Promyelocytic Leukemia Protein 4 Induces Apoptosis by Inhibition of Survivin Expression*

The promyelocytic leukemia protein (PML) plays an essential role in multiple pathways of apoptosis. Our previous study showed that PML enhances tumor necrosis factor-induced apoptosis by inhibiting the NFκB survival pathway. To continue exploring the mechanism of PML-induced apoptosis, we performed a DNA microarray screening of PML target genes using a PML-inducible stable cell line. We found that Survivin was one of the downstream target genes of PML. Cotransfection experiments demonstrated that PML4 repressed transactivation of the Survivin promoter in an isoform-specific manner. Western blot analysis demonstrated that induced PML expression down-regulated Survivin. Inversely, PML knockdown by siRNA up-regulated Survivin expression. A substantial increase in Survivin expression was found in PML-deficient cells. Re-expression of PML in PML–/– mouse embryo fibroblasts down-regulated the expression of Survivin. Furthermore, cells arrested at the G2/M cell cycle phase expressed a high level of Survivin and a significantly lower level of PML. Overexpression of PML in A549 cells reduced Survivin expression leading to massive apoptotic cell death associated with activation of procaspase 9, caspase 3, and caspase 7. Together, our results demonstrate a novel mechanism of PML-induced apoptosis by down-regulation of Survivin.

The promyelocytic leukemia protein (PML) 1 plays an important role in mediating cell death by apoptosis. Cells deficient in PML are resistant to apoptosis induced by multiple apoptotic stimuli (1). Previous studies revealed the existence of p53-dependent PML-induced apoptosis pathways, as well as a p53independent pathway (2,3). PML represses the transactivation function of Nur77, an essential protein necessary for apoptosis in thymocytes (4). Recently, PML has also been shown to interact with nuclear factor (NF)B, potentiating tumor necrosis factorinduced apoptosis by inhibition of the NFB survival pathway (5). In this study, PML was found to interact with RelA and possibly sequester it to PML nuclear bodies (NBs), thus preventing its binding to the cognate enhancer to activate its target genes. PML has also been reported to repress the NFB downstream target gene A20 by interfering with RelA binding to the promoter element (6). Furthermore, CHK2 interacts with and phosphorylates PML at Ser-117, which is required for ionizing radiation (IR)-induced apoptosis (7). This finding links PML function to a p53-independent, CHK2-mediated, IR-induced apoptosis through the ataxia telangiectasia mutated CHK2-PML pathway.
PML is the major component of the macronuclear structure designated PML NB, PML oncogenic domain (POD), or nuclear domain-10 (ND-10) (8). The PML gene is involved in the nonrandom chromosomal translocation t (15;17), which is found in more than 98% of patients with acute promyelocytic leukemia (APL) (8). In APL, the normal PML NBs are disrupted as a result of heterodimer formation with PML-retinoic acid receptor ␣ (RAR␣) (9). All-trans retinoic acid (ATRA) induces differentiation of APL cells and is associated with the reorganization of normal PML NBs (9 -11). PML is a multifunctional protein that plays essential roles in regulating several important cellular functions, including transcriptional regulation (12,13), translational regulation (14), Ras-induced premature senescence (15,16), alternative lengthening of telomere (17)(18)(19), DNA damage response, and genome stability (20 -22). PML regulates gene expression by interacting with and sequestering transcription factors to the PML NBs (4,5,23,24). PML also interacts with histone deacetylases (25,26) and the cAMPresponsive element-binding protein (27,28) thereby regulating gene expression by modification of chromatin structures. Recent studies have shown an important role of PML in genome stability and DNA-damage response (22,29). Many of the enzymes involved in double-strand-break DNA repair, including Brca1, ataxia telangiectasia mutated, Mre11, Rad51, Rad9, TopBP1, H2AX, and the product of the Bloom's syndrome gene, have been found to colocalize with the PML NBs in response to IR-induced DNA damage (20 -22,29). Interestingly, PML was shown to recruit the DNA damage response protein TopBP1 to the PML NBs and significantly stabilize the protein (29). PML and TopBP1 were shown to colocalize with the IR-induced single-stranded DNA at the sites of double-strand-break DNA damage repair. However, the mechanism of how PML is involved in DNA damage responses is not clear.
