I has previously reported that over-expression of TIS21 inhibits 293 cells growth by delayed synthesis of cyclin E and CDK4 activity independent of pRB expression (Lim et al., 1998). I, therefore, assessed the level of cyclin E and pRB in EJ cells infected with p53 and/or TIS21. The level of pRB and cyclin E1 decreased in TIS21-independent manner in EJ cell expressing p53 (Fig.4). I further analyzed the cell cycle profile of EJ cells by flow cytometry. After infection for 2 days, majority of the p53 and the p53 plus TIS21-expressing cells progressed into S phase (30.6% vs. 33.4%, respectively), whereas S phase population of TIS21-expressing cells remained similar to that of the control (Fig. 4B). Expressions of senescence marker molecules such as pErk1/2, pGSK3b and pAkt (Fig. 4C) and generation of ROS (Fig. 4D) were increased both by p53 alone and co-expression of p53 plus TIS21, whereas expression of PARP was significantly decreased. Expression of TIS21 alone, however, did not show any difference from expression of LacZ alone.
Fig.4. Expression of p53 increased the molecular markers of cellular senescence.
Expression of cell cycle-related proteins (A) and senescent markers (C) was examined after infection with or without p53 or TIS21 at the indicated time points (A) and 2 days after infection (B-D). Cells were seeded at a density of 1 x 105 per 60 mm dish and infected with p53 and/or TIS21. Cells were harvested at 2 days after infection. At least 10,000 cells were counted in each experiment and analysis of cell cycle phase was repeated 4 times by flow cytometry.
E. Effect of p53 plus TIS21 on paxillin expression in EJ cells
It has been reported that expressions of caveolin-1 and paxillin, focal adhesion protein, regulate the morphology of senescent cells (Chen et al., 2000; Cho et al., 2004; Nishio and Inoue, 2005). I therefore assessed their expression after p53 infection into EJ cells with or without TIS21 infection. Although p53 failed to regulate caveolin-1 expression (Fig. 5A), expression of paxillin was significantly increased by p53 expression (Fig. 5B), as opposed to the slight reduction by co-expression with TIS21 (Fig. 5C). Moreover, paxillin was distributed mostly at the edge of cell membrane in the cells expressing p53 alone, whereas it was cytoplasm in the other cells (Fig. 5D). These results indicate that expression of TIS21 in EJ cells attenuated p53-enhanced focal adhesion complex formation, regulated by paxillin.
Fig. 5. Expression of p53 upregulated paxillin expression in EJ cells, but co-expression with TIS21 attenuated. Cells infected with p53 and/or TIS21 were subjected to Western blot analysis (A-C) at the indicated time points. Actin and a-tubulin were used as their loading control. (D) Six days after adenovirus infection, paxillin expression was evaluated by immunocytochemistry and confocal microscopy.
F. Knockdown of p53 restored senescent phenotype and reduced the expressions of paxillin and H-ras
To determine whether p53 regulates paxillin expression, siRNA based experiment was performed. Cells were infected with p53 and then transfected with either p53-siRNA or control siRNA, and the protein expressions were assessed. Knockdown of p53 expression abrogated the expression of paxillin in concentration dependent manner, along with cellular senescence morphology (Fig. 6A and B). Additionally, expression of H-ras, a downstream of p53, was also decreased by the elimination of p53 expression. The data clearly indicate the role of p53 in morphological changes in senescence of EJ cells. In addition, transfection with p53-siRNA over 25 nM was sufficient to eliminate ROS generation by p53 expression in EJ cells (Fig. 6C).
Fig. 6. Confirmation of p53 effect on paxillin expression by transfection of p53-siRNA.
EJ cells were infected with p53 for 4 hrs and then transfected with p53-siRNA. The cells were harvested at 2 days and subjected to immunoblot analysis (A and D). Morphological change was also assessed by microscopic examination (B and E). Intracellular ROS levels were determined by staining cells with 20 mM H2-DCFDA fluorescence dye for 15 min at 37°C and the fluorescent intensities were also quantified by flow cytometry (C).
