Tsc2 Expression Increases the Susceptibility of Renal Tumor Cells to A
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《毒物学科学杂志》
Program in Toxicology and Department of Pathology, University of Maryland, School of Medicine, Baltimore, Maryland 21201
Toxicology and Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Greenfield, Indiana 46140
ABSTRACT
Although the precise role for the tuberous sclerosis complex-2 tumor suppressor gene (Tsc2) in tumor suppression is not clear, many studies have implicated Tsc2 in the regulation of cell differentiation, cell cycle control, GTPase activity, transcription, polycystin-1 localization, and translation initiation. We propose that Tsc2 also increases susceptibility to apoptosis, and that this functional role may contribute to the tumor suppressor activity of Tsc2. We previously characterized the apoptotic response of a Tsc2-null renal tumor cell line (ERC-18) to the tumor promoter okadaic acid (OKA). In the present study, we expressed Tsc2 in ERC-18 cells and compared the effect of Tsc2 expression on apoptotic induction. Tsc2 expression increased the susceptibility of ERC-18 cells to apoptosis induced by OKA and the phosphatidylinositol-3' kinase inhibitor, LY294002. In addition, Tsc2 expression abrogated OKA-induced cell detachment of ERC-18 cells. These results indicate that the OKA-induced, caspase-independent detachment previously observed in ERC-18 cells is Tsc2-dependent, and may support an additional role for the Tsc2 in regulating cell adhesion.
Key Words: tumorigenesis; Tsc2; apoptosis; okadaic acid.
INTRODUCTION
During the multistage process of neoplastic transformation, cells acquire the ability to proliferate autonomously and survive under conditions that would normally prevent survival. These abilities are acquired through heritable or exogenously induced genetic changes that activate oncogenes or inhibit tumor suppressor genes, frequently resulting in proliferation increases or a reduced apoptotic response. Cell death by apoptosis prevents neoplastic progression by balancing increased proliferation and by preventing the survival of cells carrying oncogenic mutations (reviewed in Evan and Vousden, 2001). Defects in the normal apoptotic response may promote malignant transformation by allowing the survival of mutated progeny, promoting clonal expansion and further genomic instability.
Apoptosis plays a key role in the regulation of kidney cell number. Compensatory increases in apoptosis have been correlated with increased proliferation and tumor grade, stage, and size during renal cell carcinoma (RCC) development (Todd et al., 1996). Tuberin, the product of the tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene, also appears to play an important role in renal tumorigenesis. In humans, a genetic mutation in Tsc2 results in tuberous sclerosis, a disease syndrome that consists of hamartomatous tumors in several organs and such renal manifestations as multiple, bilateral angiomyolipomas, cystic disease, and renal cell carcinoma (Cook et al., 1996). A single germline mutation in the rat homologue of Tsc2 results in the development of renal cell carcinoma with a nearly complete penetrance in the Eker rat model (Everitt et al., 1995). These rats also develop uterine leiomyomas and splenic hemangiosarcomas (Everitt et al., 1992, 1995; Howe et al., 1995) and are highly susceptible to renal cell carcinoma induced by a variety of toxicants (Hino et al., 1993a,b; Horesovsky et al., 1994; Lau et al., 2001; Walker et al., 1992; Wolf et al., 1998).
Many studies have implicated Tsc2 in the regulation of cell differentiation (Soucek et al., 1998), cell cycle control (Soucek et al., 1997, 1998), GTPase activity (Astrinidis et al., 2002; Wienecke et al., 1995; Xiao et al., 1997; Zhang et al., 2003), transcription (Henry et al., 1998; Zhang et al., 2003), polycystin-1 localization (Kleymenova et al., 2001), and translation initiation (Dan et al., 2002; Gao et al., 2002; Goncharova et al., 2002; Inoki et al., 2002; Kenerson et al., 2002; Potter et al., 2002; Tee et al., 2002). Tsc2 has recently been shown to be a downstream component of the phosphatidylinositol-3' kinase (PI3 kinase) signaling pathway, which normally functions to regulate cell growth and promote survival (reviewed in Blume-Jensen and Hunter, 2001; Datta et al., 1999). A downstream component of this signaling pathway, Akt kinase, promotes survival by phosphorylating multiple substrates. These substrates include the pro-apoptotic proteins BAD (Zha et al., 1996) and caspase-9 (Cardone et al., 1998), and the forkhead family of transcription factors (Brunet et al., 1999). Tuberin is also reported to be an Akt substrate, and phosphorylation of tuberin by Akt promotes degradation (Dan et al., 2002; Gao et al., 2002; Goncharova et al., 2002; Inoki et al., 2002; Kenerson et al., 2002; Potter et al., 2002; Tee et al., 2002). It is unknown whether Tsc2 is involved in the anti-apoptotic function of Akt via this or any other mechanism. We propose that loss of TSC2 will decrease susceptibility of renal epithelial cells to apoptosis.
To measure the effect of Tsc2 expression on the apoptotic response of renal tumor cells, we expressed Tsc2 in Tsc2-null ERC-18 cells and compared the susceptibility of Tsc2-null and Tsc2-expressing ERC-18 cells to apoptosis induced by the phosphatase inhibitor, okadaic acid (OKA), and the PI3 kinase inhibitor, LY294002. We show that Tsc2 expression increases the susceptibility of ERC-18 cells to death induced by both compounds, and that the unique morphologic response of ERC-18 cells to OKA is Tsc2-dependent. We also show that the apoptosis-promoting function of Tsc2 does not appear to be mediated through mTOR.
MATERIALS AND METHODS
Chemicals and reagents.
Okadaic acid (OKA) was purchased from Alexis Biochemicals (San Diego, CA) and prepared as a 1 mM stock solution in ethanol. LY294002 was purchased from BIOMOL (Plymouth Meeting, PA) and prepared as a 10 mM stock in dimethyl sulfoxide (DMSO). Rapamycin was purchased as a 100 μM stock solution in methanol from Cell Signaling Technology (CST; Beverly, MA). SYTOX-green was purchased from Molecular Probes (Eugene, OR) and diluted to 0.5 mM in DMSO immediately before use. Saponin was purchased from Sigma (St. Louis, MO) and prepared as a 100 mg/ml stock solution in distilled water.
Plasmid construction.
A plasmid encoding the full-length rat Tsc2 cDNA sequence (1765 amino acid splice variant; 36) in the pCDNA3 vector (pcDNA3-Tsc2) was provided by Dr. Cheryl Walker (MD Anderson Cancer Center; Smithville, TX). A 5357 base pair HindIII fragment of pcDNA3-Tsc2 containing the Tsc2 sequence was subcloned into the HindIII site of the pCMV-Tag-4 vector (Stratagene; La Jolla, CA), to create the pCMV-Tag-Tsc2 plasmid. The plasmid encodes a 1738 amino acid tuberin construct with the C-terminal 42 amino acids removed and replaced with a FLAG-epitope tag. The pCMV-Tag-Tsc2 plasmid sequence was verified by multiple restriction endonuclease digests. A similar Tsc2 construct, lacking 55 amino acids from the C-terminus, completely inhibited N-ethyl-N-nitrosurea-induced renal carcinoma formation in transgenic Eker rats (Momose et al., 2002).
Cell line development and culture.
The Tsc2-null ERC-18 cell line was derived from renal tumors of the Eker rat (38), and was provided by Dr. Cheryl Walker. ERC-18 cells were transfected with the linearized pCMV-Tag-Tsc2 plasmid or with the linearized empty pCMV-Tag vector (control) using Effectene transfection reagent (QIAGEN; Valencia, CA) according to the manufacturer's protocol. After transfection for 48 h, cells were grown in the presence of 200 μg/ml G418 (BD Biosciences Clontech; Palo Alto, CA) for 11 days. Individual G418-resistant colonies were isolated and propagated in the presence of 200 μg/ml G418 until reaching the 60-mm dish stage, then screened by immunoblot analysis for expression of FLAG with an anti-FLAG-M2 antibody (Sigma; St. Louis, MO) diluted to a concentration of 1 μg/ml. ERC18-FLAG-2B (empty plasmid vector) and ERC18-FLAG-Tsc2 (pCMV-Tag-Tsc2) cells were routinely maintained in DF-8 media, a 1:1 mixture of Dulbecco's modified Eagle media (DMEM) and Nutrient mixture F12 (Ham) supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, 1.6 μM ferrous sulfate, 50 nM sodium selenite, 12 μM vasopressin, 10 nM cholesterol, 200 nM hydrocortisone, 1 nM tri-iodothyronine (T3), 10 pg/ml transferrin, and 25 μg/ml insulin. All cell lines were grown in a humidified atmosphere of 37°C and 5% CO2/95% room air. Mycoplasmal contamination assays were not routinely performed. Cells were grown to 90% confluence in 60-mm plastic culture dishes (immunoblot analysis) or in six-well dishes (membrane permeability assay), then incubated in low-serum treatment media (DMEM/F12, 1% FBS) for 24 h prior to treatment with OKA or LY294002. Apoptotic morphology was photographed on a Leitz Diavert inverted microscope.
Determination of clone growth rate.
ERC-18, ERC18-FLAG-2B, and ERC18-FLAG-Tsc2 cells were plated in 12-well culture dishes (1 x 104 cells per well) in complete DF-8 media. On days 1, 3, 5, and 7 after plating, cells in duplicate wells were incubated with 0.5 μM SYTOX Green (Molecular Probes; Eugene, OR) and 100 μg/ml saponin (Sigma; St. Louis, MO) for 10 min at 37°C. SYTOX green fluorescence was measured on a Cytofluor 2350 (Millipore; Bedford, MA), and fluorescence values were averaged for duplicate wells at each time point. Cell number was determined using a standard curve prepared from fluorescence intensities of known cell numbers for each cell type. The mean cell number at each time point was then determined from three independent experiments. One-week growth curves were prepared for each clone and the parent ERC-18 cell line using GraphPad Prism software (Graphpad Software, Inc.; San Diego, CA) by converting to logarithms and plotting against time. Prism software was used to generate sigmoidal concentration-response curves for each cell type using nonlinear regression. Doubling times were calculated between days 3 and 5, when all cells appeared to be in a logarithmic growth phase. Doubling time was calculated for each cell line using the equation Nt = N02tf, where Nt is the number of cells at time = t, N0 is the initial number of cells, t is the time in days, and f is the frequency of cell cycles per day.
Measurement of cell death (membrane permeability assay).