Survivin is a member of the inhibitor of apoptosis family of proteins. Its expression is low or undetectable in most normal adult tissues but high in most cancer cells (30,31). Overexpression of Survivin inhibits apoptosis and promotes cancer cell survival (31,32). Inhibition of Survivin expression by antisense RNA induces cell death by apoptosis (33). The mechanism through which Survivin inhibits apoptosis has been a controversial issue (34). A recent report demonstrated that Survivin does not bind caspase 3 or inhibit its activity (35). It was convincingly demonstrated that Survivin interacts with cofactor cellular hepatitis B X-interacting protein and that the heterodimer then binds to procaspase 9 and prevents it from binding to Apaf1. This study clearly showed that Survivin inhibits apoptosis by preventing the activation of caspase 9 and downstream effector caspases (35).
Regulation of Survivin gene expression involving multiple Sp1 sites has been found along the basal Survivin promoter elements. Mutagenesis analysis of these sites revealed the importance of Sp1 in the activation of the Survivin gene (36). Tumor suppressor p53 represses transcription of the Survivin gene (37,38), and the loss of function of p53 in many cancer cells may contribute to the activation of Survivin expression. Because overexpression of Survivin is cancer cell-specific, strategies for targeted disruption of Survivin function are potential tools for cancer gene therapy (39).
To continue to explore the role of PML in apoptosis, we performed a microarray analysis and identified Survivin as one of the downstream target genes of PML. Our study demonstrated an important role of PML in the repression of Survivin expression in vivo. Consistently, PML Ϫ/Ϫ mouse embryo fibroblasts (MEFs) expressed a substantially higher level of Survivin, a finding that may partially explain why these cells are resistant to multiple pathways of apoptosis (1).

EXPERIMENTAL PROCEDURES
Plasmids-The Survivin promoter-reporter constructs SpII, SpV, and SpVI were kindly provided by Dr. M. Murphy (37). The PML expression plasmids pMEP4/PML4 and pcDNA3/PML4 and their mutant forms were constructed as described previously (5). The full-length cDNAs of PML1a and PML3 were constructed by PCR amplification of the cDNAs and subcloned into the pcDNA3 vector at the BamHI/EcoRI sites. The PML2 and PML5 isoforms were constructed by PCR amplification of the 3Ј portion of the cDNAs and replaced the MluI/EcoRI fragment of pSG5/PML4 (23).
Cell Lines, Cell Culture, and Reagents-The PML4-inducible stable cell line U2OS/PML4 and the control U2OS/pMEP4 were established and cultured as described previously (5). U2OS, Saos2, MO59K, and A549 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The immortalized PML Ϫ/Ϫ MEFs and normal MEFs were established by continuous culture over a period of 2 months in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Experiments presented in Fig. 2 were performed using the immortalized cells at passage 15.
Gene Transfer and Luciferase Reporter Assay-U2OS and Saos2 cells were cultured to semiconfluence and transfected with the plasmid constructs using FuGENE 6 transfection reagent (Roche Diagnostics). Cells continued to culture in a humidified CO 2 incubator for 48 h. Total proteins were isolated, and the reporter activity in each transfection assay was determined. Luciferase activity assay was performed using a luciferase reporter assay kit (Promega Corp., Madison, WI). In each cotransfection assay, 50 ng of the expression plasmid pCMV/␤-Gal was included, and ␤-galactosidase activity was determined to normalize transfection efficiency.
Cell Death Analysis-Cell death was determined using trypan blue exclusion assay and a cell death enzyme-linked immunosorbent assay (ELISA). The cell death ELISA kit was obtained from Roche Diagnostics Corp. A549 cells were cultured to semiconfluence and infected with recombinant PML adenovirus, Ad-PML, or control virus, Ad-ASPML, as described previously (43) at a multiplicity of infection of 25. Cell death was determined 24 h after infection with the recombinant adenovirus. Cell death by apoptosis was also determined by examining the activation of caspase 9, caspase 7, and caspase 3 by Western blotting.
