Cell proliferation is insufficient, but loss of tuberin is necessary, for chemically induced nephrocarcinogenicity

Hae-Seong Yoon1, Terrence J. Monks1, Jeffrey I. Everitt2, Cheryl L. Walker3, and Serrine S. Lau1

1 Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712-1074; 2 Chemical Industry Institute for Toxicology Centers for Health Research, Research Triangle Park, North Carolina 27709-2233; and 3 Science Park-Research Division, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ; 2.5 µmol/kg ip) markedly increased cell proliferation within the outer stripe of the outer medulla (OSOM) of the kidney in both wild-type (Tsc2+/+) and mutant Eker rats (Tsc2EK/+), only TGHQ-treated Tsc2EK/+ rats developed renal tumors, indicating that cell proliferation per se was not sufficient for tumor development. Tuberin expression was initially induced within the OSOM after TGHQ treatment but was lost within TGHQ-induced renal tumors. High extracellular signal-regulated kinase (ERK) activity occurred in the OSOM of Tsc2EK/+ rats at 4 mo and in TGHQ-induced renal tumors. Cyclin D1 was also highly expressed in TGHQ-induced renal tumors. Reexpression of Tsc2 in tuberin-negative cells decreased ERK activity, consistent with the growth-suppressive effects of this tumor suppressor gene. Thus 1) stimulation of cell proliferation after toxicant insult is insufficient for tumor formation; 2) tuberin induction after acute tissue injury suggests that Tsc2 is an acute-phase response gene, limiting the proliferative response after injury; and 3) loss of Tsc2 gene function is associated with cell cycle deregulation.

Tsc2 gene; kidney; carcinogenesis; cell cycle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE TUBEROUS SCLEROSIS-2 (Tsc2) gene is a novel tumor suppressor gene, alterations in which are responsible for the development of renal tumors (49, 50, 57). Loss of heterozygosity (LOH) at the Tsc2 locus has been demonstrated in renal tumors in humans (3, 11) and in spontaneous or chemically induced renal cell carcinomas (RCCs) in rats (25, 48, 56). The finding that Tsc2 knockout mice develop RCCs provides further compelling evidence that this gene functions as a suppressor of renal carcinogenesis (22, 35). The Eker rat (Tsc2EK/+) is a derivative of the Long-Evans strain, bearing a mutation in one allele of the Tsc2 gene, which predisposes these animals to renal cancer (8, 21, 55) and increases their susceptibility to environmental carcinogens (9, 13).

The Tsc2 gene encodes tuberin, a protein of 1,784 amino acids. A 58-amino acid region near the COOH terminus of tuberin exhibits homology with a portion of the catalytic GTPase-activating protein (GAP) for Rap1 (51). Rap1 is a member of the ras superfamily of small GTP-binding proteins and appears to function as a transducer of mitogenic signals from the cell membrane to the nucleus (32, 59, 60). The GAP homology domain of Tsc2 is highly conserved between humans and rats (23, 46). Introduction of the wild-type Tsc2 gene, or the COOH-terminal tuberin construct, including the GAP homology domain, into tuberin-negative cell lines suppresses cell proliferation and tumorigenicity. Thus the Tsc2 gene functions as a tumor suppressor, and at least a portion of this tumor-suppressing function resides at the COOH terminus (18, 36). Rap1GAP negatively regulates Rap1 activity by stimulating the hydrolysis of active, GTP-bound Rap1 to the inactive, GDP-bound form (40). Moreover, tuberin colocalizes with Rap1 in the Golgi apparatus of cultured human cell lines (53) and has similar patterns of expression with Rap1 in tissues such as the kidney, skin, and adrenal gland (52). In addition to Rap1, tuberin also exhibits GAP activity toward Rab5, and tuberin null cells exhibit increased endocytosis, implicating a role for tuberin in regulating the docking and fusion process of the endocytic pathway (54). Tuberin is also involved in cell cycle control (16, 44, 45), regulating the G0-to-G1 transition in quiescent cells. However, the precise physiological or cellular role(s) of tuberin and its role in renal carcinogenesis still remain elusive.

