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Molecular Biology and Genetics |
Departments of Radiation Medicine [M. C., S. G. Q., V. M. R.] and Microbiology and Immunology [K. M. V., V. M. R.], Graduate Center for Toxicology [V. M. R.], and L. P. Markey Cancer Center [V. M. R.], University of Kentucky, Lexington, Kentucky 40536
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activity and cell membrane trafficking of Fas and FasL that leads to the activation of the Fas-Fas-associated death domain-caspase-8 pro-death pathway. Neither Fas pathway activation alone nor inhibition of NF-B activity with IB-super repressor was sufficient to induce apoptosis of prostate cancer cells. Coregulation of these two pathways was essential and sufficient for Par-4 to induce apoptosis. On the other hand, prostate cancer cells LNCaP or normal prostatic epithelial cells that were resistant to apoptosis by Par-4 did not show Fas or FasL trafficking. These findings identify a mechanism of apoptosis by Par-4 and suggest that Par-4 may have therapeutic potential. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30% of the patients develop androgen-independent disease within 3 years of treatment (3, 4, 5)
. The development of androgen-independent prostate cancer is a consequence of a lack of an apoptotic response to androgen-ablation because of mechanisms that allow survival of the cells within primary or metastatic tumors (5, 6, 7)
.
Mammalian cells use two primary pathways to implement apoptosis (8
, 9)
. An extrinsic pathway is initiated via ligation of death receptors, such as Fas or TNF-R13
at the cell surface; this recruits a molecular complex called death-inducing signaling complex involving FADD and FLICE/caspase-8 at the cytoplasmic tail of the receptor and leads to activation of caspase-8 (8)
. An intrinsic pathway is initiated by environmental or cellular stress, resulting in dysfunction of mitochondrial function and release of cytochrome c into the cytosol (9)
. The machinery essential for both the extrinsic and intrinsic pathways of apoptosis is intact in the androgen-dependent or -independent prostate cancer cells (10
, 11)
. However, the fact that prostate cancer cells can survive therapeutic regimen implies that the apoptotic machinery fails to fully execute, most likely because of antiapoptotic "roadblocks" introduced by up-regulation of cell survival mechanisms. Several potential roadblocks have been identified in prostate tumors and cell lines; these include mutant forms of p53 (6)
, loss of the dual phosphatase PTEN function leading to up-regulation of activated Akt (12
, 13)
, and elevated expression of Bcl-2 family members (6)
. Moreover, recent studies have suggested that NF-B DNA binding activity is elevated in PC-3 and DU-145 prostate cancer cell lines (14)
.
In a differential screen for proapoptotic genes in prostate cancer cells, we identified the Par-4 gene (15)
. Par-4, the product of this gene, is a 332 amino acid protein with a leucine zipper domain at its COOH terminus; Par-4 expression is not restricted to prostate cancer cells (16, 17, 18, 19)
. The Par-4 gene maps to chromosome 12q21, a region that is often deleted in pancreatic cancers and male germ cell tumors (20)
. Par-4 is down-regulated in 75% of renal cell carcinoma specimens analyzed, and replenishment of Par-4 renders the cells susceptible to the action of vincristine and TNF-
(21)
. Consistent with a tumor suppressor role for Par-4, we (22)
and Barradas et al. (23)
have recently shown that Par-4 is down-regulated by oncogenic-Ras, -Raf, or -Src. Restoration of Par-4 levels results in abrogation of oncogene-induced cellular transformation by inhibition of extracellular signal-regulated kinase expression and activity (22)
. Moreover, stable expression of ectopic Par-4 sensitizes fibroblast cell implants to apoptosis and thereby prevents tumor growth in the presence of chemotherapeutic agents (23)
. The expression of endogenous Par-4 is up-regulated 
4-fold exclusively by apoptotic insults in diverse paradigms of cancer and neurodegenerative diseases (16
, 19)
. Abrogation of endogenous Par-4 expression with an antisense oligomer or function with a dn mutant abrogates apoptosis by exogenous insults, indicating that Par-4 induction is necessary for apoptosis (16
, 19)
. We present here the evidence that transiently introduced ectopic Par-4 was sufficient to induce prostate cancer cell apoptosis and tumor regression, despite the presence of potential protective mechanisms such as Bcl-xL or Bcl-2 or the absence of wild-type p53 or PTEN function. Importantly, this action of Par-4 was selective on prostate cancer cells that show high levels of NF-B activity and uniquely involved both Fas and FasL trafficking and coparallel inhibition of NF-B activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Constructs.
pCB6+ vector, Par-4 expression plasmid, and adenoviral Par-4 or -GFP constructs were described previously (15)
.