Small Interfering RNA Inhibition of PML Expression-Two pairs of small interfering RNA (siRNA) were synthesized (Xeragon-Qiagen, Germantown, MD) for targeting the PML mRNA: 5Ј-AAGCACGAA-GACAGACCTCTGG-3Ј and 5Ј-AACGACAGCCCAGAAGAGGAA-3Ј. Cells cultured to semiconfluence were transfected with two pairs of siRNA (3.2 g/well in a 6-well plate) using Transmessager RNA transfection reagents (Qiagen, Valencia, CA). Transfected cells continued to culture for an additional 48 h, total proteins were isolated, and the effect of siRNA on PML protein expression was determined by Western blotting.
Immunofluorescent Staining and Western Blotting-Double color immunofluorescent staining was performed using the PML polyclonal antibody raised in rabbit as described previously (29). PML and Survivin monoclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Novus (Littleton, CO), respectively.

Survivin Is a Downstream Target
Gene of PML-To identify the downstream target genes of PML, we performed a DNA microarray analysis using RNA isolated from U2OS/pMEP4 (negative control) and U2OS/PML4 established previously in our laboratory (5). Expression of PML4 was induced by treatment with 100 M Zn 2ϩ . Many genes up-regulated and downregulated by PML4 were identified. One target gene significantly down-regulated by PML4 is Survivin, which plays an important role in the inhibition of apoptosis and enhancement of cell survival. It is interesting that Survivin is not expressed in most adult tissues but is overexpressed in most cancer cells. A role of Survivin in promoting cancer cell survival and inhibition of cell death by apoptosis has been well documented (31)(32)(33)(34). We sought to further explore the effects of PML in the repression of Survivin expression and its possible function in mediating apoptosis.
Repression of Survivin Protein Expression by PML-Our previous study showed that the induced expression of PML4 in U2OS cells did not result in a significant increase in cell death in the absence of tumor necrosis factor ␣ (5). The results presented in Fig. 1A show that induced PML4 expression by Zn 2ϩ moderately decreased the protein expression of Survivin in these cells. However, a substantially lower level of Survivin protein was found after SiHa cells were treated with increasing concentrations of interferon (Fig. 1B). We next examined the effects of PML knockdown by siRNA on the expression of Survivin. The results presented in Fig. 1B show that the two sets of PML siRNAs substantially reduced the expression of PML. We found that the down-regulation of PML in SiHa cells was associated with a significant increase in Survivin expression. This result strongly supports the notion that Survivin is a direct downstream target gene of PML.
Deregulation of Survivin Gene Expression in PML-deficient Cells-We next sought to examine whether the Survivin gene is deregulated in PML-deficient cells. If PML regulates the expression of Survivin in vivo, the absence of PML should lead to increased expression of Survivin. The NB4 cell line was derived from the blast cells of a patient with APL. The PML gene is disrupted by the nonrandom chromosomal translocation t(15;17), which occurs in more than 98% of APL cases (8).
The PML gene fuses the RAR␣ gene, which encodes the leukemia-specific fusion protein PML-RAR␣. ATRA induced differentiation of NB4 cells, degradation of the PML-RAR␣ fusion protein, and reorganization of normal PML functions (8). To examine whether Survivin expression in NB4 cells was altered, Western blot analysis of Survivin expression was performed using total protein isolated from NB4 cells and cells treated with ATRA. The results presented in Fig. 1C show that the expression of Survivin protein level in NB4 cells was high and that ATRA We next examined whether expression of the Survivin gene was deregulated in PML Ϫ/Ϫ MEFs. The results presented in Fig. 2, A and B, demonstrate that the immortalized PML Ϫ/Ϫ MEFs indeed expressed substantially higher levels of Survivin protein than the normal MEFs did. Treatment of MEF cells with IR or interferon (IFN) increased the expression of PML, resulting in a reduction of Survivin expression. We next reexpressed the PML protein in PML Ϫ/Ϫ MEFs by recombinant PML adenovirus (Ad-PML)-mediated gene transfer (40). As expected, re-expression of PML in the PML Ϫ/Ϫ MEFs significantly repressed Survivin expression (Fig. 2, C and D). PML Ϫ/Ϫ cells have been shown to be resistant to multiple pathways of apoptosis (1), and the high expression of Survivin in PMLdeficient cells (Fig. 2, A-C) may have contributed to their resistance to cell death by apoptosis.