Cell membrane integrity was assessed via SYTOX-green uptake. Viable cells exclude the fluorophore, while nuclei in cells with increased membrane permeability are labeled. Cells were grown to 90% confluence in six-well culture dishes, then incubated in DMEM/F12 low-serum treatment media for 24 h. Cells were treated in low-serum media for 24 h with 0.05, 0.1, and 0.25 μM OKA or 10, 50, and 100 μM LY294002, or with an equivalent volume of vehicle (ethanol or DMSO) as a control. In rapamycin experiments, cells were pre-treated for 1 h with 10 nM rapamycin, and the inhibitor was included (at 10 nM) as a cotreatment with OKA or LY294002. After 24 h of treatment, cells were incubated with 0.5 μM SYTOX-green for 10 min prior to measuring fluorescence using a Cytofluor 2350 (Millipore; Bedford, MA). After measuring death-induced fluorescence, saponin (100 μg/ml) was added for 10 min to permeabilize all cells, and total fluorescence was measured. In each well, death-induced fluorescence values were normalized to the total cellular fluorescence, and the mean change in membrane permeability (fluorescence) from control was determined from three independent experiments. Statistically significant changes in membrane permeability from control (vehicle) were identified using randomized block analysis of variance (ANOVA) with Dunnett's multiple comparisons post-test. With each concentration, the mean change in membrane permeability was compared between cell lines using an unpaired t-test. Statistically significant differences were judged at p < 0.05.
Immunoblot analysis.
Whole cell lysates were prepared from 60-mm cultures after 10 h (OKA) or 24 h (LY294002) of treatment for analysis of caspase-3 cleavage. Floating cells were collected by centrifugation, and lysates were prepared on ice from floating and adherent cells with RIPA buffer (50 mM Tris-Cl, pH = 7.4; 150 mM NaCl; 1% Nonident P-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate; 2.5 mM sodium pyrophosphate; 1 mM -glycerolphosphate) supplemented with protease inhibitor cocktail P8340 and phosphatase inhibitor cocktails P2850 and P5726 (Sigma). Lysates were sonicated for 10 s, then clarified by centrifugation (17,000 x g, 10 min.). Protein concentrations were measured using the BCA protein assay (Pierce; Rockford, IL), and 25 μg of protein was separated by SDS–PAGE using standard techniques (Laemmli, 1970) and transferred to a PVDF membrane (Bio-Rad; Hercules, CA) in 25 mM Tris, 192 mM glycine, and 20% methanol overnight at 100 mA. Full-length and cleaved caspase-3 were detected with a caspase-3 antibody (Santa Cruz Biotechnology; Santa Cruz, CA), used at a 1:1000 dilution according to the manufacturer's protocol.
A separate lysis procedure was used to measure cytosolic BAD accumulation and phosphorylation of Akt (serine 473). Following 30 h of serum starvation (BAD) or 24 h of LY294002 treatment (phospho-Akt), cytosolic extracts were prepared as previously described (Kolb et al., 2002). Briefly, cells were placed in ice-cold MS Buffer (5 mM Tris-Cl, pH = 7.5; 210 mM mannitol; 70 mM sucrose; 1 mM EDTA; 40 μg/ml digitonin) supplemented with protease and phosphatase inhibitors, as above, and scraped into microfuge tubes. After a 10-min incubation on ice, a cytosolic extract was prepared by centrifugation at 17,000 x g for 15 min. The pellet (containing nuclei, mitochondria, and other membranes) was washed once with MS buffer, then centrifuged again (17,000 x g for 15 min ) prior to lysis with 10 volumes of RIPA buffer and sonication (10 s). Cytosolic extracts were separated by SDS–PAGE and transferred to PVDF membranes. Immunoblot analysis was performed using antibodies specific for BAD (CST; 1:500) and phospho-Akt (ser473, Santa Cruz; 1:1000) according to the manufacturers' protocol. To measure cytosolic accumulation of BAD, band intensities were quantified from scanned images using Molecular Analyst software (Bio-Rad). Mean cytosolic BAD levels from five independent experiments were compared between 2B-D and Tsc2-D cells using an unpaired t-test with statistically significant differences judged at p < 0.05.
RESULTS
Expression of Tsc2 in Tsc2-Null ERC-18 Cells
We expressed Tsc2 in an Eker renal carcinoma cell line (ERC-18) to study the effect of Tsc2 expression on the susceptibility of Eker renal tumor cells to apoptosis. The pCMV-Tag-Tsc2 construct was prepared to express the full-length rat Tsc2 cDNA (minus the C-terminal 42 amino acids) under the constitutive CMV promoter (Fig. 1A). The pCMV-Tag-Tsc2 sequence was confirmed by multiple restriction digests, and the predicted peptide sequence was analyzed for the presence of published tuberin functional domains. The product of pCMV-Tag-Tsc2 (FLAG-Tsc2, 200-kDa) includes the hamartin binding site (van Slegtenhorst et al., 1998), the Rap1GAP domain (European Chromosome 16 Tuberous Sclerosis Consortium, 1993), transcriptional activation domains AD1 and AD2 (Tsuchiya et al., 1996), the calmodulin binding site (Noonan et al., 2002), and all four required Akt phosphorylation sites (ser939, ser1086, ser1088, thr1422) (Inoki et al., 2002), but lacks the retinoid-X receptor (RXR) binding domain (Henry et al., 1998). ERC-18 cells were stably transfected with the pCMV-Tag-Tsc2 or empty pCMV-Tag plasmids, and independent clones were screened for construct expression using a FLAG-specific antibody. As shown in Figure 1B, the 200-kDa FLAG-Tsc2 construct is expressed in the ERC18-FLAG-Tsc2-D (Tsc2-D) cell line. The FLAG epitope was selected to measure construct expression in clones. The FLAG antibody had cross-reactivity with multiple bands in all cell lines tested, as evidenced by the bands between 98 and 140 kDa in Figure 1B. Similar levels of expression of these cross-reactive bands indicate equivalent protein loading between samples. The control cell line, ERC18-FLAG-2B-D (2B-D), expressing only the empty vector, was selected via resistance to G418. The 2B-D cell line does not express the 200-kDa FLAG-Tsc2 construct, as previously described (Kolb and Davis, 2004).
Expression of Tsc2 in ERC-18 Cells Alters Cell Morphology, Growth Rate, and p70S6 Kinase Phosphorylation
The characteristics of the cell lines generated above were also compared to results described in previous Tsc2-reconstitution studies. Jin et al. (1996) showed that reintroduction of Tsc2 or a C-terminal fragment in Eker renal carcinoma cells induced a flattened morphology and slowed proliferation. As shown in Figure 2A, Tsc2-D cells are less refractile and appear flatter than 2B-D cells. (The 2B-D cell line does not express the 200-kDa FLAG-Tsc2 construct, as previously described (Kolb and Davis, 2004).) One-week growth curves were prepared for each clone and the parent ERC-18 cell line (Fig. 2B), and doubling times were calculated between days 3 and 5, when all cells appeared to be in a logarithmic growth phase. The doubling time of Tsc2-D cells was 27.9 h compared to 21.4 and 20.5 h in the control 2B-D and parent ERC-18 cell lines, respectively. One-way analysis of variance showed no statistically significant differences in cell number between the cell lines on day 3, 5, or 7 (not shown). However, the shapes of the growth curves imply that Tsc2 expression may potentially slow the rate of cell turnover.
Several studies have recently shown that Tsc2-null cells have increased p70S6 kinase phosphorylation at threonine 389, and that reintroduction of Tsc2 decreases this phosphorylation (Goncharova et al., 2002; Inoki et al., 2002). As shown in Figure 2C, Tsc2-D cells showed reduced p70S6 kinase phosphorylation (thr389) when compared to 2B-D cells. Interestingly, two higher molecular weight proteins that are recognized by the phospho-p70S6 kinase antibody were also differentially phosphorylated in the cell lines (Fig. 2C). According to the antibody cross-reactivity data provided by the manufacturer, these bands most likely represent phosphorylated forms of the p85S6 kinase and protein kinase C isoforms. Taken together, these data show that expression of pCMV-Tag-Tsc2 in ERC-18 cells caused changes in cell morphology, proliferation, and p70S6 kinase phosphorylation consistent with previous studies of Tsc2 reconstitution.
Tsc2 Expression Increases the Susceptibility of ERC-18 Cells to OKA-Induced Apoptosis
In the Tsc2-null ERC-18 cell line, OKA induced extensive apoptosis with a morphologic response that was unique from the response observed in other Tsc2-expressing renal epithelial cell lines. From this observation, we inferred that there may be a difference in the susceptibility of Tsc2-null and Tsc2-expressing renal epithelial cells to OKA. To measure the effect of Tsc2 expression on susceptibility to OKA-induced apoptosis, we compared death induced in Tsc2-D cells after 24 h of exposure to 0.05, 0.1, and 0.25 μM OKA to that induced in a control cell line expressing only the empty vector (2B-D). In both cell lines, OKA induced a concentration-dependent increase in cell death after 24 h (Fig. 3A). Death was significantly increased at all concentrations when compared to cells exposed to vehicle (p < 0.01; Dunnett's multiple comparisons post-test following significant differences in randomized block ANOVA). Comparison between cell lines within each treatment group (concentration) showed that expression of Tsc2 increased susceptibility to OKA-induced death when compared to the control cell line (Fig. 3A). At the lowest concentration (0.05 μM OKA), there was no difference in the susceptibility of 2B-D and Tsc2-D cells to OKA. After 24 h of exposure with 0.1 or 0.25 μM OKA, Tsc2-D cells showed a significant increase in death when compared to the 2B-D cell line (0.1 μM, p = 0.002; 0.25 μM, p = 0.006; unpaired t-test). To confirm that apoptosis was the type of cell death caused by OKA treatment, we measured cleavage of caspase-3 after 10 h of treatment with 0.1μM and 0.25 μM OKA. We have previously shown caspase-3 is cleaved between 6 and 10 h in ERC-18 cells treated with 0.1μM OKA. As shown in Figure 3B, caspase-3 is cleaved in both cell lines after 10 h of exposure with both concentrations. The extent of cleavage was increased in the Tsc2-D cell line when compared to 2B-D cells.