Hydroquinone (HQ) is a ubiquitous environmental chemical that originates from a variety of sources, including cigarette smoke (5, 6). Major uses of HQ are as an agent in photograph-developing solutions, an antioxidant and polymerization inhibitor, and an intermediate in the synthesis of other antioxidant derivatives. HQ has been demonstrated as a rodent nephrocarcinogen in carcinogenicity bioassays and is considered a potential human nephrocarcinogen (19, 43). HQ is classified as a nongenotoxic carcinogen because of its lack of mutagenic activity in standard mutagenicity assays (10, 41). Formation of nephrotoxic glutathione conjugates of HQ contributes to the carcinogenic properties of HQ (29, 37). In particular, a potent nephrotoxic metabolite of HQ, 2,3,5-tris-(glutathion-S-yl)HQ (TGHQ), plays an important role in HQ-mediated nephrocarcinogenicity (29, 37). TGHQ is mutagenic (17), can transform primary renal epithelial cells isolated from Eker rats in vitro (58), and induces a sustained regenerative hyperplasia at sites that subsequently give rise to tumors (29). Treatment of Eker rats with TGHQ (2.5 µmol/kg ip) for 4 mo induces preneoplastic lesions, including toxic tubular dysplasias (29). Microdissection of preneoplastic lesions (toxic tubular dysplasia) has shown that LOH at the Tsc2 locus occurs at an early stage of TGHQ-induced renal tumor development (29), consistent with the view that LOH may be an initiating molecular event in renal carcinogenesis (25). Subsequent administration of TGHQ (3.5 µmol/kg ip) for a further 6 mo significantly increases the incidence of renal tumors (29).

Understanding the early events leading to TGHQ-induced renal carcinogenesis is important in dissecting the mechanism by which TGHQ causes renal cancer. Because we have previously shown that TGHQ acts at an early stage of tumor development and that the Tsc2 gene is an early target gene of TGHQ (29), a subchronic bioassay was conducted with TGHQ (2.5 µmol/kg ip) utilizing wild-type and mutant Eker rats (Tsc2+/+ and Tsc2EK/+) for 1, 2, or 4 mo to determine early biochemical changes that might contribute to TGHQ-induced renal carcinogenesis.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Chemicals and reagents. Glutathione and 1,4-benzoquinone were purchased from Boehringer Mannheim (Indianapolis, IN) and Fluka Chemical (Buchs, Switzerland), respectively. TGHQ was synthesized according to previously established methodology (28) and used at >98% purity, as determined by high-performance liquid chromatography. 5-Bromo-2'-deoxyuridine (BrdU) and protease inhibitor cocktail tablets (complete Mini) were obtained from Boehringer Mannheim. OPTI-MEM I medium and LipofectAMINE were from Life Technologies (Grand Island, NY). Anti-tuberin (C-20) and anti-cyclin D1(A-12) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies were from Kirkegaard and Perry Laboratories (Gaithersburg, MD). Vectastain ABC-alkaline phosphatase kits and Vector red for immunohistostaining were purchased from Vector Laboratories (Burlingame, CA). The mitogen-activated protein kinase (MAPK)P44/P42 assay kit was a product of Amersham Life Science (Piscataway, NJ), and [gamma -33P]ATP (3,000 Ci/mmol) was obtained from New England Nuclear Life Science (Boston, MA).

Animal dosing and tissue preparation. Male Eker rats (wild-type, Tsc2+/+, and mutant, Tsc2EK/+), 2 mo of age, were provided from a closed breeding colony maintained at the University of Texas M. D. Anderson Cancer Center (Smithville, TX). The rats were housed according to a 12:12-h light-dark cycle and allowed food and water ad libitum. The animals were divided into four subgroups: the Tsc2EK/+ control group; the Tsc2EK/+ TGHQ-treated group; the Tsc2+/+ control group; and the Tsc2+/+ TGHQ-treated group. Each subgroup consisted of 11 rats. The rats were administered either vehicle (0.5 ml saline ip; control groups) or TGHQ (2.5 µmol/kg in 0.5 ml of saline ip; treatment groups) 5 days a week for 1, 2, or 4 mo, respectively. The TGHQ dosing solution was prepared fresh in saline each day 5-10 min before dosing. The rats were euthanized by CO2 asphyxiation, and their kidneys and renal tumors were quickly removed. For histological studies, a midsagittal longitudinal section of the kidney and several renal tumors were fixed in 10% phosphate-buffered formalin and paraffin embedded. For biochemical assays, the outer stripe of the outer medulla (OSOM), cortex, and renal tumors were excised, frozen immediately in liquid nitrogen, and stored at -80°C.