DsRed1-Mito plasmid, which expresses a red fluorescent protein, was from CLONTECH Corp. (Palo Alto, CA). The RelA and p50 expression plasmids were from Nancy Rice (NIH, Bethesda, MD). Mutant IB (IB-SR, in which S32 and S36 phosphorylation sites are mutated, making it resistant to phosphorylation by IKK and subsequent degradation), dn-IKKß (SS/AA) plasmid construct, and adenoviral IB-SR and empty adenoviral constructs were from A. Baldwin (University of North Carolina at Chapel Hill, Chapel Hill, NC). The NF-B-luc, NF-B-CAT, mutant-NF-B-CAT, and cytomegalovirus-ß-galactosidase reporter plasmids have been described previously by us (24)
. The expression constructs for dn-FADD (
1–79), dn-FLICE/caspase-8 (C360S), dn-caspase-9 (C287A), and poxvirus inhibitor of apoptosis crmA were from Gabriel Nunez (University of Michigan, Ann Arbor, MI), and di-Fas (T225P) was from Jennifer Puck (NIH).
Antibodies and Chemical Reagents.
Polyclonal antibodies for Par-4, p50, RelA, FADD, FasL, Fas, and caveolin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), the monoclonal antibody for Fas was from Becton Dickinson Transduction Laboratories (Lexington, KY), and the actin antibody was from Sigma Chemical Co. (St. Louis, MO). The Fas antagonist antibody ZB4 and agonistic Fas antibody CH11 (purified IgM) were from Beckman Coulter Corp. (Miami, FL). The FasL decoy Fas-Fc and enhancer protein were from Alexis Corp. (San Diego, CA). The cell permeable inhibitor of caspases benzyloxy Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) was from Calbiochem Corp. (La Jolla, CA).
Transfection or Infection of Cells.
Cells were transfected with expression constructs (2 µg of DNA) or reporter constructs (1 µg of DNA) as described previously (24)
. Transfection mixtures included the DsRed1-Mito plasmid to monitor transfection efficiency. Cells were transduced with the adeno-GFP control virus or adeno-Par-4 virus as described previously (24)
. The cells were subjected to FITC-Annexin V staining (reagents from Boehringer Mannheim, Indianapolis, IN), and fluorescent labeling of cell membrane phosphatidyl-serine was visualized by fluorescent microscope. A total of 400 cells were scored, and the ratio of the number of Annexin V-positive cells and the total number of DsRed1-Mito plasmid-positive cells was calculated. Whole-cell lysates were subjected to CAT or luciferase and ß-galactosidase assays, and CAT or luc activity was normalized with respect to the corresponding ß-galactosidase activity. At least three experiments were always performed with three readings in each experiment. Each data point presented is a mean of values from all of the experiments, and error bars represent ±SDs.
Cells or tumor sections were subjected to terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labeling or to indirect immunostaining for Fas or FasL by using polyclonal or monoclonal antibodies followed by either FITC-conjugated secondary antibody and fluorescent microscopy or secondary antibody and 3–3'-diaminobenzidine (final brown stain) followed by light microscopy. To prevent trafficking of Fas or FasL, cells were treated with BFA (2 µg/ml; Sigma Chemical Co.). Cell fractionation was performed to isolate the membrane fraction (27) , and 30-µg amounts of membrane protein extracts were subjected to Western blot analysis.
Coimmunoprecipitation Studies.
After the cells were harvested, the cell surface proteins were cross-linked, and the cells were lysed as described (28)
. The lysates were precleared with normal rabbit IgG (Sigma Chemical Co.) and used for immunoprecipitation either with normal rabbit IgG or anti-Fas antibody by using protein A-Sepharose beads (Pharmacia-Biotech). The immunoprecipitates were subjected to Western blot analysis for Fas or FADD.
Animal Experiments.