The Unique Morphologic Response of ERC-18 Cells to OKA Is Tsc2-Dependent
In the initial characterization of the apoptotic response of ERC-18 cells to OKA, we described a unique morphologic response (Kolb et al., 2002). OKA-treated ERC-18 cells rapidly lost microvilli and focal adhesion structure, shrunk, rounded, and detached with minimal membrane budding. These early changes in morphology preceded classical apoptotic markers (nuclear fragmentation, membrane budding, cytochrome c release, caspase activation) by several hours. To determine if this unique morphologic response was Tsc2 dependent, we observed the early morphologic changes in our 2B-D and Tsc2-D cells treated with 0.05, 0.1, and 0.25 μM OKA for 2 h. As previously described, the Tsc2-null 2B-D (control) cell line showed shrinkage, rounding, and minimal membrane budding after 2 h of treatment with 0.1 μM OKA (Fig. 4). When treated with 0.25 μM OKA, greater than 50% of cells had detached after 2 h. This response is consistent with the response previously observed in ERC-18 cells. Conversely, Tsc2-expressing ERC-18 cells (Tsc2-D) showed apoptotic morphology that was similar to the response previously observed in other Tsc2-expressing renal epithelial cell lines. OKA-treated Tsc2-D cells remained adherent and did not round, and shrinkage was accompanied by extensive membrane budding (arrows in Fig. 4). No detachment was observed after 2 h of treatment with 0.25 μM OKA. No morphologic changes were observed in either cell line after 2 h of treatment with 0.05 μM OKA, and both cell lines progressed to detachment and secondary oncotic necrosis after 24 h of treatment in all concentrations.
Tsc2 Expression Increases the Susceptibility of ERC-18 Cells to Apoptosis Induced by LY294002
PI3 kinase promotes cell survival through activation of the anti-apoptotic kinase Akt, and inhibition of PI3 kinase activity induces apoptosis in most cell types (reviewed in Datta et al., 1999). Tsc2 has recently been shown to be a substrate for Akt, and we examined the effect of Tsc2 expression on PI3 kinase-mediated survival. To inhibit PI3 kinase activity, we treated cells with 10, 50, and 100 μM LY294002 (a specific, cell-permeable PI3 kinase inhibitor) for 24 h and measured cell death as described. As shown in Figure 5A, treatment of 2B-D and Tsc2-D cells with LY294002 for 24 h caused a marked decrease in Akt phosphorylation. In both cell lines, LY294002 induced a concentration-dependent increase in cell death (Fig. 5B), although the number of apoptotic cells was lower than observed following OKA treatment. Significant increases in death were measured after treatment with 50 and 100 μM LY294002 in all cell lines, as indicated in Figure 5B. Comparison between cell lines within each treatment group (concentration) showed that Tsc2 expression increased susceptibility to LY294002-induced death when compared to the control cell line (Fig. 5B). Tsc2-D cells showed increased death when compared to 2B-D cells after 24 h of treatment with 50 and 100 μM LY294002 (50 μM, p = 0.005; 100 μM, p = 0.006; unpaired t-test). Caspase-3 cleavage was measured after 24 h of LY294002 treatment to confirm that cell death was the result of apoptosis. As shown in Figure 5C, caspase activation was prominent and concentration dependent in the Tsc2-D cell line, which was the most susceptible to LY294002-induced death.
Tsc2-Dependent Increases in Apoptotic Susceptibility Do Not Require mTOR Activity
Several reports have recently indicated that Tsc2 functions to inhibit mTOR in the regulation of protein translation. Since the increased activity of mTOR in Tsc2-null cells has been observed and associated with increased p70S6 kinase activity, we hypothesized that the enhanced mTOR activities in Tsc2-null cells could contribute to decreased susceptibility to apoptosis.
To determine whether mTOR contributed to the observed Tsc2-dependent increase in apoptotic susceptibility, we inhibited mTOR by pretreating cells with 10 nM rapamycin, then treated cells with 0.25 μM OKA or 100 μM LY294002 in the presence of 10 nM rapamycin. If the pro-apoptotic effects of Tsc2 are mTOR dependent, rapamycin should functionally replace Tsc2 in null cells (2B-D) to inhibit mTOR, thereby increasing apoptotic susceptibility. As shown in Figure 6, mTOR inhibition with rapamycin had no effect on OKA-induced apoptosis in either cell line and actually inhibited the apoptotic response of 2B-D cells to LY294002 (p < 0.01; unpaired t-test).
DISCUSSION
While intensive effort has been focused on determining the function of Tsc2 in the regulation of cell proliferation and growth, little is known about the effects of the tumor suppressor on apoptosis. A decreased apoptotic response in Tsc2-null renal epithelial cells could promote tumor progression by failing to: (1) induce death in the absence of survival stimuli, (2) constrain cell proliferation in the presence of tumor promoters, or (3) eliminate mutated cells following genotoxic insult. In the present study, we compared the susceptibility of Tsc2-null and Tsc2-expressing ERC-18 cells to apoptosis induced by two different compounds and showed that Tsc2 expression increased the apoptotic response of ERC-18 cells to both treatments.
We initially characterized differences in apoptotic susceptibility of Tsc2-null and Tsc2-expressing cells using OKA, a compound that consistently induces apoptosis in renal epithelial cells in vitro (Davis et al., 1994, 1996), including the ERC-18 tumor cell line (Kolb et al., 2002). Our results show that expression of Tsc2 in ERC-18 cells increased susceptibility to OKA-induced apoptosis. OKA inhibits the activities of the phosphatases PP1 and PP2A, and OKA-induced apoptosis in renal epithelial cells appears to involve the concurrent activation of multiple kinases, including protein kinase C (PKC), Raf-1, extracellular signal-regulated kinases 1 and 2 (ERK-1/2), jun N-terminal kinase (JNK), and p38 (Davis and Carbott, 1999; Davis et al., 1996). The present studies show that OKA-induced apoptosis in renal epithelial cells can be regulated by Tsc2.
The synthetic quercetin derivative LY294002 inhibits PI3 kinase and may promote cell death via inhibition of Akt activity. Since multiple studies have shown Tsc2 to be an Akt substrate (Dan et al., 2002; Inoki et al., 2002; Tee et al., 2002), we wanted to know if expression of the tumor suppressor would influence PI3 kinase inhibitor-mediated cell death. While LY294002 induced apoptosis in a small but significant population of Tsc2-null cells, expression of Tsc2 in ERC-18 cells increased susceptibility to LY294002-induced death. These results imply that Tsc2-expressing cells may be more dependent on PI3 kinase-mediated signaling for survival than Tsc2-null renal tumor cells. The decreased apoptotic response of Tsc2-null cells to PI3 kinase inhibition may have important implications for tumor progression, since survival signals normally activating PI3 kinase may become limiting as cell proliferation and tumor growth continues. Tsc2-null cells would therefore be at a distinct survival advantage under these conditions due to their decreased apoptotic susceptibility.
Although we have not determined the precise mechanistic role played by the Tsc2 in cell death, we have data that imply the mechanism does not involve mTOR. Tuberin has been shown to inhibit mTOR activity (Gao et al., 2002; Inoki et al., 2002; Kenerson et al., 2002; Tee et al., 2002), and tuberin loss results in rapamycin-sensitive increases in p70S6 kinase activity (Goncharova et al., 2002; Inoki et al., 2002). Phosphorylation of BAD by p70S6 kinase is rapamycin-sensitive and has been reported to promote cell survival (Harada et al., 2001). Therefore, it was reasonable to expect that inhibition of mTOR by rapamycin would mimic the pro-apoptotic effects of Tsc2 in null cells if the tumor suppressor promotes the chemical-induced apoptosis observed via inhibition of mTOR. However, rapamycin did not increase the susceptibility of Tsc2-null cells to apoptosis induced by either compound. Although a recent study showed that short-term rapamycin treatment induced apoptosis in Eker rat renal tumors in vivo (Kenerson et al., 2002), the concentrations of rapamycin used in our study did not induce apoptosis in our cell lines at the times measured. However, both studies strongly support a role for a reduced apoptotic response in the pathogenesis of Tsc2-null renal tumors. Additional study is needed to reveal additional mechanistic links between tuberin and apoptosis in renal epithelial cells.
Our previous characterization of OKA-induced apoptosis in ERC-18 cells showed a unique early morphologic response that was caspase independent (Kolb et al., 2002). In the present study, we show that these early morphologic changes (shrinkage, rounding, detachment) are also Tsc2 dependent. While the Tsc2-null control cell line (2B-D) showed early morphologic changes identical to those observed in ERC-18 cells when exposed to OKA, expression of Tsc2 abrogated this effect and produced a morphological response more characteristic of that observed in other Tsc2-competent renal epithelial cells. These findings indicate that Tsc2 expression may regulate shrinkage, rounding, and detachment in renal epithelial cells. Our results also provide additional support for the hypothesis that Tsc2 plays a role in regulation of cell adhesion. Astrinidis et al. (2002) recently reported that tuberin regulates cell adhesion, migration, and activation of the Rho GTPase in renal epithelial cells and in a Tsc2-null cell line derived from an Eker rat leiomyoma. These findings may be particularly important in TSC-associated diseases of smooth muscle cell origin like malignant renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). A model for LAM pathogenesis has been proposed where Tsc2-null smooth muscle cells migrate from the kidney to the lung (Carsillo et al., 2000; Yu et al., 2001).
Our results may have important implications for the progression of both spontaneous and chemically-induced Eker rat renal carcinomas. A decreased apoptotic response in Tsc2-null or Tsc2-deficient renal epithelial cells can contribute to the progression of both types of tumors. Furthermore, decreased dependence of Tsc-2 deficient cells on PI3 kinase-mediated survival signals might potentiate renal carcinogenesis or may facilitate tumor progression when growth factors are limiting. A general decrease in the apoptotic response may also contribute to chemical carcinogenesis by preventing deletion of mutated cells following genotoxic exposures or by failing to balance proliferative increases induced by nongenotoxic chemicals. Additional studies on the mechanism of Tsc2-dependent apoptotic regulation may therefore provide a better understanding of Tsc2 tumor suppressor function.
In summary, we have shown that expression of Tsc2 in ERC-18 cells increases susceptibility to apoptosis induced by the tumor promoter OKA and the PI3 kinase inhibitor LY294002. Although the precise mechanism by which Tsc2 promotes apoptotic susceptibility to these two compounds remains unclear, our data indicate that alterations in mTOR signaling pathways are not required. In addition, we have shown that Tsc2 expression abrogates the OKA-induced–caspase-independent detachment of Tsc2-null ERC-18 cells. Tsc2-dependent changes in cell adhesion properties may have important implications in determining the cellular susceptibility to apoptosis. Additional studies are required to more specifically address the mechanistic relationship between Tsc2 expression, apoptotic susceptibility, and cell adhesion.