Measurement of cell proliferation. Six days before euthanization, Alzet osmotic minipumps (model 2ML1, Alza, Palo Alto, CA) containing 16 mg/ml BrdU in sterile PBS were surgically implanted subcutaneously in the rats over the abdominal area. The avidin-biotin-peroxidase complex method was used to immunolocalize BrdU incorporation into newly synthesized DNA (29). Labeling indices for BrdU-immunoreactive cells in renal tubular epithelium were determined in OSOM and cortical zones by counting 1,000 tubular epithelial lining cells in random fields with the aid of an eyepiece graticle in a Nikon Labophot light microscope at ×400. The labeling indices were determined independently for both the OSOM and renal cortex using a counting scheme previously described for rat nephrotoxicant studies (38). Antibody and tissue controls for BrdU immunostaining were performed (data not shown).

Cell culture and transient transfection. Tuberin-negative cell lines, quinol-thioether-transformed rat renal epithelial (QT-RRE) cells, were established from primary renal epithelial cells of the Eker rat (Tsc2EK/+) (58) and maintained in medium consisting of 50% DMEM with 4.5 g/l glucose (GIBCO BRL, Grand Island, NY) and 50% Ham's F-12 with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2. LLC-PK1 cells were purchased from the American Type Culture Collection (CL101) and cultured with DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2. Full-length Tsc2 cDNA in the pcDNA3 expression vector was kindly provided by Dr. Raymond S. Yeung (University of Washington, Seattle, WA). To restore tuberin expression in QT-RRE cells, transient transfection was performed with the Tsc2 cDNA using LipofectAMINE according to the manufacturer's protocol. Briefly, 5 × 105 cells/well were plated in six-well plates. When the cells reached 70% confluency, transient transfection was carried out. For each well, 8 µg of pcDNA3-Tsc2 cDNA were diluted into 400 µl of OPTI-MEM I medium. LipofectAMINE (15 µl) reagent was also diluted into 400 µl of OPTI-MEM I medium and incubated for 5 min at room temperature. The diluted DNA was combined with diluted LipofectAMINE reagent and incubated at room temperature for 20 min to allow DNA-reagent complexes to be formed. The solution containing the DNA-reagent complexes was directly added to each well containing QT-RRE cells, mixed gently, and incubated for 6 h at 37°C in a CO2 incubator. For controls, QT-RRE cells were transfected with only the pcDNA3 expression vector (Invitrogen). The cells were lysed ~48 h after the transfection. Tuberin expression was confirmed by Western blot analysis of the transfected cells (data not shown). Two independent transient transfection experiments (n = 3 at each time point) were carried out with the same procedure and provided similar results.

Western blot analysis. The OSOM and cortex of kidney tissue, renal tumors, or QT-RRE cells were homogenized with lysis buffer [PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS containing phenylmethylsulfonyl fluoride, aprotinin, and sodium orthovanadate as well as a protease inhibitor tablet (complete Mini, Boehringer Mannheim) containing (in µg/ml) 50 antipain dihydrochloride, 40 bestatin, 60 chymostatin, 10 E-64, 0.5 leupeptin, 0.7 pepstatin, 300 phosphoramidon, and 2 aprotinin, as well as 1 mg/ml Pefabloc SC and 0.5 mg/ml EDTA-disodium salt]. The tissue or cell lysate was centrifuged at 14,000 g for 30 min at 4°C, and protein concentrations were determined with the Bradford assay (1) using BSA as a standard. Protein (50 or 100 µg) was subject to 7 or 12% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes at a constant voltage of 100 V for 1 and 3 h for cyclin D1 or tuberin, respectively. The PVDF membranes were blocked in 5% nonfat dry milk in TBS-0.1% Tween buffer [25 mM Tris · HCl, pH 7.6; 0.2 mM NaCl; 0.1% Tween 20 (vol/vol)] (TBS-T) for 1 h and then incubated with the anti-tuberin (C-20, Santa Cruz Biotechnology) or anti-cyclin D1 (A-12, Santa Cruz Biotechnology) antibodies in TBS-T with 1% nonfat dry milk for 90 min at room temperature. After being extensively washed with TBS-T buffer, anti-rabbit or anti-mouse immunoglobulin conjugated with horseradish peroxidase was added at a 1:2,000-3,000 dilution and incubated for 1 h at room temperature. Tuberin (180 kDa) or cyclin D1 (34 kDa) was visualized by ECL (Amersham, Piscataway, NJ).