PML Represses Transactivation of the Survivin Promoter-To understand how PML4 represses expression of the Survivin gene, we analyzed the effects of PML4 on the transactivation function of the Survivin promoter. Fig. 3B shows that PML4 represses Survivin promoter activity in a dose-dependent manner. The repression effects of PML4 on the Survivin promoter and two deletion mutants (36) were analyzed in two different cell lines (Fig. 3, C and D). Significant repression of Survivin promoter activity was found in both cell lines tested. Because Saos2 cells lack p53 and retinoblastoma protein, Rb, the transcription repression of the Survivin promoter mediated by PML4 is p53-independent (Fig. 3D). To further elucidate how PML4 represses transactivation of the Survivin promoter, we performed a series of cotransfection experiments using PML4 and various PML4 mutants. The results presented in Fig. 4A demonstrate that the C-terminal portion (amino acids 555-633) of the PML4 protein is responsible for the repression effects. This finding suggests that PML repressed Survivin promoter in a PML isoform-specific manner. The cotransfection experiment was repeated using various PML isoforms (41). Expression of the respective proteins encoded from the transfected PML4 mutants and various PML isoform-specific cDNAs was verified by Western blotting (Fig. 4, B and D). The results presented in Fig. 4C show that PML4 is the only isoform that repressed transactivation of the Survivin promoter. Although p53 has been shown to be a transcriptional repressor of the Survivin gene promoter (37,38), our study revealed a PML4specific transcriptional repression of the Survivin promoter that was independent of p53 function.
Expression of PML and Survivin at Various Phases of the Cell Cycle-Survivin is not expressed in normal adult tissues but is highly expressed in many types of cancer and acute myeloid leukemia (30,42). In cancer cells, Survivin is expressed at the highest levels during the G 2 /M phase of the cell cycle (43). To further explore the inverse relationship between PML and Survivin, MO59K cells were synchronized at various phases of the cell cycle, and levels of PML and Survivin were determined by Western blotting. The results presented in Fig.  5A show that serum starving and mimosine treatment arrested 77.3 and 80.6% of the cells at the G 1 phase, hydroxyurea and thymidine arrested 86.5 and 80.1% of the cells at the S phase, and nocodazole arrested 80.5% of the cells at the G 2 /M phase. Western blot analysis demonstrated that Survivin was expressed at significantly higher levels in the G 2 /M phase, as expected (Fig. 5B). Interestingly, a significant down-regulation of PML in the G 2  phase of the cell cycle by treatment with mimosine. The cell cycle progression was monitored by flow cytometry after release from G 1 arrest (Fig. 5C). This study showed that 71.4% of the cells were at the G 2 /M phase 12 h after being released from G 1 arrest. Western blot analysis demonstrated an increase in Survivin and a significant down-regulation of PML at the G 2 /M phase. This finding provides further support for the role of PML in the negative regulation of Survivin expression in vivo.
PML Represses Survivin Expression and Induces Cell Death by Apoptosis-To further investigate the effects of PML on the expression of Survivin and apoptotic cell death, we used the cell line A549, which expresses a high level of Survivin. It has been shown previously that antisense inhibition of Survivin expression induced apoptosis in this cell line (33,44). A549 cells were infected with recombinant PML adenovirus Ad-PML (40). A significant increase in PML expression was found in these cells 36 h after infection, and a substantial reduction of Survivin expression was observed (Fig. 6A). No noticeable increase in PML or down-regulation of Survivin was found in cells infected with the adenovirus control. We next examined the effects of PML on apoptosis in these cells by trypan blue exclusion assay and by using a Cell Death Detection ELISA-plus kit (Roche Applied Science). Massive cell death was seen in PML-overexpressing cells (Fig. 6, B and C), which was associated with the activation of pro-caspase 9, caspase 7, and caspase 3. Survivin has been shown to be a potent inhibitor of pro-caspase 9 by interacting and preventing it from binding to Apaf1 (35). This study demonstrated that PML induces apoptotic cell death through the down-regulation of Survivin, a potent inhibitor of apoptosis.