ACKNOWLEDGMENTS
This work was supported by pre-doctoral fellowship ES05925 to T.M.K. from the National Institute of Environmental Health Sciences (NIEHS), and by NIH grant ES08157 to M.A.D. Conflict of interest: none declared.
REFERENCES
Astrinidis, A., Cash, T. P., Hunter, D. S., Walker, C. L., Chernoff, J., and Henske, E. P. (2002). Tuberin, the tuberous sclerosis complex 2 tumor suppressor gene product, regulates Rho activation, cell adhesion and migration. Oncogene 21, 8470–8476.
Blume-Jensen, P., and Hunter, T. (2001). Oncogenic kinase signalling. Nature 411, 355–365.
Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868.
Cardone, M. H., Roy, N., Stennicke, H. R., Salvesen, G. S., Franke, T. F., Stanbridge, E., Frisch, S., and Reed, J. C. (1998). Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318–1321.
Carsillo, T., Astrinidis, A., and Henske, E. P. (2000). Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc. Natl. Acad. Sci. U.S.A. 97, 6085–6090.
Cook, J. A., Oliver, K., Mueller, R. F., and Sampson, J. (1996). A cross sectional study of renal involvement in tuberous sclerosis. J. Med. Genet. 33, 480–484.
Dan, H. C., Sun, M., Yang, L., Feldman, R. I., Sui, X. M., Ou, C. C., Nellist, M., Yeung, R. S., Halley, D. J., Nicosia, S. V., et al. (2002). Phosphatidylinositol 3-kinase/Akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin. J. Biol. Chem. 277, 35364–35370.
Datta, S. R., Brunet, A., and Greenberg, M. E. (1999). Cellular survival: A play in three Akts. Genes Dev. 13, 2905–2927.
Davis, M. A., and Carbott, D. E. (1999). Herbimycin A and geldanamycin inhibit okadaic acid-induced apoptosis and p38 activation in NRK-52E renal epithelial cells. Toxicol. Appl. Pharmacol. 161, 59–74.
Davis, M. A., Chang, S. H., and Trump, B. F. (1996). Differential sensitivity of normal and H-ras oncogene-transformed rat kidney epithelial cells to okadaic acid-induced apoptosis. Toxicol. Appl. Pharmacol. 141, 93–101.
Davis, M. A., Smith, M. W., Chang, S. H., and Trump, B. F. (1994). Characterization of a renal epithelial cell model of apoptosis using okadaic acid and the NRK-52E cell line. Toxicol. Pathol. 22, 595–605.
European Chromosome 16 Tuberous Sclerosis Consortium (1993). Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305–1315.
Evan, G. I., and Vousden, K. H. (2001). Proliferation, cell cycle and apoptosis in cancer. Nature 411, 342–348.
Everitt, J. I., Goldsworthy, T. L., Wolf, D. C., and Walker, C. L. (1992). Hereditary renal cell carcinoma in the Eker rat: A rodent familial cancer syndrome. J. Urol. 148, 1932–1936.
Everitt, J. I., Goldsworthy, T. L., Wolf, D. C., and Walker, C. L. (1995). Hereditary renal cell carcinoma in the Eker rat: A unique animal model for the study of cancer susceptibility. Toxicol. Lett. 82–83, 621–625.
Gao, X., Zhang, Y., Arrazola, P., Hino, O., Kobayashi, T., Yeung, R. S., Ru, B., and Pan, D. (2002). Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat. Cell Biol. 4, 699–704.
Goncharova, E. A., Goncharov, D. A., Eszterhas, A., Hunter, D. S., Glassberg, M. K., Yeung, R. S., Walker, C. L., Noonan, D., Kwiatkowski, D. J., Chou, M. M., et al. (2002). Tuberin Regulates p70 S6 Kinase Activation and Ribosomal Protein S6 Phosphorylation. A Role for the TSC2 Tumor Suppressor Gene in Pulmonary Lymphangioleiomyomatosis (LAM). J. Biol. Chem. 277, 30958–30967.
Harada, H., Andersen, J. S., Mann, M., Terada, N., and Korsmeyer, S. J. (2001). p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD, Proc. Natl. Acad. Sci. U.S.A. 98, 9666–9670.
Henry, K. W., Yuan, X., Koszewski, N. J., Onda, H., Kwiatkowski, D. J., and Noonan, D. J. (1998). Tuberous sclerosis gene 2 product modulates transcription mediated by steroid hormone receptor family members. J. Biol. Chem. 273, 20535–20539.
Hino, O., Klein-Szanto, A. J., Freed, J. J., Testa, J. R., Brown, D. Q., Vilensky, M., Yeung, R. S., Tartof, K. D., and Knudson, A. G. (1993a). Spontaneous and radiation-induced renal tumors in the Eker rat model of dominantly inherited cancer. Proc. Natl. Acad. Sci. U.S.A. 90, 327–331.
Hino, O., Mitani, H., and Knudson, A. G. (1993b). Genetic predisposition to transplacentally induced renal cell carcinomas in the Eker rat. Cancer Res 53, 5856–5858.
Horesovsky, G., Ginsler, J., Everitt, J., and Walker, C. (1994). Increased susceptibility to in vitro transformation of cells carrying the Eker tumor susceptibility mutation. Carcinogenesis 15, 2183–2187.
Howe, S. R., Gottardis, M. M., Everitt, J. I., Goldsworthy, T. L., Wolf, D. C., and Walker, C. (1995). Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. Am. J. Pathol 146, 1568–1579.
Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. (2002). TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4, 648–657.
Jin, F., Wienecke, R., Xiao, G. H., Maize, J. C., Jr., DeClue, J. E., and Yeung, R. S. (1996). Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region. Proc. Natl. Acad. Sci. U.S.A. 93, 9154–9159.
Kenerson, H. L., Aicher, L. D., True, L. D., and Yeung, R. S. (2002). Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res. 62, 5645–5650.
Kleymenova, E., Ibraghimov-Beskrovnaya, O., Kugoh, H., Everitt, J., Xu, H., Kiguchi, K., Landes, G., Harris, P., and Walker, C. (2001). Tuberin-dependent membrane localization of polycystin-1: A functional link between polycystic kidney disease and the TSC2 tumor suppressor gene. Mol. Cell 7, 823–832.
Kolb, T. M., Chang, S. H., and Davis, M. A. (2002). Biochemical and morphological events during okadaic acid-induced apoptosis of Tsc2-null ERC-18 cell line. Toxicol. Pathol. 30, 235–246.
Kolb, T. M., and Davis, M. A. (2004). The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) provokes a prolonged morphologic response and ERK activation in Tsc2-null renal tumor cells. Toxicol. Sci. 80, 233–242.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lau, S. S., Monks, T. J., Everitt, J. I., Kleymenova, E., and Walker, C. L. (2001). Carcinogenicity of a nephrotoxic metabolite of the "nongenotoxic" carcinogen hydroquinone. Chem. Res. Toxicol. 14, 25–33.
Momose, S., Kobayashi, T., Mitani, H., Hirabayashi, M., Ito, K., Ueda, M., Nabeshima, Y., and Hino, O. (2002). Identification of the coding sequences responsible for Tsc2-mediated tumor suppression using a transgenic rat system. Hum. Mol. Genet. 11, 2997–3006.
Noonan, D. J., Lou, D., Griffith, N., and. Vanaman, T. C. (2002). A calmodulin binding site in the tuberous sclerosis 2 gene product is essential for regulation of transcription events and is altered by mutations linked to tuberous sclerosis and lymphangioleiomyomatosis. Arch. Biochem. Biophys. 398, 132–140.
Potter, C. J., Pedraza, L. G., and Xu, T. (2002). Akt regulates growth by directly phosphorylating Tsc2, Nat. Cell Biol. 4, 658–665.
Soucek, T., Pusch, O., Wienecke, R., DeClue, J. E., and Hengstschlager, M. (1997). Role of the tuberous sclerosis gene-2 product in cell cycle control. Loss of the tuberous sclerosis gene-2 induces quiescent cells to enter S phase. J. Biol. Chem. 272, 29301–29308.
Soucek, T., Yeung, R. S., and Hengstschlager, M. (1998). Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2. Proc. Natl. Acad. Sci. U.S.A. 95, 15653–15658.
Tee, A. R., Fingar, D. C., Manning, B. D., Kwiatkowski, D. J., Cantley, L. C., and Blenis, J. (2002). Tuberous sclerosis complex-1 and –2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc. Natl. Acad. Sci. U.S.A. 99, 13571–13578.
Todd, D., Yang, G., Brown, R. W., Cao, J., D'Agati, V., Thompson, T. S., and Truong, L. D. (1996). Apoptosis in renal cell carcinoma: Detection by in situ end-labeling of fragmented DNA and correlation with other prognostic factors. Hum. Pathol. 27, 1012–1017.
Tsuchiya, H., Orimoto, K., Kobayashi, K., and Hino, O. (1996). Presence of potent transcriptional activation domains in the predisposing tuberous sclerosis (Tsc2) gene product of the Eker rat model. Cancer Res. 56, 429–433.
van Slegtenhorst, M., Nellist, M., Nagelkerken, B., Cheadle, J., Snell, R., van den Ouweland, A., Reuser, A., Sampson, J., Halley, D., and van der Sluijs, P. (1998). Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Hum. Mol. Genet. 7, 1053–1057.
Walker, C., Goldsworthy, T. L., Wolf, D. C., and Everitt, J. (1992). Predisposition to renal cell carcinoma due to alteration of a cancer susceptibility gene. Science 255, 1693–1695.
Wienecke, R., Konig, A., and DeClue, J. E. (1995). Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity. J. Biol. Chem. 270, 16409–16414.
Wolf, D. C., Goldsworthy, T. L., Donner, E. M., Harden, R., Fitzpatrick, B., and Everitt, J. I. (1998). Estrogen treatment enhances hereditary renal tumor development in Eker rats. Carcinogenesis 19, 2043–2047.
Xiao, G. H., Shoarinejad, F., Jin, F., Golemis, E. A., and Yeung, R. S. (1997). The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. J. Biol. Chem. 272, 6097–6100.
Yu, J., Astrinidis, A., and Henske, E. P. (2001). Chromosome 16 loss of heterozygosity in tuberous sclerosis and sporadic lymphangiomyomatosis. Am. J. Respir. Crit. Care Med. 164, 1537–1540.
Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14–3–3 not BCL-X(L). Cell 87, 619–628.