PC-3 cells (5 x 106) were implanted into both flanks of 6–7-week-old male immunodeficient (nu/nu) mice by s.c. injection, and tumors were allowed to develop to a size of 0.35 cm in diameter. The tumors were injected with the Par-4 adenovirus or control adenovirus, and the tumor volume was monitored for 
21 days.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, top panel). Western blot analysis indicated comparable levels of ectopic Par-4 expression in PC-3 and LNCaP cells (Fig. 1a
, bottom panel). Cotreatment of the cells with caspase inhibitor Z-VAD-fmk or cotransfection with an expression construct for the poxvirus inhibitor of apoptosis crmA prevented apoptosis by Par-4 (Fig. 1b)
. These findings indicated that Par-4 induced apoptosis in PC-3 cells by a p53- or PTEN-independent and caspase-dependent pathway.
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. The transfection efficiency and expression of Par-4 in each cell line was comparable, as judged by ß-galactosidase assays and Western blot analysis, respectively (Fig. 1d
, top and bottom panels). Furthermore, Par-4 did not induce apoptosis in primary human prostate stromal cells or mouse embryo fibroblast cells and immortalized NIH 3T3 fibroblast cells (data not shown). These findings indicated that Par-4 induced apoptosis in androgen-independent prostate cancer cell lines but not in androgen-sensitive prostate cancer cells or normal/nontransformed cells.
Next, we determined whether Par-4 induces apoptosis and regression of solid tumors arising from PC-3 cell implants. A single injection of the Par-4 adenoviral construct caused remarkable reduction in tumor volume in 2–3 weeks (Fig. 1e)
in all of the 20 tumors tested (Table 1)
. By contrast, tumors injected with an adenovirus-producing GFP or IB-SR or an empty adenovirus continued to grow (Table 1)
. Moreover, intratumoral injection of Par-4 adenovirus, but not the control adenovirus, caused apoptosis in the tumor cells (Fig. 1f)
. These findings indicated that the apoptotic action of Par-4 was associated with tumor regression.
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B Transcription Activity.B transcription activity was the target of Par-4 action. The prioritization of NF-B transcription activity as a potential target of Par-4 was guided by our recent finding that NIH 3T3 cells expressing oncogenic Ras undergo apoptosis by ectopic Par-4 because of inhibition of NF-B transcription activity (24)
. Unlike NIH 3T3/Ras cells in which oncogenic Ras causes up-regulation of NF-B activity, the prostate cancer cells (LNCaP, PC-3, and DU-145) do not show oncogenic Ras mutations (30)
and constitute a thus far uncharacterized background for studying the underlying cause of sensitivity to Par-4. PC-3, DU-145, and TSU-Pr cells showed elevated levels of NF-B transcription activity compared with LNCaP or PrE cells (Fig. 2a
, left panel). By contrast, transfection with a mutant NF-B-CAT construct, in which the NF-B site had been scrambled, produced relatively low but equal levels of basal CAT expression in all cell lines (data not shown). Together with the comparable expression of ß-galactosidase activity in the different cell lines (Fig. 1d)
, these data suggested differential NF-B transcription activity in the different cell lines.
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B activity in PC-3, DU-145, and TSU-Pr cells but only marginally inhibited that in LNCaP or PrE cells (Fig. 2a
, right panel). Moreover, Z-VAD-fmk, which inhibited apoptosis by Par-4, did not abrogate the ability of Par-4 to inhibit NF-B activity in PC-3, DU-145, or TSU-Pr cells (data not shown). These data suggested that apoptosis by Par-4 in androgen-independent prostate cancer cells correlated with high endogenous NF-B transcription activity and inhibition of this activity by Par-4.
Inhibition of RelA-dependent Transcription Is Essential but not Sufficient for Apoptosis by Par-4.
Classical NF-B is a heterodimer consisting of p65/RelA and NF-B1/p50 subunits (31)
. To determine whether inhibition of NF-B transcription activity was necessary for Par-4-induced apoptosis in prostate cancer cells, we tested whether ectopic RelA (which can activate transcription) or ectopic p50 (which is unable to induce transcription on its own) could override the effect of Par-4. Expression of the constructs was confirmed by Western blot analysis (Fig. 2b)
. Coexpressed RelA, but not coexpressed p50, prevented apoptosis by Par-4 (Fig. 2b)
. These findings implied that inhibition of NF-B transcription activity was essential for the apoptotic action of Par-4 in prostate cancer cells.
