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J Biol Chem, Vol. 274, Issue 42, 29976-29983, October 15, 1999

Oncogenic Ras Sensitizes Cells to Apoptosis by Par-4^*

Aysegul Nalca^§, Shirley Guofang Qiu, Nadia El-Guendy^§, Sumathi Krishnan, and Vivek M. Rangnekar^§¶**

From the  Department of Surgery, Division of Urology, ^§ Department of Microbiology and Immunology, ^¶ Graduate Center for Toxicology, and  Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Certain mutations in the mammalian ras gene are oncogenic and are often detected in human cancers. Oncogenic Ras induces the transcription activity of NF-B 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-B transcription activity but does not interfere with cytoplasmic activation, or the DNA binding activity, of NF-B. Because abrogation of NF-B 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-B function at the transcription level and suggest a mechanism by which Par-4 expression may selectively induce apoptosis in oncogenic Ras-expressing cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 (1-4). 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 (3-6). Certain mutations in the ras gene occur at high frequency in mammalian cells resulting in transformation and malignant progression to cancer (7, 8). In fact, ras is the most commonly occurring oncogene in about 30% of human cancers (7, 8). 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 (9). 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-B (10-13).

NF-B, a key regulator of cytokine-inducible gene expression (14-17), serves to block the process of apoptosis (18-20). NF-B is also essential for focus formation, a hallmark of transformation, by oncogenic Ras (12). The most common form of NF-B is a heterodimer consisting of p50 and RelA/p65 protein subunits (14-17). When in an inactive state, this heterodimer is bound to an inhibitory molecule IB and restricted to the cytoplasm (14). Treatment with cytokines, chemotherapeutic agents, or ionizing radiation activates a second messenger cascade that causes phosphorylation of IB (14). This modification event is required for the dissociation, and subsequent ubiquitination and degradation, of IB (14). RelA contains a nuclear localization signaling sequence that is exposed upon dissociation of IB, thereby allowing translocation of the heterodimer to the nucleus, where it executes its transcription regulatory functions (14). Interestingly, NF-B transcription activity can be induced by oncogenic Ras or -Raf, and this induction of activity is not preceded by cytoplasmic activation of NF-B or an increase in the amount of nuclear NF-B bound to its target response site in the DNA (12, 13). Most importantly, inhibition of oncogenic Ras-inducible NF-B activation by a super-repressor form of IB (IB-SR) is sufficient to induce apoptosis (13).

Par-4 is the product of the prostate apoptosis response-4 (par-4) gene that shows widespread expression in human and rodent tissues (21-24). The deduced amino acid sequence of par-4 predicts a protein with a leucine zipper domain and nuclear localization sequences (21-27). 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 (27). 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 (22, 23) by inhibition of downstream targets that include protein kinase C (23, 28) or Bcl-2 (29). 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.¹ 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-B 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-B. Because inhibition of NF-B by ectopic IB-SR is sufficient to induce apoptosis in NIH 3T3/iRas cells when oncogenic Ras is induced (13), we sought to examine whether transient expression of Par-4, which results in inhibition of oncogenic Ras-inducible NF-B 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-B 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-B transcription activity and to induce apoptosis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Lines, Plasmid and Adenoviral Constructs, and Chemical Reagents-- 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--D-thiogalactopyranoside (IPTG)²-inducible promoter.¹ The luciferase (luc) reporter construct empty luc (pGL2 from Promega Corp.), or NF-B-luc that contained two copies, in tandem, of the NF-B-responsive element from the  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 (31) 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 (13, 32).¹ TNF- was purchased from R & D Systems (Minneapolis, MN). IPTG was from Promega Corp. (Madison, WI).

The adeno-Par-4 recombinant adenoviral construct containing the EcoRI fragment of Par-4 cDNA downstream of the tetracycline operator and the CMV promoter was constructed by using the Cre-lox recombination system (33). First, the EcoRI fragment of Par-4 cDNA from pCB6+/Par-4 was subcloned into the EcoRI 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 5 adenovirus and the ptet-lox-Par-4 shuttle construct in CRE8 cells, which are human embryonic kidney 293 cells containing the cre recombinase gene (33). 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 (33), 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.

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared from cells, and 10-µg amounts were used in reaction mixtures along with a radiolabeled NF-B probe made from the  light chain enhancer sequence and subjected to EMSA as described previously (34). 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).