G. Co-infected cells with p53 and TSI21 increased the apoptosis markers related with intrinsic pathway
It has been reported that p53-dependent apoptosis is dependent on Apaf-1/Caspase-9 pathway (Soengas et al., 1999) and involves mitochondrial cytochrome c release (Schuler et al., 2000). A number of p53-regulated genes with p53 response elements have been identified, and some of these represent the potential downstream mediators of p53-dependent apoptosis.
Therefore, I investigated the expressions of the p53 target genes in the apoptotic cells by p53 plus TIS21 co-expression, and found the cleaved Apaf1 and increased the expression of p53AIP1 in the cells (Fig. 7). Expressions of DR5, Fas and IGFBP3 were not changed, suggesting that p53 plus TIS21 may not induce apoptosis via intrinsic pathway. Among the known pro-apoptotic proteins, Bax expression was significantly increased in the co-infected cells (Fig. 8A). Activity of caspase 3/7 was induced by over-expression of p53 alone, however, it was higher in cells co-expressing p53 plus TIS21 (Fig. 8B). Indeed, expression of cleaved caspase 3 was more abundant in cells expressing p53 plus TIS21 along with loss of p21WAF1 expression (Fig. 8C). These findings collectively indicate that co-expression of p53 plus TIS21 induced apoptosis.
Fig. 7. Co-expression of p53 plus TIS21 increased the expression of pro-apoptotic protein, Apaf-1 and p53AIP1 in EJ cells. After infection for 2 days, cells were harvested and immunoblot analysis was performed.
Fig. 8. Co-expression of wild-type p53 with TIS21 enhanced apoptosis in EJ cells. (A) After infection for 2 days, cell lysates were prepared for detection of pro-apoptotic proteins.
Specific antibodies were used to detect the proteins of interest. (B) To determine caspase 3/7 activity, caspase-Glo 3/7 assay was performed in p53, TIS21 and p53 plus TIS21 infected EJ cells. Five thousand cells which were infected with adenovirus for 2 days were seeded in 96 wells plates. 100 ml of caspase-Glo 3/7 reagent was added directly to the cells in 96-well plates and incubated for 1 hr before recording luminescence using TD-20/20 luminometer.
For detection of cleaved caspase 3, immunoblot analysis was employed.
H. TIS21 posttranslationally modificated p53 and changes the cellular localization of p53
Protein levels and activity of p53 as a transcription factor are regulated by numerous stress-induced posttranslational modifications that converge on two distinct domains of the protein: p53 NH2-terminal region (phosphorylation site) and p53 COOH terminus (acetylation site, (Tang et al., 2006). Distinct p53 acetylation cassettes differentially influence gene-expression pattern and fate of the cells (Knights et al., 2006). So I assessed acetylation of p53 on lysine residues. Interestingly, acetylation status of p53 increased in the cells infected with p53 plus TIS21 (Fig. 9). I also observed that co-expression with p53 plus TIS21 significantly enhanced accumulation of p53 in the nuclei, as compared with that of the p53 alone (Fig. 10). Next I attempted to find out the mechanism of nuclear translocation of p53 in the co-infected cells. Two recent studies have identified that kinetic stabilization of microtubule enhances microtubule-mediated transport of p53 into the nuclei (Giannakakou et al., 2000; Rathinasamy and Panda, 2008). I also speculated a possible involvement of acetylated a-tubulin (Ac-tubulin) in the translocation of p53 in the TIS21-dependent manner.
When I measured the expression of Ac-tubulin in EJ cells co-infected with p53 plus TIS21, I observed that expression of Ac-tubulin was not changed, thus ruled out the potential (Fig. 11).
Next, I checked the localization of p53 and acetylated p53. Acetylated p53 in the cells expressing p53 did not localize in the nucleus. In cells infected with p53 plus TIS21, I observed that total p53 and acetylated p53 existed in the nucleus (Fig. 12.).
Fig. 9. Co-expression of p53 plus TIS21 in EJ cells significantly increased the acetylation of p53 on the several lysine residues. EJ cell were infected with adenovirus and harvested after 48 hrs. Acetylation level of p53 was determined by immunoblot analysis.
Fig. 10. Differential localization of p53 in the cells expressing p53 alone and cells expressing p53 plus TIS21. For visualization of localization of p53 plus TIS21, cells were fixed with 4% paraformaldehyde solution and stained with anti-p53 and anti-Flag antibodies.