To examine whether inhibition of NF-B transcription activity was sufficient to account for the apoptotic action of Par-4 in prostate cancer cells, we transiently transfected PC-3 cells with two different inhibitors of NF-B activity: IB-SR or dn-IKKß. Expression of IB-SR or dn-IKKß was ascertained by Western blot analysis (Fig. 2c)
. As expected, IB-SR or dn-IKKß blocked the transcription activity of NF-B (Fig. 2c)
. However, neither IB-SR nor dn-IKK induced apoptosis of PC-3 cells (Fig. 2c)
. These findings indicated that inhibition of NF-B transcription activity alone is not sufficient to cause apoptosis of prostate cancer cells.
Apoptosis by Par-4 Is Inhibited by Abrogation of the Fas Signaling Pathway.
Because IB-SR did not induce apoptosis on its own, it was obvious that in addition to inhibition of NF-B function, either activation of a pro-death pathway or inactivation of another survival pathway was essential for Par-4 to induce apoptosis. We sought to address this question by examining whether Par-4 activated a pro-death pathway. Because NF-B activity can block the apoptotic cascade induced by the death receptors, and because previous studies (10)
have shown that PC-3 are susceptible to apoptosis only by very high concentrations of anti-Fas antibody, we examined the involvement of the Fas death receptor signaling pathway in Par-4 action. PC-3 or DU-145 cells were cotransfected with vector or Par-4 and dn-FADD, dn-FLICE/caspase-8, dn-caspase-9, di-mutant of Fas, or RelA expression plasmid. The cells were left untreated or treated with Fas antagonist antibody ZB4 or FasL decoy Fas-Fc, and after 48 h, the cultures were scored for apoptosis. Par-4-inducible apoptosis was inhibited by RelA, dn-FADD, di-Fas, dn-FLICE/caspase-8, ZB4, or Fas-Fc but not by dn-caspase-9 (Fig. 3a)
. As Par-4-inducible apoptosis was inhibited by the Fas-ligand decoy Fas-Fc, a productive interaction between FasL and Fas was essential for Par-4 action. Moreover, the observations with the dn- or di-mutants indicated that the Fas/FADD/caspase-8 pathway was crucial for Par-4 action in both PC-3 and DU-145 cells. Similar to observations in PC-3 cells, RelA rescued the DU-145 cells from Par-4 action (Fig. 3a)
. These findings indicated that Par-4 induced apoptosis by inhibition of NF-B function and activation of the Fas pro-death signaling pathway.
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B inhibition and Fas activation was essential and sufficient for apoptosis by Par-4. PC-3 or DU-145 cells were transfected with vector or IB-SR construct and then either left untreated or treated with various concentrations of anti-Fas antibody CH11 to ligate Fas and trigger death receptor signaling. DU-145 cells, which are known to be resistant to Fas ligation (10)
, did not undergo apoptosis with high concentrations (100 or 200 ng/ml) of anti-Fas antibody; however, a combination of CH11 and IB-SR induced apoptosis that was comparable with that seen with Par-4 alone (Fig. 3b)
. The transfection efficiency with IB-SR was 40–50%, and this imposed an upper limit on the number of cells undergoing apoptosis with CH11 and IB-SR. In PC-3 cells, which are sensitive only to very high concentrations of Fas antibody (10)
, low concentrations of CH11 failed to induce apoptosis unless combined with IB-SR (Fig. 3b)
. Moreover, neither ectopic Fas expression nor IB-SR alone was able to induce apoptosis; cotransfection with Fas and IB-SR constructs induced apoptosis (Fig. 3b)
. Together, our findings imply that activation of Fas signaling pathway and coparallel inhibition of NF-B activity are critical for prostate cancer cell apoptosis and that both of these actions are accomplished by Par-4.
Par-4 Increases Fas and FasL Trafficking to the Cell Membrane.
FasL-dependent or -independent activation of Fas requires Fas multimerization at the cell surface (28)
. In the inactive monomeric form, Fas is heterogeneously distributed in the cytosol and at the cell surface, but on multimerization, bright staining aggregates of Fas can be detected at the cell surface. To determine whether Fas expression was enhanced at the cell surface in response to Par-4, we tested vector- and Par-4-transfected PC-3 or LNCaP cells for cellular distribution of Fas by indirect immunofluorescent analysis. PC-3 cells transfected with vector showed cytoplasmic expression of Fas, but PC-3 cells transfected with Par-4 showed expression of Fas at the cell surface (Fig. 4a)
. Par-4 did not alter the localization of Fas in LNCaP cells (Fig. 4a)
. Moreover, pretreatment of PC-3 cells with BFA, a protein-secretion inhibitor that redistributes Golgi proteins to the endoplasmic reticulum or near the microtubular organizing center (32)
, prevented cell surface translocation of Fas by Par-4 (Fig. 4b)
. We also tested tumors injected with adenoviral Par-4 or GFP constructs for trafficking of Fas. Par-4 but not the control adenovirus caused cell surface trafficking of Fas in the regressing tumors (Fig. 4c)
.