Transfection and Reporter Assays-- Cells were transfected transiently with the luc reporter and various driver plasmids, along with CMV--galactosidase expression construct for an internal control. Transfections were performed for 48 h as described previously (34) and whole-cell protein extracts from the transfectants were examined for luc activity or -galactosidase activity. The luc activity in each reaction was normalized with respect to the corresponding -galactosidase activity and expressed as relative luc activity or response.

Apoptosis Assays-- 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.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Par-4 Inhibits Ras- or Raf-dependent NF-B Activation Pathway-- 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-B activation that is considered an important mediator of focus formation and cell survival functions of oncogenic Ras (12, 13). These experiments used transient cotransfection of NIH 3T3 cells with reporter construct NF-B-luc or empty luc (for control), and oncogenic Ha-Ras (V12) with either pCB6+/Par-4 or vector, and CMV--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-B transcription activity (Fig. 1A). Par-4 inhibited oncogenic Ras-inducible expression of NF-B activity in a dose-dependent manner (Fig. 1A). Oncogenic Ras did not induce luc activity from the empty luc construct that lacked the NF-B-binding site (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Par-4 inhibits oncogenic Ras- or activated Raf-inducible activation of NF-B. A, NIH 3T3 cells were cotransfected with NF-B-luc (4 µg) and oncogenic Ras (4 µg), with vector (16 µg), or with different amounts (4, 8, or 16 µg) of pCB6+/Par-4 and CMV--galactosidase (to normalize the transfection frequency). Whole-cell protein extracts were prepared from the transfectants after 48 h, and luc activity was determined. B, NIH 3T3 cells were transiently cotransfected with the NF-B-luc reporter (4 µg), vector (16 µg), activated Raf (4 µg), and Par-4 (4, 8, or 16 µg) and examined for luc activity 48 h after transfection. Cotransfection with -galactosidase plasmid was used to normalize transfection efficiency. Ratios of oncogenic Ras or activated Raf plasmid DNA to Par-4 plasmid DNA are indicated. C, NIH 3T3 cells were cotransfected with the CMV--galactosidase reporter plasmid (4 µg) and with vector (4 µg), oncogenic Ras (4 µg) with vector (16 µg), oncogenic Ras (4 µg) with pCB6+/Par-4 (16 µg), activated Raf (4 µg) with vector (16 µg), activated Raf (4 µg) and pCB6+/Par-4 (16 µg), or vector (4 µg) with pCB6+/Par-4 (16 µg) as indicated. Whole-cell protein extracts were prepared from the transfectants after 48 h, and -galactosidase activity was determined. Each data bar is a mean of nine observations and from three independent experiments; error bars indicate ± S.D. (A-C).

Because Raf mediates the oncogenic action of Ras, we next determined whether activated Raf induced NF-B transcription activity and whether this pathway was susceptible to Par-4 action. NIH 3T3 cells were transiently cotransfected with the NF-B-luc reporter and with activated Raf and Par-4, vector alone, or activated Raf and vector, and -galactosidase plasmid to normalize the transfection efficiency. As seen in Fig. 1B, 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. 1B). Moreover, the expression of -galactosidase from the CMV--galactosidase construct was unaffected by Par-4 cotransfection (Fig. 1C), 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-B-luc reporter, and -galactosidase plasmid, and then luc or -galactosidase activity was determined. NIH 3T3 cells stably expressing oncogenic Ras or activated Raf showed strong induction of luc activity from the NF-B-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-B transcription activity.

Activated Raf Does Not Induce Degradation of IB-- Because activated Raf enhanced NF-B transcription activity, we determined whether activated Raf caused enhanced degradation of IB. Parent NIH 3T3 cells or NIH 3T3 cells stably expressing activated Raf were left untreated or treated with TNF- for various intervals of time, and whole-cell extracts were subjected to Western blot analysis for IB expression. Treatment with TNF-, which causes activation of NF-B by phosphorylation, dissociation, and degradation of its inhibitory partner IB in the cytoplasm, served as a positive control. As seen in Fig. 2, although the IB basal levels in cells expressing activated Raf were higher relative to those in parent NIH 3T3 cells, and the kinetics of degradation of IB seen at 10 or 20 min treatment with TNF- were somewhat different in the parent cells and in those expressing activated Raf, the loss of IB 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 IB degradation. These studies indicated that transiently transfected activated Raf does not cause degradation of IB (data not shown). These findings suggested that activated Raf, which strongly induces the transcription activity of NF-B, does not contribute to the degradation of IB.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Activated Raf does not induce IB degradation. Parent NIH 3T3 cells or NIH 3T3/Raf cells were left untreated (UT) or treated with TNF- for various intervals of time, and whole-cell extracts were processed for Western blot analysis for IB expression. The blots were probed with -actin antibody for a loading control (left panel). The signals were subjected to densitometric scanning, and the IB signal was normalized with respect to the corresponding -actin signal. The normalized data are expressed as relative IB levels (right panel).