Zhang, Y., Gao, X., Saucedo, L. J., Ru, B., Edgar, B. A., and Pan, D. (2003). Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat. Cell Biol. 5, 578–581.(Todd M. Kolb, Ling Duan and Myrtle A. Da)
Toxicology and Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Greenfield, Indiana 46140
ABSTRACT
Although the precise role for the tuberous sclerosis complex-2 tumor suppressor gene (Tsc2) in tumor suppression is not clear, many studies have implicated Tsc2 in the regulation of cell differentiation, cell cycle control, GTPase activity, transcription, polycystin-1 localization, and translation initiation. We propose that Tsc2 also increases susceptibility to apoptosis, and that this functional role may contribute to the tumor suppressor activity of Tsc2. We previously characterized the apoptotic response of a Tsc2-null renal tumor cell line (ERC-18) to the tumor promoter okadaic acid (OKA). In the present study, we expressed Tsc2 in ERC-18 cells and compared the effect of Tsc2 expression on apoptotic induction. Tsc2 expression increased the susceptibility of ERC-18 cells to apoptosis induced by OKA and the phosphatidylinositol-3' kinase inhibitor, LY294002. In addition, Tsc2 expression abrogated OKA-induced cell detachment of ERC-18 cells. These results indicate that the OKA-induced, caspase-independent detachment previously observed in ERC-18 cells is Tsc2-dependent, and may support an additional role for the Tsc2 in regulating cell adhesion.
Key Words: tumorigenesis; Tsc2; apoptosis; okadaic acid.
INTRODUCTION
During the multistage process of neoplastic transformation, cells acquire the ability to proliferate autonomously and survive under conditions that would normally prevent survival. These abilities are acquired through heritable or exogenously induced genetic changes that activate oncogenes or inhibit tumor suppressor genes, frequently resulting in proliferation increases or a reduced apoptotic response. Cell death by apoptosis prevents neoplastic progression by balancing increased proliferation and by preventing the survival of cells carrying oncogenic mutations (reviewed in Evan and Vousden, 2001). Defects in the normal apoptotic response may promote malignant transformation by allowing the survival of mutated progeny, promoting clonal expansion and further genomic instability.
Apoptosis plays a key role in the regulation of kidney cell number. Compensatory increases in apoptosis have been correlated with increased proliferation and tumor grade, stage, and size during renal cell carcinoma (RCC) development (Todd et al., 1996). Tuberin, the product of the tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene, also appears to play an important role in renal tumorigenesis. In humans, a genetic mutation in Tsc2 results in tuberous sclerosis, a disease syndrome that consists of hamartomatous tumors in several organs and such renal manifestations as multiple, bilateral angiomyolipomas, cystic disease, and renal cell carcinoma (Cook et al., 1996). A single germline mutation in the rat homologue of Tsc2 results in the development of renal cell carcinoma with a nearly complete penetrance in the Eker rat model (Everitt et al., 1995). These rats also develop uterine leiomyomas and splenic hemangiosarcomas (Everitt et al., 1992, 1995; Howe et al., 1995) and are highly susceptible to renal cell carcinoma induced by a variety of toxicants (Hino et al., 1993a,b; Horesovsky et al., 1994; Lau et al., 2001; Walker et al., 1992; Wolf et al., 1998).
Many studies have implicated Tsc2 in the regulation of cell differentiation (Soucek et al., 1998), cell cycle control (Soucek et al., 1997, 1998), GTPase activity (Astrinidis et al., 2002; Wienecke et al., 1995; Xiao et al., 1997; Zhang et al., 2003), transcription (Henry et al., 1998; Zhang et al., 2003), polycystin-1 localization (Kleymenova et al., 2001), and translation initiation (Dan et al., 2002; Gao et al., 2002; Goncharova et al., 2002; Inoki et al., 2002; Kenerson et al., 2002; Potter et al., 2002; Tee et al., 2002). Tsc2 has recently been shown to be a downstream component of the phosphatidylinositol-3' kinase (PI3 kinase) signaling pathway, which normally functions to regulate cell growth and promote survival (reviewed in Blume-Jensen and Hunter, 2001; Datta et al., 1999). A downstream component of this signaling pathway, Akt kinase, promotes survival by phosphorylating multiple substrates. These substrates include the pro-apoptotic proteins BAD (Zha et al., 1996) and caspase-9 (Cardone et al., 1998), and the forkhead family of transcription factors (Brunet et al., 1999). Tuberin is also reported to be an Akt substrate, and phosphorylation of tuberin by Akt promotes degradation (Dan et al., 2002; Gao et al., 2002; Goncharova et al., 2002; Inoki et al., 2002; Kenerson et al., 2002; Potter et al., 2002; Tee et al., 2002). It is unknown whether Tsc2 is involved in the anti-apoptotic function of Akt via this or any other mechanism. We propose that loss of TSC2 will decrease susceptibility of renal epithelial cells to apoptosis.
To measure the effect of Tsc2 expression on the apoptotic response of renal tumor cells, we expressed Tsc2 in Tsc2-null ERC-18 cells and compared the susceptibility of Tsc2-null and Tsc2-expressing ERC-18 cells to apoptosis induced by the phosphatase inhibitor, okadaic acid (OKA), and the PI3 kinase inhibitor, LY294002. We show that Tsc2 expression increases the susceptibility of ERC-18 cells to death induced by both compounds, and that the unique morphologic response of ERC-18 cells to OKA is Tsc2-dependent. We also show that the apoptosis-promoting function of Tsc2 does not appear to be mediated through mTOR.
MATERIALS AND METHODS
Chemicals and reagents.
Okadaic acid (OKA) was purchased from Alexis Biochemicals (San Diego, CA) and prepared as a 1 mM stock solution in ethanol. LY294002 was purchased from BIOMOL (Plymouth Meeting, PA) and prepared as a 10 mM stock in dimethyl sulfoxide (DMSO). Rapamycin was purchased as a 100 μM stock solution in methanol from Cell Signaling Technology (CST; Beverly, MA). SYTOX-green was purchased from Molecular Probes (Eugene, OR) and diluted to 0.5 mM in DMSO immediately before use. Saponin was purchased from Sigma (St. Louis, MO) and prepared as a 100 mg/ml stock solution in distilled water.
Plasmid construction.
A plasmid encoding the full-length rat Tsc2 cDNA sequence (1765 amino acid splice variant; 36) in the pCDNA3 vector (pcDNA3-Tsc2) was provided by Dr. Cheryl Walker (MD Anderson Cancer Center; Smithville, TX). A 5357 base pair HindIII fragment of pcDNA3-Tsc2 containing the Tsc2 sequence was subcloned into the HindIII site of the pCMV-Tag-4 vector (Stratagene; La Jolla, CA), to create the pCMV-Tag-Tsc2 plasmid. The plasmid encodes a 1738 amino acid tuberin construct with the C-terminal 42 amino acids removed and replaced with a FLAG-epitope tag. The pCMV-Tag-Tsc2 plasmid sequence was verified by multiple restriction endonuclease digests. A similar Tsc2 construct, lacking 55 amino acids from the C-terminus, completely inhibited N-ethyl-N-nitrosurea-induced renal carcinoma formation in transgenic Eker rats (Momose et al., 2002).
Cell line development and culture.
The Tsc2-null ERC-18 cell line was derived from renal tumors of the Eker rat (38), and was provided by Dr. Cheryl Walker. ERC-18 cells were transfected with the linearized pCMV-Tag-Tsc2 plasmid or with the linearized empty pCMV-Tag vector (control) using Effectene transfection reagent (QIAGEN; Valencia, CA) according to the manufacturer's protocol. After transfection for 48 h, cells were grown in the presence of 200 μg/ml G418 (BD Biosciences Clontech; Palo Alto, CA) for 11 days. Individual G418-resistant colonies were isolated and propagated in the presence of 200 μg/ml G418 until reaching the 60-mm dish stage, then screened by immunoblot analysis for expression of FLAG with an anti-FLAG-M2 antibody (Sigma; St. Louis, MO) diluted to a concentration of 1 μg/ml. ERC18-FLAG-2B (empty plasmid vector) and ERC18-FLAG-Tsc2 (pCMV-Tag-Tsc2) cells were routinely maintained in DF-8 media, a 1:1 mixture of Dulbecco's modified Eagle media (DMEM) and Nutrient mixture F12 (Ham) supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, 1.6 μM ferrous sulfate, 50 nM sodium selenite, 12 μM vasopressin, 10 nM cholesterol, 200 nM hydrocortisone, 1 nM tri-iodothyronine (T3), 10 pg/ml transferrin, and 25 μg/ml insulin. All cell lines were grown in a humidified atmosphere of 37°C and 5% CO2/95% room air. Mycoplasmal contamination assays were not routinely performed. Cells were grown to 90% confluence in 60-mm plastic culture dishes (immunoblot analysis) or in six-well dishes (membrane permeability assay), then incubated in low-serum treatment media (DMEM/F12, 1% FBS) for 24 h prior to treatment with OKA or LY294002. Apoptotic morphology was photographed on a Leitz Diavert inverted microscope.
Determination of clone growth rate.
ERC-18, ERC18-FLAG-2B, and ERC18-FLAG-Tsc2 cells were plated in 12-well culture dishes (1 x 104 cells per well) in complete DF-8 media. On days 1, 3, 5, and 7 after plating, cells in duplicate wells were incubated with 0.5 μM SYTOX Green (Molecular Probes; Eugene, OR) and 100 μg/ml saponin (Sigma; St. Louis, MO) for 10 min at 37°C. SYTOX green fluorescence was measured on a Cytofluor 2350 (Millipore; Bedford, MA), and fluorescence values were averaged for duplicate wells at each time point. Cell number was determined using a standard curve prepared from fluorescence intensities of known cell numbers for each cell type. The mean cell number at each time point was then determined from three independent experiments. One-week growth curves were prepared for each clone and the parent ERC-18 cell line using GraphPad Prism software (Graphpad Software, Inc.; San Diego, CA) by converting to logarithms and plotting against time. Prism software was used to generate sigmoidal concentration-response curves for each cell type using nonlinear regression. Doubling times were calculated between days 3 and 5, when all cells appeared to be in a logarithmic growth phase. Doubling time was calculated for each cell line using the equation Nt = N02tf, where Nt is the number of cells at time = t, N0 is the initial number of cells, t is the time in days, and f is the frequency of cell cycles per day.
Measurement of cell death (membrane permeability assay).