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. Moreover, tumors injected with Par-4 adenovirus showed enhanced FasL expression at the cell membrane relative to those injected with the GFP-control adenovirus (Fig. 5b)
. Pretreatment of PC-3 cells with BFA prevented FasL trafficking by Par-4 (Fig. 5a)
and also prevented apoptosis by Par-4 (Fig. 5c)
. We additionally confirmed that Par-4 causes Fas and FasL trafficking in DU-145 and TSU-Pr cells but not in PrE cells (data not shown). Together, these findings suggest that Par-4 triggers the pro-death pathway by trafficking of Fas and FasL and that resistance to Par-4-inducible apoptosis correlates with the inability of Par-4 to cause trafficking of Fas and FasL.
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, corroborating the findings of immunofluorescent analysis.
Fas activation involves the formation of a Fas-FADD complex at the cell membrane for a productive caspase-8-dependent pro-death pathway. To ascertain that Par-4 caused activation of Fas, PC-3 cells were transfected with vector or Par-4 construct, and 48 h later, the cells were subjected to immunoprecipitation of Fas, and the immunoprecipitates were examined by Western blot analysis for Fas and FADD expression. Transfection of cells with Par-4 but not vector resulted in coimmunoprecipitation of Fas and FADD (Fig. 5e)
. These findings indicated that Par-4 caused activation of the Fas-FADD pro-death pathway in PC-3 cells.
Fas Pathway Activation and Inhibition of NF-B Activity Are Both Essential for Tumor Regression by Par-4.
To determine whether Fas pathway activation and inhibition of RelA were critical in tumor regression by Par-4, we established stable cell populations of PC-3 cells transfected with dn-FADD, RelA, or vector. The cells were implanted in the flanks of nude mice, and the resultant tumors were injected with either the Par-4 adenovirus or the control adenovirus. As seen in Table 2
, adenoviral Par-4 caused regression of the tumors that arose from vector transfectants but not of those from dn-FADD or RelA transfectants. These findings indicated that tumor regression by Par-4 required activation of the Fas pro-death pathway and abrogation of NF-B activity.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B transcription activity in the cells blocked progression of the pro-apoptotic signal. NF-B transcription activity regulates the expression of inhibitor-of-apoptosis proteins, which can abrogate apoptosis by inhibition of effector caspases (33)
. Interestingly, Par-4 is equipped with NF-B inhibitory potential, and the coparallel events of inhibition of NF-B transcription activity and Fas and FasL trafficking and activation were sufficient for Par-4 to induce apoptosis. This mechanism of apoptosis induction was recapitulated in tumor regression by adenoviral delivery and expression of Par-4. Thus, unlike other proapoptotic molecules, such as TNF- or TNF-related apoptosis-inducing ligand, that signal via the death receptors but coactivate a parallel NF-B cell survival and inflammatory response that impedes apoptosis and restricts their therapeutic potential (34, 35, 36, 37, 38, 39, 40, 41, 42)
, Par-4 uniquely triggers a pro-death Fas pathway and an antisurvival NF-B inhibition pathway. Because elevated NF-B activity correlates with an increase in the aggressive traits of several cancers (34, 35, 36, 37, 38, 39, 40, 41, 42)
and because Par-4 does not cause apoptosis of normal or nontransformed cells, the findings of this study identify Par-4 as a mechanistically and functionally novel molecule with therapeutic potential.