Activated Raf Does Not Increase the DNA Binding Activity of NF-B-- To determine whether activated Raf caused increased NF-B binding to DNA and whether inhibition of NF-B transcription activity by Par-4 was a reflection of inhibition of NF-B binding to DNA, we performed EMSAs. Nuclear extracts were prepared from NIH 3T3 cells or NIH 3T3/Raf cells that were exposed to TNF- for 1 h or left untreated and subjected to EMSA by using a radiolabeled probe prepared from the NF-B binding sequence. As seen in Fig. 3, treatment of NIH 3T3 or NIH 3T3/Raf cells with TNF- caused increased binding of NF-B to DNA as judged by the increased intensity of the bound complex. By contrast, activated Raf itself did not increase the binding of NF-B 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-B binding to DNA over basal levels in NIH 3T3 cells. Moreover, because activated Raf does not increase NF-B binding to DNA, Par-4 is not expected to inhibit activated Raf-induced NF-B transcription activity by blocking NF-B binding to DNA. Similarly, oncogenic Ras did not increase IB degradation or NF-B binding to DNA (data not shown), consistent with the fact that oncogenic Ras induces NF-B transcription activity via its downstream mediator Raf.

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Raf does not induce binding of NF-B to DNA. NIH 3T3 cells or NIH 3T3/Raf cells were exposed to TNF- or vehicle (i.e. no TNF-) for 1 h. Nuclear extracts were prepared from the cells and aliquots (10 µg of protein) were subjected to EMSA by using a radiolabeled probe prepared from the NF-B binding sequence of the -light chain promoter. Supershift experiments were performed by incubating 1 µl of the appropriate antibody with the nuclear extract and radiolabeled probe in the reaction mixture. The unbound (free) probe, NF-B bound complex, and the supershifted complex are indicated.

Par-4 Inhibits RelA-dependent Transcription-- To determine whether Par-4 blocked the activation of NF-B 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-B. 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. 4A) or activated Raf (Fig. 4B) 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. 4C, 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-B.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Par-4 blocks oncogenic Ras or activated Raf-inducible NF-B transcription activity. A and B, NIH 3T3 cells were transiently cotransfected with Gal4-RelA and Gal4-luc reporter in the presence or absence of constructs expressing vector (A and B), oncogenic Ras (A), activated Raf (B), or Par-4 (A and B). C, Gal4-Elk driver plasmid and Gal4-luc reporter plasmid were transiently introduced into NIH 3T3 cells in the presence of control vector and constructs expressing oncogenic Ras or activated Raf or adeno-Par-4. Transfection with -galactosidase plasmid was used to normalize the transfection. Whole-cell extracts were prepared 48 h after transfection and processed for luc activity. Each data bar is a mean of nine observations and from three independent experiments; error bars indicate ± S.D.

Transient Expression of Par-4 Induces Apoptosis-- 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 (22). 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-B transcription activity, and the fact that inhibition of oncogenic Ras inducible NF-B activity by IB in the NIH 3T3 cell background has been unequivocally shown to be sufficient for induction of apoptosis (13). 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. 5A, 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 48 h (Fig. 5B). However, at 72 and 96 h, cells transfected with Par-4 and grown in the presence of IPTG showed about 20 and 30% apoptosis, respectively, whereas cells transfected with Par-4 but grown in the absence of IPTG showed less than 10% apoptosis. Cells transfected with vector showed about 5% apoptosis regardless of whether they were grown in the presence or absence of IPTG. These findings suggested that Par-4 expression enhances apoptosis in cells expressing oncogenic Ras.