Cell membrane integrity was assessed via SYTOX-green uptake. Viable cells exclude the fluorophore, while nuclei in cells with increased membrane permeability are labeled. Cells were grown to 90% confluence in six-well culture dishes, then incubated in DMEM/F12 low-serum treatment media for 24 h. Cells were treated in low-serum media for 24 h with 0.05, 0.1, and 0.25 μM OKA or 10, 50, and 100 μM LY294002, or with an equivalent volume of vehicle (ethanol or DMSO) as a control. In rapamycin experiments, cells were pre-treated for 1 h with 10 nM rapamycin, and the inhibitor was included (at 10 nM) as a cotreatment with OKA or LY294002. After 24 h of treatment, cells were incubated with 0.5 μM SYTOX-green for 10 min prior to measuring fluorescence using a Cytofluor 2350 (Millipore; Bedford, MA). After measuring death-induced fluorescence, saponin (100 μg/ml) was added for 10 min to permeabilize all cells, and total fluorescence was measured. In each well, death-induced fluorescence values were normalized to the total cellular fluorescence, and the mean change in membrane permeability (fluorescence) from control was determined from three independent experiments. Statistically significant changes in membrane permeability from control (vehicle) were identified using randomized block analysis of variance (ANOVA) with Dunnett's multiple comparisons post-test. With each concentration, the mean change in membrane permeability was compared between cell lines using an unpaired t-test. Statistically significant differences were judged at p < 0.05.
Immunoblot analysis.
Whole cell lysates were prepared from 60-mm cultures after 10 h (OKA) or 24 h (LY294002) of treatment for analysis of caspase-3 cleavage. Floating cells were collected by centrifugation, and lysates were prepared on ice from floating and adherent cells with RIPA buffer (50 mM Tris-Cl, pH = 7.4; 150 mM NaCl; 1% Nonident P-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate; 2.5 mM sodium pyrophosphate; 1 mM -glycerolphosphate) supplemented with protease inhibitor cocktail P8340 and phosphatase inhibitor cocktails P2850 and P5726 (Sigma). Lysates were sonicated for 10 s, then clarified by centrifugation (17,000 x g, 10 min.). Protein concentrations were measured using the BCA protein assay (Pierce; Rockford, IL), and 25 μg of protein was separated by SDS–PAGE using standard techniques (Laemmli, 1970) and transferred to a PVDF membrane (Bio-Rad; Hercules, CA) in 25 mM Tris, 192 mM glycine, and 20% methanol overnight at 100 mA. Full-length and cleaved caspase-3 were detected with a caspase-3 antibody (Santa Cruz Biotechnology; Santa Cruz, CA), used at a 1:1000 dilution according to the manufacturer's protocol.
A separate lysis procedure was used to measure cytosolic BAD accumulation and phosphorylation of Akt (serine 473). Following 30 h of serum starvation (BAD) or 24 h of LY294002 treatment (phospho-Akt), cytosolic extracts were prepared as previously described (Kolb et al., 2002). Briefly, cells were placed in ice-cold MS Buffer (5 mM Tris-Cl, pH = 7.5; 210 mM mannitol; 70 mM sucrose; 1 mM EDTA; 40 μg/ml digitonin) supplemented with protease and phosphatase inhibitors, as above, and scraped into microfuge tubes. After a 10-min incubation on ice, a cytosolic extract was prepared by centrifugation at 17,000 x g for 15 min. The pellet (containing nuclei, mitochondria, and other membranes) was washed once with MS buffer, then centrifuged again (17,000 x g for 15 min ) prior to lysis with 10 volumes of RIPA buffer and sonication (10 s). Cytosolic extracts were separated by SDS–PAGE and transferred to PVDF membranes. Immunoblot analysis was performed using antibodies specific for BAD (CST; 1:500) and phospho-Akt (ser473, Santa Cruz; 1:1000) according to the manufacturers' protocol. To measure cytosolic accumulation of BAD, band intensities were quantified from scanned images using Molecular Analyst software (Bio-Rad). Mean cytosolic BAD levels from five independent experiments were compared between 2B-D and Tsc2-D cells using an unpaired t-test with statistically significant differences judged at p < 0.05.
RESULTS
Expression of Tsc2 in Tsc2-Null ERC-18 Cells
We expressed Tsc2 in an Eker renal carcinoma cell line (ERC-18) to study the effect of Tsc2 expression on the susceptibility of Eker renal tumor cells to apoptosis. The pCMV-Tag-Tsc2 construct was prepared to express the full-length rat Tsc2 cDNA (minus the C-terminal 42 amino acids) under the constitutive CMV promoter (Fig. 1A). The pCMV-Tag-Tsc2 sequence was confirmed by multiple restriction digests, and the predicted peptide sequence was analyzed for the presence of published tuberin functional domains. The product of pCMV-Tag-Tsc2 (FLAG-Tsc2, 200-kDa) includes the hamartin binding site (van Slegtenhorst et al., 1998), the Rap1GAP domain (European Chromosome 16 Tuberous Sclerosis Consortium, 1993), transcriptional activation domains AD1 and AD2 (Tsuchiya et al., 1996), the calmodulin binding site (Noonan et al., 2002), and all four required Akt phosphorylation sites (ser939, ser1086, ser1088, thr1422) (Inoki et al., 2002), but lacks the retinoid-X receptor (RXR) binding domain (Henry et al., 1998). ERC-18 cells were stably transfected with the pCMV-Tag-Tsc2 or empty pCMV-Tag plasmids, and independent clones were screened for construct expression using a FLAG-specific antibody. As shown in Figure 1B, the 200-kDa FLAG-Tsc2 construct is expressed in the ERC18-FLAG-Tsc2-D (Tsc2-D) cell line. The FLAG epitope was selected to measure construct expression in clones. The FLAG antibody had cross-reactivity with multiple bands in all cell lines tested, as evidenced by the bands between 98 and 140 kDa in Figure 1B. Similar levels of expression of these cross-reactive bands indicate equivalent protein loading between samples. The control cell line, ERC18-FLAG-2B-D (2B-D), expressing only the empty vector, was selected via resistance to G418. The 2B-D cell line does not express the 200-kDa FLAG-Tsc2 construct, as previously described (Kolb and Davis, 2004).
Expression of Tsc2 in ERC-18 Cells Alters Cell Morphology, Growth Rate, and p70S6 Kinase Phosphorylation
The characteristics of the cell lines generated above were also compared to results described in previous Tsc2-reconstitution studies. Jin et al. (1996) showed that reintroduction of Tsc2 or a C-terminal fragment in Eker renal carcinoma cells induced a flattened morphology and slowed proliferation. As shown in Figure 2A, Tsc2-D cells are less refractile and appear flatter than 2B-D cells. (The 2B-D cell line does not express the 200-kDa FLAG-Tsc2 construct, as previously described (Kolb and Davis, 2004).) One-week growth curves were prepared for each clone and the parent ERC-18 cell line (Fig. 2B), and doubling times were calculated between days 3 and 5, when all cells appeared to be in a logarithmic growth phase. The doubling time of Tsc2-D cells was 27.9 h compared to 21.4 and 20.5 h in the control 2B-D and parent ERC-18 cell lines, respectively. One-way analysis of variance showed no statistically significant differences in cell number between the cell lines on day 3, 5, or 7 (not shown). However, the shapes of the growth curves imply that Tsc2 expression may potentially slow the rate of cell turnover.
Several studies have recently shown that Tsc2-null cells have increased p70S6 kinase phosphorylation at threonine 389, and that reintroduction of Tsc2 decreases this phosphorylation (Goncharova et al., 2002; Inoki et al., 2002). As shown in Figure 2C, Tsc2-D cells showed reduced p70S6 kinase phosphorylation (thr389) when compared to 2B-D cells. Interestingly, two higher molecular weight proteins that are recognized by the phospho-p70S6 kinase antibody were also differentially phosphorylated in the cell lines (Fig. 2C). According to the antibody cross-reactivity data provided by the manufacturer, these bands most likely represent phosphorylated forms of the p85S6 kinase and protein kinase C isoforms. Taken together, these data show that expression of pCMV-Tag-Tsc2 in ERC-18 cells caused changes in cell morphology, proliferation, and p70S6 kinase phosphorylation consistent with previous studies of Tsc2 reconstitution.
Tsc2 Expression Increases the Susceptibility of ERC-18 Cells to OKA-Induced Apoptosis
In the Tsc2-null ERC-18 cell line, OKA induced extensive apoptosis with a morphologic response that was unique from the response observed in other Tsc2-expressing renal epithelial cell lines. From this observation, we inferred that there may be a difference in the susceptibility of Tsc2-null and Tsc2-expressing renal epithelial cells to OKA. To measure the effect of Tsc2 expression on susceptibility to OKA-induced apoptosis, we compared death induced in Tsc2-D cells after 24 h of exposure to 0.05, 0.1, and 0.25 μM OKA to that induced in a control cell line expressing only the empty vector (2B-D). In both cell lines, OKA induced a concentration-dependent increase in cell death after 24 h (Fig. 3A). Death was significantly increased at all concentrations when compared to cells exposed to vehicle (p < 0.01; Dunnett's multiple comparisons post-test following significant differences in randomized block ANOVA). Comparison between cell lines within each treatment group (concentration) showed that expression of Tsc2 increased susceptibility to OKA-induced death when compared to the control cell line (Fig. 3A). At the lowest concentration (0.05 μM OKA), there was no difference in the susceptibility of 2B-D and Tsc2-D cells to OKA. After 24 h of exposure with 0.1 or 0.25 μM OKA, Tsc2-D cells showed a significant increase in death when compared to the 2B-D cell line (0.1 μM, p = 0.002; 0.25 μM, p = 0.006; unpaired t-test). To confirm that apoptosis was the type of cell death caused by OKA treatment, we measured cleavage of caspase-3 after 10 h of treatment with 0.1μM and 0.25 μM OKA. We have previously shown caspase-3 is cleaved between 6 and 10 h in ERC-18 cells treated with 0.1μM OKA. As shown in Figure 3B, caspase-3 is cleaved in both cell lines after 10 h of exposure with both concentrations. The extent of cleavage was increased in the Tsc2-D cell line when compared to 2B-D cells.