Immunohistochemistry. Tuberin expression was examined using the alkaline phosphatase method. Sections of formalin-fixed and paraffin-embedded tissues were deparaffinized in three changes of xylene and rehydrated in a graded ethanol series. Nonspecific binding sites were blocked with 10% normal goat serum (Sigma) in PBS for 30 min. The tissues were then incubated with anti-tuberin antibody (C-20, Santa Cruz Biotechnology) at a 1:300 dilution in PBS for 1 h at room temperature in a humidified chamber. After being extensively washed with PBS, the tissues were incubated with biotinylated anti-rabbit secondary antibody (Vector Laboratories) at a 1:200 dilution in PBS for 30 min at room temperature. After being washed with PBS, the tissues were incubated with ABC-alkaline phosphatase reagents containing avidin and biotinylated alkaline phosphatase H (Vector Laboratories) for 30 min. After being washed in PBS, the tissue sections were incubated in alkaline phosphatase substrate solution (Vector red) for 20-30 min to visualize the staining.

Measurement of MAPKp42/p44/ERK activity. MAPKp42/p44/ERK (mitogen-activated protein kinase/extracellular-signal-regulated kinase) activity was determined using MAPKp42/p44 enzyme assay kits (Amersham). OSOM, renal tumors, or QT-RRE cells were homogenized and lysed with a buffer containing (in mM) 50 Tris · HCl (pH 7.5), 100 NaCl, 10 sodium fluoride, 5 EDTA, 40 beta -glycerophosphate, 0.5 sodium orthovanadate, and 0.25 phenylmethylsulfonyl fluoride as well as 1% Triton X-100 and protease inhibitors (complete Mini). The tissue or the cell lysates were clarified by centrifugation, and 2 µg of protein were utilized for the kinase reaction according to the manufacturer's instructions. Briefly, 2 µg of protein lysate were added to a substrate buffer containing a synthetic peptide (KRELVEPLTPAGEAPNQALLR) highly specific for ERK phosphorylation. The reaction was started by the addition of Mg2+-ATP buffer supplemented with 1 µCi of [gamma -33P]ATP. The reaction mixtures were incubated at 30°C for 30 min, and the reaction was terminated by addition of orthophosphoric acid. Reaction mixtures were then spotted onto phosphocellulose paper to separate phosphorylated peptide, and the peptide binding papers were washed with 75 mM phosphoric acid and water. The incorporated [gamma -33P]ATP was quantified by liquid scintillation spectroscopy (Beckman LS5000TD).

Statistics. Data are expressed as means ± SE. Mean values were compared using a two-way ANOVA, followed by the Student-Newman-Kuels test of statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TGHQ-induced nephrotoxicity and cell proliferation. TGHQ-induced nephrotoxicity is manifested as site-specific damage within the OSOM (29, 37). In the present study, TGHQ (2.5 µmol/kg ip) induced a mild nephrotoxicity (data not shown), followed by cell proliferation within the OSOM (Figs. 1, B and D, and 2, A and B). The increased proliferative response was also site specific and limited to the OSOM (Figs. 1 and 2). Cell proliferation in both Tsc2EK/+ and Tsc2+/+ rats was maximal after 2 mo of TGHQ treatment (Fig. 2A). At 4 mo, although cell proliferation in treated rats was still significantly increased over vehicle controls, it was less than that observed after either 1 or 2 mo of treatment (Fig. 2A). Consistent with decreased cell proliferation, TGHQ-induced nephrotoxicity was less severe morphologically in the 4-mo group compared with that in groups treated for 1 or 2 mo (data not shown). Interestingly, although there was no difference in the pattern or level of cell proliferation between Tsc2EK/+ and Tsc2+/+ rats, only TGHQ-treated Tsc2EK/+ rats developed renal tumors (see the table in the legend for Fig. 2). Histological examination revealed that the nephrotoxicity mediated by TGHQ (2.5 µmol/kg 5 times/wk for 1, 2, and 4 mo) was identical in both Tsc2EK/+ and Tsc2+/+ animals. At this dose, only mild cell injury was noted and an identical rate of cell proliferation was observed. These results suggest that cell proliferation is necessary, but not sufficient, for tumor development and that additional genetic alterations (i.e., loss of the wild-type allele) play an important role in TGHQ-induced nephrocarcinogenesis.


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Fig. 1.   2,3,5-Tris-(glutathion-S-yl)hydroquinone (TGHQ)-induced cell proliferation in the outer stripe of the outer medulla (OSOM) of Eker rats. Cell proliferation was determined by 5-bromo-2'-deoxyuridine (BrdU) immunostaining as described in EXPERIMENTAL PROCEDURES. Kidney slices from Eker rats treated with saline (A and C) or 2.5 µmol TGHQ/kg ip (B and D) for 1 mo. Magnification: ×100 (A and B); ×300 (C and D). BrdU-immunostained cells were detected only within the OSOM of the kidney and not in the cortex (B and D). The pattern of cell proliferation was identical in tuberous sclerosis-2 mutant (Tsc2EK/+) and wild-type (Tsc2+/+) rats.