Images were captured with LSM510 confocal microscope (Magnification x1600).
Fig. 11. No difference in a-tubulin acetylation the cells expressing p53 alone and the cells co-expressing p53 plus TIS21. EJ cells were harvested after infection with p53 and/or TIS21 for 48 hrs, and the cell lysates were subjected to immunoblot analysis with anti Ac-tubulin and a-Ac-tubulin antibodies.
Fig. 12. Expression of TIS21 increased the translocation of p53 into the nuclei and its acetylation. (A) To show intracellular location of total p53 and acetylated p53, cells were fixed with 4% paraformaldehyde solution and stained with anti-p53 and anti-Ac-p53K382 antibodies. Images were captured with LSM510 confocal microscope (Magnification x400).
(B) Quantification of nuclear p53 was determined by counting cells more than 1000.
I. TIS21 siRNA abrogated p53 plus TIS21-mediated apoptosis.
To confirm the contribution of TIS21 in the determination of cellular fate into apoptosis by TIS21 plus p53, exogenous TIS21 was down-regulated by transfection of TIS21-siRNAs.
EJ cells were transfected with either TIS21-siRNA (#1 and/or #2) or control siRNA, and the efficiency of knockdown was measured by RT-PCR. As shown in Fig. 13B, expression of TIS21 mRNA was downregulated in the transfected cells, whereas the expression of glyceraldehydes-3-phosphate dehydrogenase was unaffected. Notably, transfection of TIS21-siRNA exhibited significant recovery of p53-induced senescent morphology in addition to reduction of cell death (Fig. 13A). Tranfection of EJ cells co-expressing p53 plus TIS21 with
#1 and/or #2 significantly reduced the effects of p53 plus TIS21 on the cleavage of caspase 3, acetylations of p53, and loss of p21WAF1 expression (Fig. 13C). These results clearly confirmed the pro-apoptotic activity of TIS21 in the p53 plus TIS21 infected EJ cells.
Knockdown of TIS21 switches the apoptosis induced by p53 plus TIS21 into senescence.
Fig. 13. TIS21-siRNAs inhibited apoptosis in EJ cells infected with p53 plus TIS21. EJ cells were co-infected with p53 plus TIS21 for 4 h, and then transfected with TIS21-siRNAs (#1 and/or #2) for 4 h. In 48 h of the infection, cells were harvested and subjected to RT-PCR and immunoblot analyses. (A) Microscopic images of cells were taken at 2 days after p53 plus TIS21 infection. (B) RT-PCR analysis. (C) Cleaved form of caspase 3, acetylation of p53, and expression of p21WAF1 were observed 48 h of infection by immunoblot analysis.
J. p53 localized at the cytoplasm in the doxorubicin-induced senescent cells
p53 is localized at the cytoplasm in senescent EJ cells (Figs. 10 and 12). To clarify whether the cytoplasmic localization of p53 in senescent cells is a general phenomenon of senescence, I employed replicative senescent (by cell division of 70-80 doublings in culture) or doxorubicin-induced senescent human diploid fibroblasts (HDF). First, I confirmed characteristics of cell senescence such as high expressions of b-gal (Fig.14A) and SA-pERK1/2 (Fig.14B). Expression of p53 protein was not observed in the replicatively senescent cells, however, it was easily observed in the cytoplasm of the senescent cells induced by treatment with 200 ng/ml of doxorubicin for 4 hrs and then cultured for 6 days in fresh medium (Fig.14C), supporting that cytoplasmic localization of p53 may be common phenomenon observed in p53- or doxorubicin-induced senescent cells. Accumulation of p53 protein in the nuclei was about 5.4% in the cells (Fig. 14D).
Fig. 14. p53 localization in the replicative or the doxorubicin-induced senescence of HDF cells. Cells were incubated at 90 % confluency in 60 mm culture dish and then treated with 200 ng/ml doxorubicin for 4 hr. Cells were reseeded and incubated for 6 another days.