DISCUSSION
Our study convincingly demonstrated that the apoptosis inhibitor Survivin gene is a direct target of PML. Survivin overexpression has been found in many types of cancers, promoting survival and contributing to the continued proliferation of these cells (45)(46)(47). Cotransfection experiments demonstrated that PML represses transactivation of the Survivin promoter. Overexpression of PML significantly reduced the expression of the Survivin protein. Inhibition of PML expression by siRNA was associated with a significant increase in Survivin expression. Furthermore, PML-deficient cells express high levels of Survivin. Adenovirus-mediated re-expression of PML in PML Ϫ/Ϫ MEFs significantly down-regulated Survivin expression in these cells. Altogether, our study demonstrated that Survivin is a downstream target gene of PML, and inhibition of the Survivin function by PML contributes to its pro-apoptotic function.
It is interesting to note, however, that in the APL-derived NB4 cells in which PML function is disrupted as a result of the t(15;17) translocation, the expression of Survivin is high. After ATRA-induced differentiation of NB4 cells, PML reorganizes into the normal functional NB (8,29), and the expression Survivin is reduced significantly (Fig. 3C). A similar high level of Survivin was also found in the bone marrow of patients with APL and responded to ATRA in a similar fashion to what we saw in NB4 (42). However, it was not clear why Survivin was also overexpressed in 16 of the 18 AML cases tested (42). There was no indication of whether PML expression was down-regulated in these cells, and it was not clear whether the overexpression of Survivin in cancer cells was related to PML. Our study showed that the variable C-terminal region of PML4 is necessary for transcriptional repression of the Survivin gene. It is therefore likely that PML represses Survivin gene expression in an isoform-specific manner. We have tested four other PML isoforms and have found that none of these isoforms repressed the Survivin gene promoter. Although it was reported previously that the mRNAs of all the PML isoforms are expressed in the same cell line, no information is currently available concerning the expression of various PML isoforms. Our Western blot analysis using the PML antibody against the common domain of all PML isoforms demonstrated that PML was expressed in cell lines expressing Survivin (data not shown). However, it is not known whether PML4 was expressed in these cells or not. The availability of PML isoformspecific antibodies will facilitate our understanding of how Survivin is regulated at the endogenous levels. Analysis of PML expression in various primary tumors has been reported. In hepatocellular carcinoma, PML was found to be delocalized to the cytoplasm, indicating a loss of PML function in these cells (48,49). It was also reported that the expression of PML was lost when malignant cells became invasive (50). Low PML expression was also found in several solid tumors (51), and it would be interesting to examine whether high levels of Survivin exist in these cells. It is important to keep in mind that Survivin expression could also be regulated in vivo by other mechanisms unrelated to PML.
It is clear that Survivin plays an important role in the inhibition of cell death, prolonging survival of these cancer cells (32,33). Inhibition of Survivin expression and its function induced cell death by apoptosis. Our findings demonstrated that PML inhibits the expression of Survivin in A549 cells and induces apoptosis (Fig. 6). This apoptotic event involved the activation of pro-caspase 9 and other effector caspases, including caspase 3 and caspase 7. Recent studies have suggested that Survivin plays a major role in inhibiting the mitochondriadependent apoptotic pathway (34,35). This inhibitory effect requires interaction of Survivin with the cofactor hepatitis B X-interacting protein. The heterodimer complex then binds to pro-caspase 9, preventing its binding to Apaf1 and inhibiting pro-caspase 9 activation. In Survivin-overexpressing cancer cells, the mitochondria-dependent apoptotic pathway is therefore impaired. Numerous reports have found that overexpression of PML in cancer cells inhibited cell/tumor growth and induced apoptosis (40,52,53). Our findings demonstrated a new mechanism of PML-induced apoptosis by repressing Survivin expression in these cancer cells. PML is one of the primary targets of IFNs, which induce apoptosis in many types of cancer cells (54). Our findings raise the possibility that IFNinduced apoptosis may be executed through PML-mediated repression of Survivin expression in cancer cells.