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Fig. 2.   Quantitation of TGHQ-induced cell proliferation in Eker rat kidney in the OSOM (A) and cortex (B). Positively immunostained nuclei of tubular epithelial cells were counted. Values are means ± SE (n = 4-5) expressed as the no. of proliferating cells/1,000 cells. Significantly different between control and TGHQ-treatment groups, * P < 0.01. 
No. of Tumors/Animal At 4 Mo
Tsc2EK/+ Tsc2+/+
Control       1/11 0/11
TGHQ 3/11 0/11


TGHQ-induced tuberin expression. Western blot analysis demonstrated that Tsc2EK/+ rats tended to express less tuberin than Tsc2+/+ rats (Fig. 3), consistent with the hemizygosity of the functional allele of the Tsc2 gene. In normal kidneys, tuberin expression within the OSOM, the target region for TGHQ-induced toxicity, was undetectable in untreated naïve animals (Fig. 3, A and B). However, administration of saline to control animals slightly increased tuberin expression, more so in Tsc2+/+ than in Tsc2EK/+ animals. Exposure to TGHQ induced tuberin expression in the OSOM (Fig. 3, A and B). Tuberin induction was observed in both groups of animals treated with TGHQ for 1 and 4 mo (Fig. 3, A and B); the increases at 1 mo were substantially less than those observed at 4 mo. In contrast to the OSOM, induction of tuberin was not observed in the cortex (Fig. 3C) in animals treated with TGHQ for 4 mo.


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Fig. 3.   TGHQ induces tuberin expression. Total protein (50 µg) was loaded onto a 7% SDS-polyacrylamide gel. Subsequently, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, and the membrane was incubated with anti-tuberin primary antibody (C-20) followed by anti-rabbit secondary antibody. Tuberin was visualized by the ECL system in OSOM of rats from 1 (A)- or 4-mo (B) treatment with TGHQ and cortex of rats treated with TGHQ for 4 mo (C). Quinol-thioether-transformed rat renal epithelial (QT-RRE) cells as well as LLC-PK1 cells and brain tissues were used as a negative and positive controls, respectively.

Loss of tuberin expression in renal tumors. LOH at the Tsc2 locus occurs with a high frequency in preneoplastic lesions and renal tumors derived from TGHQ-treated Tsc2EK/+ rats (29). Consistent with these findings, kidney tissue adjacent to TGHQ-induced renal tumors expressed high levels of tuberin after treatment with TGHQ for 4 mo, but loss of tuberin expression was observed in the tumors and preneoplastic dysplasias. (Fig. 4).


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Fig. 4.   Loss of tuberin expression in renal tumors. Immunostaining of renal tissues and adjacent tumor tissues was performed as described in EXPERIMENTAL PROCEDURES. a: Control. b and c: Tumors with adjacent renal tissues. T and Tx, renal tumor and toxic tubular dysplasia, respectively. Magnification: ×300 (A and B); ×100 (C ).

High ERK activity and cyclin D1 expression in renal tumors. MAPKp42p44/ERK plays a pivotal role in mitogenesis (7, 39) and participates in cell transformation and tumor development (14, 31, 33, 34). Constitutive activation of the MAPK cascade also occurs in human renal cell carcinomas (14, 33). To determine whether ERK activation occurs during TGHQ-induced nephrocarcinogenicity, ERK activity was determined in renal tumors derived from TGHQ-treated Tsc2EK/+ rats and in the OSOM. During periods of high TGHQ-induced cell proliferation (1 and 2 mo), there was no significant difference in ERK activity between control (Tsc2EK/+ and Tsc2+/+) rats or rats treated with TGHQ for either 1 or 2 mo (Fig. 5). In contrast, after 4 mo of TGHQ treatment, Tsc2EK/+ rats exhibited higher ERK activity than did control Tsc2+/+ rats (Fig. 5). ERK activity was also examined in renal tumors. Renal tumors derived from TGHQ-treated Tsc2EK/+ rats showed significantly higher ERK activity than in the OSOM of all other groups (Fig. 5). To determine the effect of restoration of tuberin expression on ERK activity, we transiently transfected full-length Tsc2 cDNA into a tuberin-negative cell line, QT-RRE, derived from TGHQ-transformed primary renal epithelial cells of Eker Tsc2EK/+ rats (58). Tuberin expression in QT-RRE cells significantly decreased ERK activity (Fig. 6), concomitant with decreases in growth rate ([3H]thymidine incorporation; Yoon HS and Lau SS, unpublished observations). Thus the decreased ERK activity is consistent with the growth-suppression function of tuberin, suggesting that either directly or indirectly by modulating the cell cycle, it acts as a negative regulator of ERK activity. Similarly, in tumors and QT-RRE cells, cyclin D1, a cell-cycle-checkpoint protein involved in the G1-to-S phase transition and a downstream effector of ERK (30), was highly expressed (Fig. 7).