(A) SA-b-galactosidase activity (B) Senescent markers were examined. (C) Intracellular localizations of p53 protein were visualized by immunofluorescence staining using anti-p53 antibody and observed under a fluorescence microscope. (D) Quantification of nuclear p53 was determined by counting the cells. DIS: doxorubicin-induced senescence
K. TIS21 induced p53-mediated apoptosis
To confirm and elucidate induction of apoptosis by TIS21 plus p53, I infected EJ cells with p53 for 4 hr and subsequently imposed TIS21 infection at 0, 6, 12, 24 and 36 hr after p53 infection. The cells were harvested 48 hr after p53 infection. As shown in Fig. 15, expression of TIS21 induced cell death of the p53-expressing EJ cells, but not cellular senescence (Fig. 15). I also observed the increased level of cleaved caspase 3 and lysine acetylation in p53 molecule following the co-expression of TIS21 (Fig. 15C).
Fig. 15. Co-infection with p53 plus TIS21 induced apoptosis in EJ cells. EJ cells were infected with p53 for 4 hr, and the cells were subsequently infected with TIS21 for 4 hrs in 6, 12, 24, and 36 hrs of the p53 infection. All of the cells were harvested 48 hr after the p53 infection. (A) Diagram showing time for TIS21 infection. (B) Cell morphology was assessed under inverted microscope. (C) Cleaved caspase 3 and acetylated p53 were increased in the cells infected with TIS21 plus p53
L. Regulation of TIS21 stability by p53
Protein level of TIS21 in cells co-infected with p53 plus TIS21 was found to be more than that in cells transfected with TIS21 alone (Figs. 4A and 4C). I have previously reported that skp2, a downstream target of FoxM1, enhances polyubiquitnation and degradation of TIS21 (Park et al., 2009). Here, I observed that p53 decreased both protein and RNA expressions of skp2 (Figs. 16A-C). I also found that p53 downregulated the expression of FoxM1, a transcription factor for skp2 (Fig. 16D), thus p53 may contribute to the stabilization of TIS21 expression through repression of FoxM1.
Fig. 16. Inhibition of FoxM1 and Skp2 expression by p53 in EJ cells, which can stabilize TIS21 expression in the cells. Cells were harvested at the indicated times (A, B and D) and expressions of Skp2 and FoxM1 were analyzed by immunoblot analyses. (C) Total RNAs were isolated from the cells and mRNA expressions were determined by reverse-transcription PCR. GAPDH was used as an internal control.
IV. DISCUSSION
I evaluated the effect of TIS21 on senescence program induced by p53 expression in EJ cells. Considering the report that TIS21 enhances cell death in HeLa (Lim et al., 2008) and U937 (Hong et al., 2005) cells, I assumed that TIS21 expression might induce apoptosis in cancer cells which express p53-induced senescence phenotypes. The phenomenon was confirmed in the present study by subsequent infections of EJ cells, they were already expressing p53, with TIS21 virus and TIS21-siRNAs (Figs. 14 and 15). To the best of our knowledge, this is the first report indicating that the activity of TIS21 that can switch the cellular fates from p53-dependent senescence to p53-dependent apoptosis.
First, I observed that over-expression of p53 in EJ cells induced senescence; however, simultaneous infection with p53 plus TIS21 failed to manifest senescence phenotypes (Fig.
1). Senescent morphology by p53 was accompanied by the increased expression of paxillin in the cells. However, paxillin expression was significantly reduced in the co-infected cells.
Our data showed that knockdown of p53 lost senescent morphology and reduced paxillin and H-ras expressions (Fig. 6A). These results suggest that paxillin might be an important in senescent morphology. However further work is needed to get a deep insight regarding the correlation between paxillin expression and senescence phenomenon. It has been reported that degradation of focal adhesion proteins, paxillin and p130CAS, were related with caspase 3 activation (Kook et al., 2000; Shim et al., 2001), suggesting that paxillin might act as a potential substrate of active caspase 3 in the process of apoptosis.