View larger version (64K): [in this window] [in a new window]   Fig. 5.   Par-4 plasmid or adenoviral construct induces apoptosis in oncogenic Ras-expressing cells. NIH 3T3/iRas cells were transiently transfected with pCB6+/Par-4 or vector (A and B) or infected with control or Par-4-adenoviral construct (A-D), and the cells were maintained in the presence or absence of IPTG for various time intervals. A, whole-cell protein extracts were prepared at 48 h post-transfection or post-infection and examined for Par-4, oncogenic Ras, or -actin expression by Western blot analysis. B and C, the cells were subjected to Annexin V staining; and the number of Annexin V-positive cells was quantified. Each data bar is a mean of six observations; error bars indicate ± S.D. (C, data for 96-h time point after Par-4 or control adenoviral infection are shown). D, the cells were examined by light microscopy for morphological changes; data for the 96-h post-infection time point are shown.

Because of the relatively low level of transfection efficiency expected with transient transfection of plasmid DNA, we used an adenoviral Par-4 construct to ascertain expression of Par-4 in a large number of cells in the population and tested for direct apoptosis by Par-4. NIH 3T3/iRas cells were infected either with adenovirus Par-4 or with control adenovirus and plated in the absence or presence of IPTG to induce oncogenic Ras. The adeno-Par-4 construct produced about 8-fold induction of Par-4 in the presence or absence of IPTG relative to that with the control adenovirus (Fig. 5A). Immunocytochemical analysis of the infected populations indicated that over 80% of the cells infected with the adeno-Par-4 construct expressed Par-4 (data not shown). Although the percentage of cells infected with the adeno-Par-4 construct was almost twice as high as that of cells transfected with the Par-4 expression plasmid, the amount of Par-4 expression with the infection and transfection was comparable. This suggested that the Par-4-adenovirus actually caused a lower amount of Par-4 expression, but in a higher percentage of cells, relative to the Par-4 expression plasmid. When the cells were examined for apoptosis by Annexin V staining, it became apparent that in cells grown in the presence of IPTG, the adeno-Par-4 construct did not cause more than 5% apoptosis in 24 or 48 h of infection but caused about 35% apoptosis in 72 h and about 80% apoptosis in 96 h after infection (Fig. 5, B and C). By contrast, in cells grown in the absence of IPTG, the adeno-Par-4 construct did not cause more than 10% apoptosis (Fig. 5, B and C). The control virus did not cause more than 5% apoptosis in the absence or presence of IPTG (Fig. 5, B and C). Light microscopy of the infected populations indicated that the cells infected with the control adenovirus showed focus formation in the presence but not in the absence of IPTG (Fig. 5D). Cells infected with the adeno-Par-4 construct failed to show focus formation in the presence of IPTG and showed a clear decrease in cell numbers and morphological features consistent with induction of apoptosis (Fig. 5D). These studies indicated that adeno-Par-4 caused apoptosis selectively in cells grown in the presence of IPTG to induce oncogenic Ras and not in those grown in the absence of IPTG.

Because the NIH 3T3/iRas cells transiently expressing ectopic Par-4 showed apoptosis upon induction of oncogenic Ras (Fig. 5), whereas the NIH 3T3/iRas cell populations that were selected for stable expression of Par-4 did not undergo apoptosis upon induction of oncogenic Ras,¹ we examined the NF-B transcription activity in the transiently or stably transfected NIH 3T3/iRas cells in the presence of IPTG. NIH 3T3/iRas cells or NIH 3T3/iRas/Par-4 and NIH 3T3/iRas/vector stable transfectants were transiently transfected with either vector or pCB6+/Par-4 and Gal4-RelA, Gal4-luc reporter, and -galactosidase plasmid for an internal control and grown in the presence of IPTG for 48 h. As seen in Fig. 6, the NIH 3T3/iRas cells transiently transfected with Par-4 showed reduced luc activity relative to those transfected with vector. By contrast, there was no change in the relative levels of luc activity in the stable transfectants regardless of whether they constitutively expressed Par-4 or vector (Fig. 6). However, transient transfection of the stable transfectants with Par-4 but not with vector resulted in inhibition of luc activity (Fig. 6). These findings indicate that the ectopic levels of Par-4 in the stable transfectants are insufficient for inhibition of NF-B transcription activity. Consistent with this observation on a lack of inhibition of IPTG- (or oncogenic Ras) inducible NF-B transcription activity in the NIH 3T3/iRas/Par-4-stable transfectants, the cells showed no indication of apoptosis as judged by Annexin V staining when grown in the presence or absence of IPTG (negative data; hence not shown). The Par-4-stable transfectants have not undergone any selection for resistance to inhibition of NF-B transcription activity or to apoptosis by Par-4 as evident from the fact that transient transfection of Par-4 into these cells produces inhibition of NF-B transcription activity (Fig. 6) and apoptosis (data not shown). These findings indicate a correlation between inhibition of NF-B activity and induction of apoptosis by Par-4 in cells expressing oncogenic Ras.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   The ability of Par-4 to induce apoptosis correlates with suppression of oncogenic Ras-inducible NF-B transcription activity. NIH 3T3/iRas, NIH 3T3/iRas/Par-4, or NIH 3T3/iRas/vector cells were transiently transfected with pCB6+/Par-4 or vector and Gal4-RelA, Gal4-luc reporter, and -galactosidase plasmid. After transfection, the cells were maintained in the presence of IPTG to induce oncogenic Ras. Whole-cell protein extracts were prepared after 48 h, and luc activity was determined. Each data bar is a mean of six observations; error bars indicate ± S.D.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A key finding of this study is that oncogenic Ras expression sensitizes cells to apoptosis by Par-4. The oncogenic Ras-inducible cell survival pathways confer resistance to the action of apoptosis-producing cellular insults including growth factor depletion, ionizing radiation, and anti-cancer chemotherapy (34-38). In the light of the function of oncogenic Ras in cell survival, and the fact that oncogenic mutations in the ras gene are encountered in about 30% of human cancers (7, 8), the finding that oncogenic Ras-expressing cells are sensitized to Par-4-inducible apoptosis is of potential therapeutic significance.