The Unique Morphologic Response of ERC-18 Cells to OKA Is Tsc2-Dependent
In the initial characterization of the apoptotic response of ERC-18 cells to OKA, we described a unique morphologic response (Kolb et al., 2002). OKA-treated ERC-18 cells rapidly lost microvilli and focal adhesion structure, shrunk, rounded, and detached with minimal membrane budding. These early changes in morphology preceded classical apoptotic markers (nuclear fragmentation, membrane budding, cytochrome c release, caspase activation) by several hours. To determine if this unique morphologic response was Tsc2 dependent, we observed the early morphologic changes in our 2B-D and Tsc2-D cells treated with 0.05, 0.1, and 0.25 μM OKA for 2 h. As previously described, the Tsc2-null 2B-D (control) cell line showed shrinkage, rounding, and minimal membrane budding after 2 h of treatment with 0.1 μM OKA (Fig. 4). When treated with 0.25 μM OKA, greater than 50% of cells had detached after 2 h. This response is consistent with the response previously observed in ERC-18 cells. Conversely, Tsc2-expressing ERC-18 cells (Tsc2-D) showed apoptotic morphology that was similar to the response previously observed in other Tsc2-expressing renal epithelial cell lines. OKA-treated Tsc2-D cells remained adherent and did not round, and shrinkage was accompanied by extensive membrane budding (arrows in Fig. 4). No detachment was observed after 2 h of treatment with 0.25 μM OKA. No morphologic changes were observed in either cell line after 2 h of treatment with 0.05 μM OKA, and both cell lines progressed to detachment and secondary oncotic necrosis after 24 h of treatment in all concentrations.
Tsc2 Expression Increases the Susceptibility of ERC-18 Cells to Apoptosis Induced by LY294002
PI3 kinase promotes cell survival through activation of the anti-apoptotic kinase Akt, and inhibition of PI3 kinase activity induces apoptosis in most cell types (reviewed in Datta et al., 1999). Tsc2 has recently been shown to be a substrate for Akt, and we examined the effect of Tsc2 expression on PI3 kinase-mediated survival. To inhibit PI3 kinase activity, we treated cells with 10, 50, and 100 μM LY294002 (a specific, cell-permeable PI3 kinase inhibitor) for 24 h and measured cell death as described. As shown in Figure 5A, treatment of 2B-D and Tsc2-D cells with LY294002 for 24 h caused a marked decrease in Akt phosphorylation. In both cell lines, LY294002 induced a concentration-dependent increase in cell death (Fig. 5B), although the number of apoptotic cells was lower than observed following OKA treatment. Significant increases in death were measured after treatment with 50 and 100 μM LY294002 in all cell lines, as indicated in Figure 5B. Comparison between cell lines within each treatment group (concentration) showed that Tsc2 expression increased susceptibility to LY294002-induced death when compared to the control cell line (Fig. 5B). Tsc2-D cells showed increased death when compared to 2B-D cells after 24 h of treatment with 50 and 100 μM LY294002 (50 μM, p = 0.005; 100 μM, p = 0.006; unpaired t-test). Caspase-3 cleavage was measured after 24 h of LY294002 treatment to confirm that cell death was the result of apoptosis. As shown in Figure 5C, caspase activation was prominent and concentration dependent in the Tsc2-D cell line, which was the most susceptible to LY294002-induced death.
Tsc2-Dependent Increases in Apoptotic Susceptibility Do Not Require mTOR Activity
Several reports have recently indicated that Tsc2 functions to inhibit mTOR in the regulation of protein translation. Since the increased activity of mTOR in Tsc2-null cells has been observed and associated with increased p70S6 kinase activity, we hypothesized that the enhanced mTOR activities in Tsc2-null cells could contribute to decreased susceptibility to apoptosis.
To determine whether mTOR contributed to the observed Tsc2-dependent increase in apoptotic susceptibility, we inhibited mTOR by pretreating cells with 10 nM rapamycin, then treated cells with 0.25 μM OKA or 100 μM LY294002 in the presence of 10 nM rapamycin. If the pro-apoptotic effects of Tsc2 are mTOR dependent, rapamycin should functionally replace Tsc2 in null cells (2B-D) to inhibit mTOR, thereby increasing apoptotic susceptibility. As shown in Figure 6, mTOR inhibition with rapamycin had no effect on OKA-induced apoptosis in either cell line and actually inhibited the apoptotic response of 2B-D cells to LY294002 (p < 0.01; unpaired t-test).
DISCUSSION
While intensive effort has been focused on determining the function of Tsc2 in the regulation of cell proliferation and growth, little is known about the effects of the tumor suppressor on apoptosis. A decreased apoptotic response in Tsc2-null renal epithelial cells could promote tumor progression by failing to: (1) induce death in the absence of survival stimuli, (2) constrain cell proliferation in the presence of tumor promoters, or (3) eliminate mutated cells following genotoxic insult. In the present study, we compared the susceptibility of Tsc2-null and Tsc2-expressing ERC-18 cells to apoptosis induced by two different compounds and showed that Tsc2 expression increased the apoptotic response of ERC-18 cells to both treatments.
We initially characterized differences in apoptotic susceptibility of Tsc2-null and Tsc2-expressing cells using OKA, a compound that consistently induces apoptosis in renal epithelial cells in vitro (Davis et al., 1994, 1996), including the ERC-18 tumor cell line (Kolb et al., 2002). Our results show that expression of Tsc2 in ERC-18 cells increased susceptibility to OKA-induced apoptosis. OKA inhibits the activities of the phosphatases PP1 and PP2A, and OKA-induced apoptosis in renal epithelial cells appears to involve the concurrent activation of multiple kinases, including protein kinase C (PKC), Raf-1, extracellular signal-regulated kinases 1 and 2 (ERK-1/2), jun N-terminal kinase (JNK), and p38 (Davis and Carbott, 1999; Davis et al., 1996). The present studies show that OKA-induced apoptosis in renal epithelial cells can be regulated by Tsc2.
The synthetic quercetin derivative LY294002 inhibits PI3 kinase and may promote cell death via inhibition of Akt activity. Since multiple studies have shown Tsc2 to be an Akt substrate (Dan et al., 2002; Inoki et al., 2002; Tee et al., 2002), we wanted to know if expression of the tumor suppressor would influence PI3 kinase inhibitor-mediated cell death. While LY294002 induced apoptosis in a small but significant population of Tsc2-null cells, expression of Tsc2 in ERC-18 cells increased susceptibility to LY294002-induced death. These results imply that Tsc2-expressing cells may be more dependent on PI3 kinase-mediated signaling for survival than Tsc2-null renal tumor cells. The decreased apoptotic response of Tsc2-null cells to PI3 kinase inhibition may have important implications for tumor progression, since survival signals normally activating PI3 kinase may become limiting as cell proliferation and tumor growth continues. Tsc2-null cells would therefore be at a distinct survival advantage under these conditions due to their decreased apoptotic susceptibility.
Although we have not determined the precise mechanistic role played by the Tsc2 in cell death, we have data that imply the mechanism does not involve mTOR. Tuberin has been shown to inhibit mTOR activity (Gao et al., 2002; Inoki et al., 2002; Kenerson et al., 2002; Tee et al., 2002), and tuberin loss results in rapamycin-sensitive increases in p70S6 kinase activity (Goncharova et al., 2002; Inoki et al., 2002). Phosphorylation of BAD by p70S6 kinase is rapamycin-sensitive and has been reported to promote cell survival (Harada et al., 2001). Therefore, it was reasonable to expect that inhibition of mTOR by rapamycin would mimic the pro-apoptotic effects of Tsc2 in null cells if the tumor suppressor promotes the chemical-induced apoptosis observed via inhibition of mTOR. However, rapamycin did not increase the susceptibility of Tsc2-null cells to apoptosis induced by either compound. Although a recent study showed that short-term rapamycin treatment induced apoptosis in Eker rat renal tumors in vivo (Kenerson et al., 2002), the concentrations of rapamycin used in our study did not induce apoptosis in our cell lines at the times measured. However, both studies strongly support a role for a reduced apoptotic response in the pathogenesis of Tsc2-null renal tumors. Additional study is needed to reveal additional mechanistic links between tuberin and apoptosis in renal epithelial cells.
Our previous characterization of OKA-induced apoptosis in ERC-18 cells showed a unique early morphologic response that was caspase independent (Kolb et al., 2002). In the present study, we show that these early morphologic changes (shrinkage, rounding, detachment) are also Tsc2 dependent. While the Tsc2-null control cell line (2B-D) showed early morphologic changes identical to those observed in ERC-18 cells when exposed to OKA, expression of Tsc2 abrogated this effect and produced a morphological response more characteristic of that observed in other Tsc2-competent renal epithelial cells. These findings indicate that Tsc2 expression may regulate shrinkage, rounding, and detachment in renal epithelial cells. Our results also provide additional support for the hypothesis that Tsc2 plays a role in regulation of cell adhesion. Astrinidis et al. (2002) recently reported that tuberin regulates cell adhesion, migration, and activation of the Rho GTPase in renal epithelial cells and in a Tsc2-null cell line derived from an Eker rat leiomyoma. These findings may be particularly important in TSC-associated diseases of smooth muscle cell origin like malignant renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). A model for LAM pathogenesis has been proposed where Tsc2-null smooth muscle cells migrate from the kidney to the lung (Carsillo et al., 2000; Yu et al., 2001).
Our results may have important implications for the progression of both spontaneous and chemically-induced Eker rat renal carcinomas. A decreased apoptotic response in Tsc2-null or Tsc2-deficient renal epithelial cells can contribute to the progression of both types of tumors. Furthermore, decreased dependence of Tsc-2 deficient cells on PI3 kinase-mediated survival signals might potentiate renal carcinogenesis or may facilitate tumor progression when growth factors are limiting. A general decrease in the apoptotic response may also contribute to chemical carcinogenesis by preventing deletion of mutated cells following genotoxic exposures or by failing to balance proliferative increases induced by nongenotoxic chemicals. Additional studies on the mechanism of Tsc2-dependent apoptotic regulation may therefore provide a better understanding of Tsc2 tumor suppressor function.
In summary, we have shown that expression of Tsc2 in ERC-18 cells increases susceptibility to apoptosis induced by the tumor promoter OKA and the PI3 kinase inhibitor LY294002. Although the precise mechanism by which Tsc2 promotes apoptotic susceptibility to these two compounds remains unclear, our data indicate that alterations in mTOR signaling pathways are not required. In addition, we have shown that Tsc2 expression abrogates the OKA-induced–caspase-independent detachment of Tsc2-null ERC-18 cells. Tsc2-dependent changes in cell adhesion properties may have important implications in determining the cellular susceptibility to apoptosis. Additional studies are required to more specifically address the mechanistic relationship between Tsc2 expression, apoptotic susceptibility, and cell adhesion.
ACKNOWLEDGMENTS
This work was supported by pre-doctoral fellowship ES05925 to T.M.K. from the National Institute of Environmental Health Sciences (NIEHS), and by NIH grant ES08157 to M.A.D. Conflict of interest: none declared.
