. Weinstein M.H. Strange R. Rangnekar V.M. Cell Growth Differ. 1997; 8: 881-890PubMed Google Scholar) by inhibition of downstream targets that include protein kinase Cζ (23Boghaert E.R. Sells S.F. Walid A.-J. Malone P. Williams N.M. Weinstein M.H. Strange R. Rangnekar V.M. Cell Growth Differ. 1997; 8: 881-890PubMed Google Scholar, 28Berra E. Municio M.M. Sanz L. Frutos S. Diaz-Meco M.T. Moscot J. Mol. Cell. Biol. 1997; 17: 4346-4354Crossref PubMed Scopus (160) Google Scholar) or Bcl-2 (29Qiu G. Ahmed M. Sells S.F. Mohiuddin M. Weinstein M. Rangnekar V.M. Oncogene. 1999; 18: 623-631Crossref PubMed Scopus (76) Google Scholar). In the course of studies performed to determine the effect of oncogenes on Par-4 expression, we found that oncogenic Ras, -Raf, or -Src cause down-regulation of Par-4 in immortalized fibroblasts. 1Qiu, S. G., Krishnan, S., and Rangnekar, V. M. (1999) Oncogene, in press.Similarly, regulated induction of oncogenic Ras causes down-regulation of Par-4 in NIH 3T3/iRas fibroblast cells.1 Stable expression of Par-4 in NIH 3T3/iRas transfectants inhibits cellular transformation by oncogenic Ras indicating that Par-4 is a negative regulator that has to be down-regulated for cellular transformation.1 These stable NIH 3T3/iRas/Par-4 transfectants show neither inhibition of NF-κB transcription activity nor apoptosis when oncogenic Ras is induced. However, in parallel transient transfection studies, we noted that Par-4 acts to inhibit the transcriptional activity of NF-κB. Because inhibition of NF-κB by ectopic IκBα-SR is sufficient to induce apoptosis in NIH 3T3/iRas cells when oncogenic Ras is induced (13Mayo M.W. Wang C.-Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (507) Google Scholar), we sought to examine whether transient expression of Par-4, which results in inhibition of oncogenic Ras-inducible NF-κB activation, also causes apoptosis in the absence of another death signal. We present here evidence that oncogenic Ras-expressing cells but not those that lack oncogenic Ras expression show apoptosis when transfected with a Par-4 expression plasmid or an adenoviral construct. Thus, unlike Par-4 stable transfectants that neither show inhibition of NF-κB transcription activity nor undergo apoptosis when oncogenic Ras is induced, transient expression of Par-4 in cells containing oncogenic Ras is sufficient to inhibit NF-κB transcription activity and to induce apoptosis. NIH 3T3 parent and NIH 3T3/Raf cells expressing activated Raf were from Marty Mayo and Albert Baldwin, Jr., University of North Carolina, Chapel Hill, NC. NIH 3T3/iRas/Par-4 cells and NIH 3T3/iRas/vector cells, which were made by stably transfecting pCB6+/Par-4 or vector, respectively, into NIH 3T3/iRas cells, have been described.1 The NIH 3T3:iRas cell line contains a stably integrated oncogenic Ha-ras (VI2) gene under the control of an isopropyl-β-d-thiogalactopyranoside (IPTG) 2The abbreviations used are: IPTGisopropyl-β-d-thiogalactopyranosideEMSAelectrophoretic mobility shift assayTNF-αtumor necrosis factor-αCMVcytomegaloviruslucluciferase-inducible promoter.1 The luciferase (luc) reporter construct empty luc (pGL2 from Promega Corp.), or NF-κB-luc that contained two copies, in tandem, of the NF-κB-responsive element from the κ light chain enhancer placed upstream of the SV40 promoter in pGL2 were from Brett Spear, University of Kentucky. The oncogenic Ha-Ras (V12 mutant) and activated Raf (N-terminal truncated) expression constructs (31Devary Y. Rossette C. DiDinato J.A. Karin M. Science. 1993; 261: 1442-1445Crossref PubMed Scopus (577) Google Scholar) were from Michael Karin (University of California, San Diego, La Jolla, CA). The Gal4-RelA and Gal4 transactivation-deficient mutant (Gal4-TDM) driver plasmids (from M. Lienhard Schmitz, German Cancer Research Center, Heidelberg, Germany) and Gal4-Elk and Gal4-luc plasmids (from Marty W. Mayo, University of North Carolina, Chapel Hill, NC) have been described (13Mayo M.W. Wang C.-Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (507) Google Scholar, 32Schmitz M.L. dos Santos Silva M.A. Baeuerle P. J. Biol. Chem. 1995; 270: 15576-15584Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar).1 TNF-α was purchased from R & D Systems (Minneapolis, MN). IPTG was from Promega Corp. (Madison, WI). isopropyl-β-d-thiogalactopyranoside electrophoretic mobility shift assay tumor necrosis factor-α cytomegalovirus luciferase The adeno-Par-4 recombinant adenoviral construct containing theEcoRI fragment of Par-4 cDNA downstream of the tetracycline operator and the CMV promoter was constructed by using the Cre-lox recombination system (33Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar). First, the EcoRI fragment of Par-4 cDNA from pCB6+/Par-4 was subcloned into theEcoRI site of ptet-lox shuttle vector (a derivative of pCMV-Ad5 that contains the tetracycline operator). The adeno-Par-4 virus was then prepared by using the Ψ5 adenovirus and the ptet-lox-Par-4 shuttle construct in CRE8 cells, which are human embryonic kidney 293 cells containing the cre recombinase gene (33Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar). Similarly, the control adeno-green fluorescent protein virus was made after incorporating the cDNA for green fluorescent protein into the ptet-lox shuttle vector. High titers of the adenoviral constructs were prepared in 293 cells as described (33Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar), and NIH 3T3/iRas cells were co-infected with the adeno-green fluorescent protein control virus or adeno-Par-4 virus and a helper virus that expresses the chimeric transcriptional activator, composed of the tetracycline repressor and the VP16 transactivator, which can be repressed by tetracycline. Nuclear extracts were prepared from cells, and 10-μg amounts were used in reaction mixtures along with a radiolabeled NF-κB probe made from the κ light chain enhancer sequence and subjected to EMSA as described previously (34Joshi-Barve S.S. Rangnekar V.V. Sells S.F. Rangnekar V.M. J. Biol. Chem. 1993; 268: 18018-18029Abstract Full Text PDF PubMed Google Scholar). Supershift experiments were performed by using Par-4, RelA/p65, or Egr-1 polyclonal antibodies (1 μg/reaction) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cells were transfected transiently with the luc reporter and various driver plasmids, along with CMV-β-galactosidase expression construct for an internal control. Transfections were performed for 48 h as described previously (34Joshi-Barve S.S. Rangnekar V.V. Sells S.F. Rangnekar V.M. J. Biol. Chem. 1993; 268: 18018-18029Abstract Full Text PDF PubMed Google Scholar) and whole-cell protein extracts from the transfectants were examined for luc activity or β-galactosidase activity. The luc activity in each reaction was normalized with respect to the corresponding β-galactosidase activity and expressed as relative luc activity or response. Cells were infected with adenoviral-Par-4 and helper adenovirus or with helper adenovirus alone for control, or transfected with pCB6+/Par-4 or pCB6+ control plasmid, and subjected to Annexin V staining (with ApoAlert Annexin V-fluorescein isothiocyanate from CLONTECH Laboratories, Palo Alto, CA). Fluorescent labeling of membrane phosphatidylserine was visualized by using a fluorescent microscope. Our recent studies1 have shown that Par-4 is down-regulated by oncogenic Ras, and stable expression of ectopic Par-4 abrogates the ability of oncogenic Ras to form foci in monolayer cultures. To identify the molecular targets of Par-4 in the signal transduction pathway evoked by oncogenic Ras, we tested the effect of Par-4 on NF-κB activation that is considered an important mediator of focus formation and cell survival functions of oncogenic Ras (12Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 13Mayo M.W. Wang C.-Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (507) Google Scholar). These experiments used transient cotransfection of NIH 3T3 cells with reporter construct NF-κB-luc or empty luc (for control), and oncogenic Ha-Ras (V12) with either pCB6+/Par-4 or vector, and CMV-β-galactosidase. The cells were harvested 48 h after transfection and processed for luc activity. Oncogenic Ras but not vector caused strong up-regulation of NF-κB transcription activity (Fig. 1 A). Par-4 inhibited oncogenic Ras-inducible expression of NF-κB activity in a dose-dependent manner (Fig. 1 A). Oncogenic Ras did not induce luc activity from the empty luc construct that lacked the NF-κB-binding site (data not shown). Because Raf mediates the oncogenic action of Ras, we next