Previously, we (22, 39, 40)¹ and others (23) have shown that Par-4 is not sufficient on its own to induce apoptosis but that it strongly enhances the effect of coactivating apoptotic signals such as those provided by intracellular calcium elevation, -amyloid, or presinelin-1 mutant protein, TNF-, doxorubicin, or serum growth factor depletion. None of the cell lines previously used expressed oncogenic Ras, and consequently none of them was directly induced to undergo apoptosis by Par-4. The expression of oncogenic Ras results in the induction of both pro- and anti-apoptotic pathways, and the anti-apoptotic functions, which include those conferred by NF-B transcription activity, are necessary to sustain cell survival (13). Abrogation of NF-B transcription activity is sufficient to abrogate survival and induce apoptosis in NIH 3T3 cells expressing oncogenic Ras (13). In the present study, Par-4 abrogated oncogenic Ras-inducible NF-B activation, and this may explain why Par-4 is sufficient on its own to induce apoptosis in the oncogenic Ras-expressing cells. Moreover, activated Raf also induced NF-B transcription activity, and Par-4 abrogated activated Raf-inducible NF-B activity (this study) and rendered activated Raf-expressing cells susceptible to apoptosis.³ Because Raf, a direct downstream target of Ras, mediates the function of oncogenic Ras, this finding further reinforces the ability of Par-4 to antagonize the protective function of the oncogenic Ras-induced pathway. Several NF-B-inducible anti-apoptotic genes such as cIAPs and TRAFs have been identified, and cIAP has been shown to complex directly with caspases and inhibit apoptosis (30). Thus, this study has identified the NF-B pathway for anti-apoptosis as a target of Par-4 action. Studies are underway to identify the NF-B-inducible genes that are affected by Par-4 action.

Unlike TNF-, which causes degradation of IB in the cytoplasm leading to activation of NF-B as evident by an increase in both DNA binding and transcription activity, oncogenic Ras or activated Raf expressed after stable or transient transfection did not cause activation of NF-B in the cytoplasm and consequently did not increase the DNA binding activity of NF-B. Consistent with previous studies (12, 13), oncogenic Ras or its downstream functional mediator Raf induced the transcription activity of NF-B. Par-4 selectively abrogated the transcription activity of NF-B by oncogenic Ras or by its downstream mediator Raf but did not affect the basal NF-B activity in cells lacking oncogenic Ras or activated Raf. Previous studies have shown that cells expressing oncogenic Ras are dependent on NF-B transcription activity for survival (13). This dependence is attributed to the fact that oncogenic Ras evokes both apoptotic and anti-apoptotic pathways, and NF-B contributes a key function in the anti-apoptotic pathway that sustains survival of cells expressing oncogenic Ras. By contrast, cells that lack oncogenic Ras expression (i.e. NIH 3T3/iRas cells not treated with IPTG) do not require high levels of NF-B activity for survival; these cells show only a basal level of NF-B transcription activity, and Par-4 does not inhibit this basal level and consequently does not induce apoptosis. The relevance of inhibition of NF-B activation by Par-4 to induction of apoptosis in oncogenic Ras-expressing cells is further substantiated by the finding that NIH 3T3/iRas/Par-4 cell populations that were obtained by selection for stable expression of ectopic Par-4 failed to inhibit NF-B activation or to undergo apoptosis in the presence of IPTG-inducible oncogenic Ras. Lack of inhibition of NF-B transcription activity by Par-4 in the stably transfected NIH 3T3/iRas/Par-4 cells exposed to IPTG is likely due to the fact that these cells express low levels (2-3-fold over basal levels) of Par-4, because transient transfection of these cells with the Par-4 expression construct, which produces an 8-10-fold higher Par-4 over basal level, resulted in inhibition of NF-B transcription activity.

