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 p21^WAF1 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 p21^WAF1 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 p130^CAS, 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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