determined whether activated Raf induced NF-κB transcription activity and whether this pathway was susceptible to Par-4 action. NIH 3T3 cells were transiently cotransfected with the NF-κB-luc reporter and with activated Raf and Par-4, vector alone, or activated Raf and vector, and β-galactosidase plasmid to normalize the transfection efficiency. As seen in Fig. 1 B, activated Raf but not the control vector caused an induction of luc activity. Cotransfection with Par-4 abrogated activated Raf-inducible expression of luc activity in a dose-dependent manner (Fig. 1 B). Moreover, the expression of β-galactosidase from the CMV-β-galactosidase construct was unaffected by Par-4 cotransfection (Fig. 1 C), indicating that Par-4 did not cause generalized inhibition of gene expression in the transfectants. Also, to ascertain that the above effects of Par-4 were not restricted to cells transiently transfected with oncogenic Ras or activated Raf, we performed experiments in NIH 3T3 cells stably expressing oncogenic Ras or activated Raf. The cells were cotransfected with vector or pCB6+/Par-4, NF-κB-luc reporter, and β-galactosidase plasmid, and then luc or β-galactosidase activity was determined. NIH 3T3 cells stably expressing oncogenic Ras or activated Raf showed strong induction of luc activity from the NF-κB-luc reporter construct relative to parent cells, and Par-4 but not vector cotransfection resulted in >90% inhibition of the luc activity (data similar to those in Fig. 1 and hence not shown). These findings suggest that Par-4 blocks Ras- and Raf-inducible signals that trigger NF-κB transcription activity. Because activated Raf enhanced NF-κB transcription activity, we determined whether activated Raf caused enhanced degradation of IκBα. Parent NIH 3T3 cells or NIH 3T3 cells stably expressing activated Raf were left untreated or treated with TNF-α for various intervals of time, and whole-cell extracts were subjected to Western blot analysis for IκBα expression. Treatment with TNF-α, which causes activation of NF-κB by phosphorylation, dissociation, and degradation of its inhibitory partner IκBα in the cytoplasm, served as a positive control. As seen in Fig. 2, although the IκBα basal levels in cells expressing activated Raf were higher relative to those in parent NIH 3T3 cells, and the kinetics of degradation of IκBα seen at 10 or 20 min treatment with TNF-α were somewhat different in the parent cells and in those expressing activated Raf, the loss of IκBα protein expression with activated Raf was comparable to that in the parent cells. Because the above experiments were performed with cells stably expressing activated Raf, we also performed experiments with NIH 3T3 cells that were transiently transfected with activated Raf or vector for control and examined the effect on IκBα degradation. These studies indicated that transiently transfected activated Raf does not cause degradation of IκBα (data not shown). These findings suggested that activated Raf, which strongly induces the transcription activity of NF-κB, does not contribute to the degradation of IκBα. To determine whether activated Raf caused increased NF-κB binding to DNA and whether inhibition of NF-κB transcription activity by Par-4 was a reflection of inhibition of NF-κB binding to DNA, we performed EMSAs. Nuclear extracts were prepared from NIH 3T3 cells or NIH 3T3/Raf cells that were exposed to TNF-α for 1 h or left untreated and subjected to EMSA by using a radiolabeled probe prepared from the NF-κB binding sequence. As seen in Fig.3, treatment of NIH 3T3 or NIH 3T3/Raf cells with TNF-α caused increased binding of NF-κB to DNA as judged by the increased intensity of the bound complex. By contrast, activated Raf itself did not increase the binding of NF-κB to DNA (Fig. 3). The supershift reactions with the various antibodies indicated that p65 but not Par-4 or Egr-1 was present in the bound complex (Fig. 3). These findings suggest that activated Raf does not increase NF-κB binding to DNA over basal levels in NIH 3T3 cells. Moreover, because activated Raf does not increase NF-κB binding to DNA, Par-4 is not expected to inhibit activated Raf-induced NF-κB transcription activity by blocking NF-κB binding to DNA. Similarly, oncogenic Ras did not increase IκB degradation or NF-κB binding