Although data primarily from experiments performed in the NIH 3T3 cell background are presented here, similar observations relating inhibition of NF-B transcription activity to apoptosis by Par-4 were made in transfection experiments performed in immortalized rat fibroblast Rat 6 cells.⁴ Most interestingly, however, the use of IPTG-inducible oncogenic Ras-expressing cells allowed us to determine whether apoptosis was induced before or after the cells became transformed. Because treatment with IPTG for 3 h is sufficient to cause strong induction of oncogenic Ras,¹ but prolonged treatment with IPTG (at least 72 h) is necessary for the cells to form foci, and because apoptosis was induced by Par-4 after 72 h, cellular transformation (as judged by focus formation) by oncogenic Ras seems essential for sensitization to Par-4. This view is consistent with the fact that NIH 3T3 or Rat 6 cells constitutively expressing oncogenic Ras or activated Raf show apoptosis within 24-36 h of infection with the Par-4-expressing adenovirus (data not shown). Thus, it appears that the delay in apoptosis of NIH 3T3/iRas cells after exposure to Par-4 is a likely consequence of the time required for the cells to transform after induction of oncogenic Ras. Studies to decipher the components of the pathway by which oncogenic Ras selectively activates NF-B transcription function are currently underway, and the findings may provide valuable insights into the potential target(s) of Par-4 inhibitory action. Moreover, our studies are being expanded to determine the role of NF-B anti-apoptosis and Par-4-inducible apoptosis in oncogenic Ras function in epithelial cells, especially those from cancers of the pancreas, breast, colon, and the lung, wherein oncogenic Ras mutations are important prognostic indicators. Our present observations are of considerable relevance because a number of physiological or pharmacological regulators of Par-4 cause 5-10-fold induction of Par-4 (21-23, 25, 26, 39), and these amounts may serve to block the NF-B-dependent cell survival functions of oncogenic Ras and prevent transformation by induction of apoptosis. Moreover, down-regulation of Par-4 by oncogenic Ras is essential for transformation but not for activation of NF-B, because replenishment of Par-4 (i.e. 2-3-fold over basal level) by stable transfection blocks oncogenic Ras-induced transformation but fails to block NF-B transcription activity. Interestingly, these oncogenic Ras-expressing Par-4-stable transfectants are sensitized to the action of serum growth factor withdrawal and readily undergo apoptosis relative to cells expressing control vector.⁵ Based on these observations, we hypothesize that, unlike induction of apoptosis via an NF-B-dependent mechanism by transiently introduced Par-4 (this study), an NF-B-independent mechanism may be involved in the transformation-suppressor function¹ and apoptosis-sensitizing function¹ of Par-4 in oncogenic Ras-expressing cells. This hypothesis has to be addressed directly in in vivo models of oncogenic Ras-mediated cell survival and tumorigenesis to determine the physiological significance of the observations.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant R01 CA60872 (to V. M. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Combs Research Bldg., Rm. 303, University of Kentucky, 800 Rose St., Lexington, KY 40536. Tel.: 606-257-2677; Fax: 606-257-9608; E-mail: vmrang01@pop. uky.edu.

Contributed equally to this work.

¹ Qiu, S. G., Krishnan, S., and Rangnekar, V. M. (1999) Oncogene, in press.

³ S. G. Qiu, S. Krishnan, and V. M. Rangnekar, unpublished results.

⁴ A. Nalca and V. M. Rangnekar, unpublished data.

⁵ A. Nalca, S. Krishnan, and V. M. Rangnekar, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: IPTG, isopropyl--D-thiogalactopyranoside; EMSA, electrophoretic mobility shift assay; TNF-, tumor necrosis factor-; CMV, cytomegalovirus; luc, luciferase.

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