Here, I found that TIS21 enhances posttranslational modification and nuclear accumulation of p53 in p53-infected EJ cells (Figs. 10-12). Next, I tested the mechanism of
nuclear translocation of p53 in the co-infected cells. Nuclear localization of p53 also depends on the ability of p53 to interact with microtubules, and it appears that p53 uses the microtubule networks and the molecular motor dynein to move through the cytoplasm toward the nucleus and the nuclear import machinery (Giannakakou et al., 2000;
Rathinasamy and Panda, 2008). However, in our present model, I could not observe any change in acetylation of a-tubulin (Fig.11). p53 selectively triggers either senescence or apoptosis depending on the posttranslational modification, such as acetylation or phosphorylation. Among them, lysine acetylation of p53 is very important in apoptosis, because acetylation of p53 C-terminal domain can activate the genes containing p53-specific DNA binding elements (Gu and Roeder, 1997), thus acetylation of the p53 DNA-binding domain regulates induction of apoptosis (Sykes et al., 2006). Therefore, I focused on posttranslational modification of p53, specifically, acetylation of p53. Our data showed that acetylation of p53 was increased in EJ cells infected with p53 plus TIS21 (Fig. 9). In our study, p53 is more acetylated in cells co-expressed with p53 plus TIS21 than in cells expressed with p53 alone (Fig. 12). Therefore, localization of p53 in the induced senescent cells existed mostly in the cytoplasm, whereas higher amount of p53 was observed in the nuclei of cells co-infected with p53 plus TIS21 (Fig. 10 and 11). I also examined whether our observations were general phenomenon in cellular senescence program by employing HDF cells; p53 protein was not detected by immunocytochemistry in the replicatively senescent HDF cells, whereas p53 protein was mostly present in the cytoplasm of the induced senescence of HDF cells with 5% in the nuclei (Fig.12C). These data showed possibility to have additional function of cytoplasmic p53. An emerging area of research unravels additional activities of p53 in the cytoplasm, where it triggers apoptosis (Mihara et al., 2003;
Leu et al., 2004) and inhibits autophagy (Tasdemir et al., 2008).
Histone deacetylases (HDACAs) down-regulate p53-dependent transactivation by deacetylation of p53 protein. It has been reported that HDAC regulates Apaf-1 and caspase 3 expression in the developing mouse retina (Wallace et al., 2006). Deacetylation of p53 by HDACs is likely to be a part of the mechanisms that control the physiological activity of p53 (Juan et al., 2000; Wallace and Cotter, 2009). Mammalian HDAC-1, HDAC-2, and HDAC-3 are all capable of down-regulating p53 function (Juan et al., 2000).
Co-expression of p53 plus TIS21 strongly inhibited the colony formation as compared with the expression of p53 alone (Fig. 3). Induction of either senescence or apoptosis is very important for growth inhibition of cancer cells and protects from tumor cell development. It has been reported that senescence environment contributes to alter epithelial cell, alveolar epithelial morphogenesis, functional differentiation and branching morphogenesis, via MMP3 (Parrinello et al., 2005). In other words, secretion of senescent cells is able to induce growth of cancer cells. These results suggest that induction of senescence in cancer cells may not contribute effectively to the suppression of cancer cell growth, in this respect, apoptosis may be more efficient inhibition mechanism with regard to cancer associated therapeutic actions.
In conclusion, our data showed that p53 alone induced senescence in EJ bladder carcinoma cells, however, TIS21 induced up-regulation of pro-apoptotic gene expression such as Apaf-1, p53AIP1, cleaved caspase 3, and Bax in p53-dependent manner. Taken together, acetylation of p53 was increased in EJ cells co-expressing p53 plus TIS21. Based on the evidences presented herein, I conclude that TIS21 shifts the p53-induced cellular
response from senescence to apoptosis in EJ cells, and thus acts and one of the downstream targets.
V. CONCLUSION
TIS21/BTG2/PC3 has many functions: control of cell differentiation, cell cycle regulator, transcriptional co-regulator, and apoptosis or cell death. In the present study, I showed that expression of TIS21 induced the apoptosis through p53-dependent manner. In EJ human bladder carcinoma cells, TIS21 upregulated the expression of the pro-apoptotic genes such as Aparf-1, p53AIP1, cleavage caspase 3, and Bax in a p53-dependent manner. Moreover, TIS21 induces posttranslational modifications of p53, which may be involved in the induction of apoptosis and nuclear translocation of p53 protein.
This is the first report to indicate that co-expression of TIS21 and p53 induces apoptosis of EJ human bladder carcinoma cells, whereas expression of p53 induces senescence.
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