to DNA (data not shown), consistent with the fact that oncogenic Ras induces NF-κB transcription activity via its downstream mediator Raf. To determine whether Par-4 blocked the activation of NF-κB transcription activity, NIH 3T3 cells were cotransfected with Gal4-RelA and Gal4-luc in the presence or absence of oncogenic Ras, activated Raf, or Par-4 expression constructs. Gal4-luc cotransfection with vector or with Gal4-TDM (which contained the DNA binding sequence but lacked the transactivation sequence of Gal4; data not shown) was used for controls. The Gal4-RelA fusion protein contained the DNA binding sequence but lacked the transactivation sequence of the yeast Gal4 transcription factor and contained the transactivation sequence but lacked the DNA binding sequence of RelA/p65 subunit of NF-κB. The rationale here was that because binding to the Gal4 reporter was solely mediated by the Gal4 component and transactivation was solely mediated by the RelA component of the Gal4-RelA fusion protein, the effect of oncogenic Ras, activated Raf, or Par-4 on the ability of RelA to cause transcriptional activation could be directly assessed by using this reporter-driver system. The transient cotransfection experiments indicated that the Gal4-luc reporter showed a low basal level expression with Gal4-RelA or vector (Fig.4). Oncogenic Ras (Fig. 4 A) or activated Raf (Fig. 4 B) enhanced the ability of Gal4-RelA to cause expression of the Gal4-luc reporter. Par-4 did not affect the basal level of reporter expression by Gal4-RelA but blocked the ability of oncogenic Ras or activated Raf to induce the Gal4-RelA-mediated luc expression (Fig. 4, A and B). We also tested the effect of Par-4 on activation of Elk transcription activity by oncogenic Ras or activated Raf. NIH 3T3 cells were transiently transfected with Gal4-Elk and Gal4-luc reporter in the presence of vector and constructs expressing oncogenic Ras, activated Raf, or Par-4. As seen in Fig. 4 C, both oncogenic Ras and activated Raf induced Gal4-luc reporter expression by Gal4-Elk, and Par-4 did not abrogate this induction. These findings suggest that Par-4 specifically blocks the oncogenic Ras- or activated Raf-inducible transcription activity of nuclear NF-κB. Our previous studies performed with Par-4-stable transfectants suggested that Par-4 was necessary for stimulus-dependent apoptosis but not sufficient on its own to induce apoptosis (22Sells S.F. Han S.-S. Muthukkumar S. Maddiwar N. Johnstone R. Boghaert E. Gillis D. Liu G. Nair P. Monnig S. Collini P. Mattson M.P. Sukhatme V.P. Zimmer S.G. Wood D.P. McRoberts J.W. Shi Y. Rangnekar V.M. Mol. Cell. Biol. 1997; 17: 3823-3832Crossref PubMed Scopus (180) Google Scholar). However, because the stable transfectants express a maximum of about 4-fold higher Par-4 relative to basal levels and are resistant to direct apoptosis by Par-4, we considered testing the effect of transient transfection of cells that express oncogenic Ras with Par-4 constructs. These experiments were primarily motivated by the observations described above suggesting that transient transfection of Par-4 caused inhibition of NF-κB transcription activity, and the fact that inhibition of oncogenic Ras inducible NF-κB activity by IκBα in the NIH 3T3 cell background has been unequivocally shown to be sufficient for induction of apoptosis (13Mayo M.W. Wang C.-Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (507) Google Scholar). These experiments used NIH 3T3/iRas cells that were transiently transfected with either vector or the pCB6+/Par-4 expression construct and then grown in the presence or absence of IPTG to induce oncogenic Ras for 24, 48, 72, or 96 h, and apoptosis was quantified by Annexin V staining. As seen in Fig.5 A, transient transfection with pCB6+/Par-4 led to an 8–10-fold increase in Par-4 expression in cells grown in the presence or absence of IPTG over basal levels in cells transfected with vector. Immunocytochemical analysis for Par-4 expression indicated that about 40% of the cells were transfected with the Par-4 expression construct (data not shown). Annexin V staining indicated that all transfectants grown in the presence or absence of IPTG showed less than 5% apoptotic cells at 24 or" @default.
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