TIG3 Tumor Suppressor Figure 1. TIG3 decreases cell survival. Subconfluent cultures of SCC-13 cells, growing in 3.8 cm2 wells, were infected with 10 MOI tAd5-EV or tAd5-TIG3. At 0, 24, 48, and 72 h post-infection, cells were counted and lysates prepared. A TIG3 expression decreases cell number. Values are mean 6 SEM, n = 3. Those comparisons marked by an asterisk are significantly different as determined by Student’s t-test (p,0.001). B TIG3 is detected by immunoblot in tAd5-TIG3 infected SCC-13 cells. The monomer is visible at 18 kDa and the bracket indicates higher molecular weight crosslinked TIG3 [10,11]. Molecular weights are indicated to the left of the blot in kDa. doi:10.1371/journal.pone.0023230.g001 72 h, but that cell number is significantly reduced at 48 and 72 h in TIG3-expressing cells. Fig. 1B shows that TIG3 level is maximal in the infected cells by 24 and 48 h post-infection and is reduced by 72 h. In addition to the TIG3 monomer, we observe accumulation of high molecular weight forms which are thought to be covalently-crosslinked TIG3 [10–12]. As previously reported, TIG3 is expressed at low levels in most transformed cells [10,11] and therefore is not detected at time zero. TIG3 decreases cell proliferation by inhibiting cell cycle progression We next monitored cell cycle progression. We began by assessing the percentage of cells in S-phase using BrdU labeling. SCC-13 cells were infected with TIG3-expressing virus and after 24 h labeled with BrdU for 2 h before detection of BrdU and TIG3. As shown in Fig. 2A, 5462.8% (mean 6 SEM) of EV- infected cells were positive for BrdU as compared to 23%64% of TIG3-expressing cells. As shown in Fig. 2B, the most prominent cell cycle changes are a reduction in G1 and increase in subG1 events. To investigate the mechanism responsible for these changes, we measured the level of key cell cycle regulatory proteins. Fig. 2C shows that cyclin-dependent kinase (cdk1, cdk2, cdk6, cdk4) levels are not altered by TIG3, but cyclin D1 and cyclin E levels are decreased and p21 level is increased. These changes are consistent with reduced progression through the G1 phase and the G1/S transition. Fig. 2D shows that p21 mRNA level increases in parallel with the increase in p21 protein. TIG3 induces apoptosis The presence of subG1 DNA content can be associated with cell apoptosis. We therefore assessed the impact of TIG3 on apoptosis. As shown in Fig. 3A, TIG3 expression activates cleavage of procaspase 9 and procaspase 3 and generates cleaved PARP. In addition, the pro-apoptotic regulator, Bax, is increased and the prosurvival regulator, Bcl-XL, is reduced. Increased apoptosis can also be observed in situ. Fig. 3B shows that very few cleaved PARP-positive cells are observed in control cultures (161%, mean 6 SD), but that the number is markedly increased in TIG3- positive (2368%) cultures. These findings, together with the increase in subG1 DNA content (Fig. 2B), suggest that TIG3 induces apoptotic cell death. TIG3 localizes at a pericentrosomal location and causes organelle redistribution To gain insight into the TIG3 mechanism of action we monitored TIG3 subcellular location in tAd5-TIG3 virus-infected cells. As shown in Fig. 4A, TIG3 (green) accumulates along the cell periphery near the plasma membrane (arrowheads) and in a perinuclear location (arrows), and the nuclei in TIG3-expressing cells are reduced in size. To further assess the TIG3 location, cells were stained to detect pericentrin, a centrosome marker. The images in Fig. 4B reveal TIG3 staining juxtaposed with pericentrin staining (white arrow) adjacent to the nucleus (n). TIG3 (green) localizes in the general vicinity of pericentrin (red) suggesting accumulation in the vicinity of the centrosome. The centrosome is a control point for a wide range of cell functions. It functions as the microtubule organizing center (MTOC) which is the site of microtubule assembly [24,25]. In addition, it replicates during cell division and the daughter centrosomes serve to organize the mitotic spindles that guide chromosome separation [24,26]. Importantly, the MTOC serves as a control point for movement of molecular motor-carried cargo, including organelles, along the microtubules [27]. Moreover, centrosome dysfunction is associated with cell apoptosis [28]. We therefore examined whether pericentrosomal TIG3 accumulation alters microtubule distribution. As shown in Fig. 5A, the microtubules in TIG3-negative cells are spread throughout the cell in a typical lattice-type network that radiates out from the centrosome (white arrow). In contrast, in TIG3-expressing cells (black arrow indicates pericentrosomal TIG3), the microtubules localize as a band (red arrows) at the cell periphery and do not PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23230