Progression of prostate cancer is attributed to loss of apoptotic mechanisms and up-regulation of antiapoptotic mechanisms that serve to hamper the effectiveness of therapeutic protocols, including androgen-ablation, ionizing radiation, or chemotherapy (2, 3, 4, 5, 6, 7)
. Lack of wild-type p53, PTEN activity, and the presence of Bcl-2, Bcl-xL, or aberrant NF-B activity are examples of such mechanisms (6
, 12, 13, 14)
. However, PC-3 cells, which lack both wild-type p53 and PTEN activity and show aberrantly elevated NF-B activity (10
, 14
, 29)
were susceptible to apoptosis by Par-4. Moreover, PC-3/vector, PC-3/Bcl-2 (25)
, or PC-3/Bcl-xL (26)
cell populations stably expressing vector, Bcl-2, or Bcl-xL, respectively, were found to be similarly susceptible to apoptosis by Par-4 (data not shown). These findings indicated that none of the cell survival factors prevented ectopic Par-4 from producing an apoptotic response in the androgen-independent prostate cancer cells. Wild-type p53 promotes trafficking of Fas to the cell membrane and induces apoptosis (32)
. However, in cells lacking wild-type p53, Par-4 can provide similar functional outcomes. The precise molecular and biochemical mechanism(s) involved in trafficking of Fas or FasL from the Golgi to the cell membrane is currently under investigation.
Prostate cancer cells are generally resistant to anti-Fas antibody CH11-induced apoptosis (Refs. 10
and 43
and this study). The present study linked the underlying cause of Fas resistance to aberrantly elevated NF-B activity, as inhibition of the activity with IB-SR resulted in induction of apoptosis after Fas pathway activation. However, inhibition of aberrantly elevated NF-B activity alone with IB-SR, in the absence of Fas pathway activation, was not sufficient to induce apoptosis in the prostate cancer cells. This observation is in sharp contrast to that in other types of tumors in which IB-SR alone is sufficient to induce apoptosis (34, 35, 36)
. Combined activation of the Fas pathway and inhibition of NF-B activity were essential and sufficient to induce apoptosis of the androgen-independent prostate cancer cells, and Par-4 accomplished both of these functions on its own. Thus, this study has advanced our understanding of the underlying mechanism of apoptosis resistance in prostate cancer cells and identified Par-4 as a pro-death and antisurvival molecule that can cause tumor regression.
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FOOTNOTES |
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1 Supported by NIH Grants CA60872 and CA84511 (to V. M. R.). 
2 To whom requests for reprints should be addressed, at Combs Building, Room 303, University of Kentucky, 800 Rose Street, Lexington, KY 40536. Phone: (859) 257-2677; Fax: (859) 257-9608; E-mail: vmrang01{at}pop.uky.edu
3 The abbreviations used are: TNF, tumor necrosis factor; FADD, Fas-associated death domain; FLICE, FADD-like interleukin-1 converting enzyme; Par-4, prostate apoptosis response-4; dn, dominant-negative; PrE, prostate primary epithelial; di, dominant-interfering; CAT, chloramphenicol acetyltransferase; BFA, brefeldin A; GFP, green fluorescent protein.
Received 6/15/01. Accepted 7/26/01.
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< |
, PC-3/vector; •, PC-3/Par-4; 
, LNCaP/vector; 
, LNCaP/Par-4. In b, Par-4 induces caspase activation. Cells were transiently cotransfected with expression constructs for crmA, Par-4, or vector, either left untreated or treated with Z-VAD-fmk (2 µM) for 48 h, and subjected to Annexin V staining. In c, Par-4 induces apoptosis in DU-145 and TSU-Pr cells but not normal prostatic cells PrE. Cells were transfected with vector or Par-4 construct for 48 h and subjected to Annexin V staining. 
, vector; 
, Par-4. In d, ectopic Par-4 expression is comparable in the cell lines. Cells were transfected with vector or Par-4 construct and ß-galactosidase expression plasmid for 48 h and subjected to ß-galactosidase activity measurements (top panel). ß-galactosidase activity is expressed relative to that of PC-3 cells arbitrarily set at 100. Whole-cell extracts prepared from the transfected cells were examined by Western blot analysis for Par-4 or actin (bottom panel). In e, Par-4 causes regression of solid tumors. PC-3 cells were implanted by s.c. injections in both the flanks of nude mice to form solid tumors (left panel). The tumors were injected with the adenoviral Par-4 construct or with control adenoviral construct. Tumor volumes were measured at day 21 after the adenoviral injections (right panel). In f, tumor regression by Par-4 is associated with apoptosis. Tumors injected with the Par-4 adenovirus or control adenovirus were sectioned at day 16 and subjected to terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labeling.