REFERENCES
Astrinidis, A., Cash, T. P., Hunter, D. S., Walker, C. L., Chernoff, J., and Henske, E. P. (2002). Tuberin, the tuberous sclerosis complex 2 tumor suppressor gene product, regulates Rho activation, cell adhesion and migration. Oncogene 21, 8470–8476.
Blume-Jensen, P., and Hunter, T. (2001). Oncogenic kinase signalling. Nature 411, 355–365.
Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868.
Cardone, M. H., Roy, N., Stennicke, H. R., Salvesen, G. S., Franke, T. F., Stanbridge, E., Frisch, S., and Reed, J. C. (1998). Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318–1321.
Carsillo, T., Astrinidis, A., and Henske, E. P. (2000). Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc. Natl. Acad. Sci. U.S.A. 97, 6085–6090.
Cook, J. A., Oliver, K., Mueller, R. F., and Sampson, J. (1996). A cross sectional study of renal involvement in tuberous sclerosis. J. Med. Genet. 33, 480–484.
Dan, H. C., Sun, M., Yang, L., Feldman, R. I., Sui, X. M., Ou, C. C., Nellist, M., Yeung, R. S., Halley, D. J., Nicosia, S. V., et al. (2002). Phosphatidylinositol 3-kinase/Akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin. J. Biol. Chem. 277, 35364–35370.
Datta, S. R., Brunet, A., and Greenberg, M. E. (1999). Cellular survival: A play in three Akts. Genes Dev. 13, 2905–2927.
Davis, M. A., and Carbott, D. E. (1999). Herbimycin A and geldanamycin inhibit okadaic acid-induced apoptosis and p38 activation in NRK-52E renal epithelial cells. Toxicol. Appl. Pharmacol. 161, 59–74.
Davis, M. A., Chang, S. H., and Trump, B. F. (1996). Differential sensitivity of normal and H-ras oncogene-transformed rat kidney epithelial cells to okadaic acid-induced apoptosis. Toxicol. Appl. Pharmacol. 141, 93–101.
Davis, M. A., Smith, M. W., Chang, S. H., and Trump, B. F. (1994). Characterization of a renal epithelial cell model of apoptosis using okadaic acid and the NRK-52E cell line. Toxicol. Pathol. 22, 595–605.
European Chromosome 16 Tuberous Sclerosis Consortium (1993). Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305–1315.
Evan, G. I., and Vousden, K. H. (2001). Proliferation, cell cycle and apoptosis in cancer. Nature 411, 342–348.
Everitt, J. I., Goldsworthy, T. L., Wolf, D. C., and Walker, C. L. (1992). Hereditary renal cell carcinoma in the Eker rat: A rodent familial cancer syndrome. J. Urol. 148, 1932–1936.
Everitt, J. I., Goldsworthy, T. L., Wolf, D. C., and Walker, C. L. (1995). Hereditary renal cell carcinoma in the Eker rat: A unique animal model for the study of cancer susceptibility. Toxicol. Lett. 82–83, 621–625.
Gao, X., Zhang, Y., Arrazola, P., Hino, O., Kobayashi, T., Yeung, R. S., Ru, B., and Pan, D. (2002). Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat. Cell Biol. 4, 699–704.
Goncharova, E. A., Goncharov, D. A., Eszterhas, A., Hunter, D. S., Glassberg, M. K., Yeung, R. S., Walker, C. L., Noonan, D., Kwiatkowski, D. J., Chou, M. M., et al. (2002). Tuberin Regulates p70 S6 Kinase Activation and Ribosomal Protein S6 Phosphorylation. A Role for the TSC2 Tumor Suppressor Gene in Pulmonary Lymphangioleiomyomatosis (LAM). J. Biol. Chem. 277, 30958–30967.
Harada, H., Andersen, J. S., Mann, M., Terada, N., and Korsmeyer, S. J. (2001). p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD, Proc. Natl. Acad. Sci. U.S.A. 98, 9666–9670.
Henry, K. W., Yuan, X., Koszewski, N. J., Onda, H., Kwiatkowski, D. J., and Noonan, D. J. (1998). Tuberous sclerosis gene 2 product modulates transcription mediated by steroid hormone receptor family members. J. Biol. Chem. 273, 20535–20539.
Hino, O., Klein-Szanto, A. J., Freed, J. J., Testa, J. R., Brown, D. Q., Vilensky, M., Yeung, R. S., Tartof, K. D., and Knudson, A. G. (1993a). Spontaneous and radiation-induced renal tumors in the Eker rat model of dominantly inherited cancer. Proc. Natl. Acad. Sci. U.S.A. 90, 327–331.
Hino, O., Mitani, H., and Knudson, A. G. (1993b). Genetic predisposition to transplacentally induced renal cell carcinomas in the Eker rat. Cancer Res 53, 5856–5858.
Horesovsky, G., Ginsler, J., Everitt, J., and Walker, C. (1994). Increased susceptibility to in vitro transformation of cells carrying the Eker tumor susceptibility mutation. Carcinogenesis 15, 2183–2187.
Howe, S. R., Gottardis, M. M., Everitt, J. I., Goldsworthy, T. L., Wolf, D. C., and Walker, C. (1995). Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. Am. J. Pathol 146, 1568–1579.
Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. (2002). TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4, 648–657.
Jin, F., Wienecke, R., Xiao, G. H., Maize, J. C., Jr., DeClue, J. E., and Yeung, R. S. (1996). Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region. Proc. Natl. Acad. Sci. U.S.A. 93, 9154–9159.
Kenerson, H. L., Aicher, L. D., True, L. D., and Yeung, R. S. (2002). Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res. 62, 5645–5650.
Kleymenova, E., Ibraghimov-Beskrovnaya, O., Kugoh, H., Everitt, J., Xu, H., Kiguchi, K., Landes, G., Harris, P., and Walker, C. (2001). Tuberin-dependent membrane localization of polycystin-1: A functional link between polycystic kidney disease and the TSC2 tumor suppressor gene. Mol. Cell 7, 823–832.
Kolb, T. M., Chang, S. H., and Davis, M. A. (2002). Biochemical and morphological events during okadaic acid-induced apoptosis of Tsc2-null ERC-18 cell line. Toxicol. Pathol. 30, 235–246.
Kolb, T. M., and Davis, M. A. (2004). The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) provokes a prolonged morphologic response and ERK activation in Tsc2-null renal tumor cells. Toxicol. Sci. 80, 233–242.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lau, S. S., Monks, T. J., Everitt, J. I., Kleymenova, E., and Walker, C. L. (2001). Carcinogenicity of a nephrotoxic metabolite of the "nongenotoxic" carcinogen hydroquinone. Chem. Res. Toxicol. 14, 25–33.
Momose, S., Kobayashi, T., Mitani, H., Hirabayashi, M., Ito, K., Ueda, M., Nabeshima, Y., and Hino, O. (2002). Identification of the coding sequences responsible for Tsc2-mediated tumor suppression using a transgenic rat system. Hum. Mol. Genet. 11, 2997–3006.
Noonan, D. J., Lou, D., Griffith, N., and. Vanaman, T. C. (2002). A calmodulin binding site in the tuberous sclerosis 2 gene product is essential for regulation of transcription events and is altered by mutations linked to tuberous sclerosis and lymphangioleiomyomatosis. Arch. Biochem. Biophys. 398, 132–140.
Potter, C. J., Pedraza, L. G., and Xu, T. (2002). Akt regulates growth by directly phosphorylating Tsc2, Nat. Cell Biol. 4, 658–665.
Soucek, T., Pusch, O., Wienecke, R., DeClue, J. E., and Hengstschlager, M. (1997). Role of the tuberous sclerosis gene-2 product in cell cycle control. Loss of the tuberous sclerosis gene-2 induces quiescent cells to enter S phase. J. Biol. Chem. 272, 29301–29308.
Soucek, T., Yeung, R. S., and Hengstschlager, M. (1998). Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2. Proc. Natl. Acad. Sci. U.S.A. 95, 15653–15658.
Tee, A. R., Fingar, D. C., Manning, B. D., Kwiatkowski, D. J., Cantley, L. C., and Blenis, J. (2002). Tuberous sclerosis complex-1 and –2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc. Natl. Acad. Sci. U.S.A. 99, 13571–13578.
Todd, D., Yang, G., Brown, R. W., Cao, J., D'Agati, V., Thompson, T. S., and Truong, L. D. (1996). Apoptosis in renal cell carcinoma: Detection by in situ end-labeling of fragmented DNA and correlation with other prognostic factors. Hum. Pathol. 27, 1012–1017.
Tsuchiya, H., Orimoto, K., Kobayashi, K., and Hino, O. (1996). Presence of potent transcriptional activation domains in the predisposing tuberous sclerosis (Tsc2) gene product of the Eker rat model. Cancer Res. 56, 429–433.
van Slegtenhorst, M., Nellist, M., Nagelkerken, B., Cheadle, J., Snell, R., van den Ouweland, A., Reuser, A., Sampson, J., Halley, D., and van der Sluijs, P. (1998). Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Hum. Mol. Genet. 7, 1053–1057.
Walker, C., Goldsworthy, T. L., Wolf, D. C., and Everitt, J. (1992). Predisposition to renal cell carcinoma due to alteration of a cancer susceptibility gene. Science 255, 1693–1695.
Wienecke, R., Konig, A., and DeClue, J. E. (1995). Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity. J. Biol. Chem. 270, 16409–16414.
Wolf, D. C., Goldsworthy, T. L., Donner, E. M., Harden, R., Fitzpatrick, B., and Everitt, J. I. (1998). Estrogen treatment enhances hereditary renal tumor development in Eker rats. Carcinogenesis 19, 2043–2047.
Xiao, G. H., Shoarinejad, F., Jin, F., Golemis, E. A., and Yeung, R. S. (1997). The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. J. Biol. Chem. 272, 6097–6100.
Yu, J., Astrinidis, A., and Henske, E. P. (2001). Chromosome 16 loss of heterozygosity in tuberous sclerosis and sporadic lymphangiomyomatosis. Am. J. Respir. Crit. Care Med. 164, 1537–1540.
Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14–3–3 not BCL-X(L). Cell 87, 619–628.
Zhang, Y., Gao, X., Saucedo, L. J., Ru, B., Edgar, B. A., and Pan, D. (2003). Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat. Cell Biol. 5, 578–581.(Todd M. Kolb, Ling Duan and Myrtle A. Da)