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Fig. 5.   Extracellular signal-regulated kinase (ERK) activity is elevated in the OSOM and in renal tumors of TGHQ-treated Tsc2EK/+ rats. ERK activity was measured utilizing a p42/p44 mitogen-activated protein kinase enzyme assay kit (Amersham) with 2 µg of protein isolated from OSOM of Eker rats treated with saline (control) or 2.5 µmol/kg TGHQ for 1 mo (solid bars), 2 mo (open bars), and 4 mo (gray bars) or from renal tumors after 4 mo of TGHQ treatment (hatched bar). Values are means ± SE expressed as pmol Pi · min-1 · µg protein-1 ; n = 3 and 9 for tumor and OSOM samples, respectively. Significantly different between control and TGHQ-treatment groups, * P < 0.05.



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Fig. 6.   Tuberin modulates ERK activity in QT-RRE cells. Transient transfection of QT-RRE cells, tuberin-negative cell lines, was performed in 70% confluent cells in 6-well plates using LipofectAMINE. Cells were transfected with full-length Tsc2 cDNA in the pcDNA3 expression vector or with pcDNA3 vector as a control. ERK activity was quantitated utilizing a p42/p44 mitogen-activated protein kinase enzyme assay kit (Amersham). Values are means ± SE (n = 3) expressed as pmol Pi · min-1 · µg protein-1. Significantly different between QT-RRE and Tsc2-transfected QT-RRE cells, * P < 0.01.



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Fig. 7.   Cyclin D1 is upregulated in QT-RRE cells and in renal tumors from TGHQ-treated Tsc-2EK/+ rats. Total protein (100 µg) was loaded onto 12% SDS-polyacrylamide gels. After electorphoresis, the proteins were transferred to PVDF membranes and the membranes were incubated with anti-cyclin D1 primary antibody (A-12), followed by anti-mouse secondary antibody. Cyclin D1 was visualized by the enhanced chemiluminescence system. LLC-PK1 cells were used as a negative control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Site-specific cell proliferation in response to TGHQ-induced injury was identical in both Tsc2+/+ and Tsc2EK/+ rats (Figs. 1 and 2), but only TGHQ-treated Tsc2EK/+ rats developed renal tumors. Thus in this model of chemical carcinogenesis, cell proliferation may be necessary, but is certainly not sufficient, for tumor development. Genetic alterations must therefore also be an important factor in TGHQ-induced nephrocarcinogenesis. However, it is unclear whether TGHQ causes genetic alterations directly, because in Tsc2EK/+ rats a highly sustained mitotic environment might indirectly lead to inactivation of the remaining wild-type allele of the Tsc2 gene via mechanisms such as chromosome nondisjunction. In this respect, TGHQ-induced sustained regenerative cell proliferation within the OSOM, without direct interaction with the genetic machinery, may therefore promote LOH at the Tsc2 locus in Tsc2EK/+ rats. Alternatively, TGHQ may be a complete carcinogen (29), because a single treatment with TGHQ increases the mutation frequency in the supF gene when replicated in human AD293 and Escherichia coli MBL50 cells (17) and causes transformation of primary renal epithelial cells from Tsc2EK/+ rats (58). The reactivity of TGHQ lies in its ability to generate reactive oxygen species and to arylate cellular macromolecules (20, 47). Both oxidative DNA damage and arylation of macromolecules may contribute to TGHQ-mediated nephrocarcinogenicity.

Basal tuberin expression was not observed in the OSOM of naïve animals. In contrast, intraperitoneal saline injections to otherwise "untreated" animals slightly increased tuberin expression (Fig. 3, A and B), suggesting that physiological stress may be able to induce a modest upregulation of tuberin. Furthermore, while TGHQ significantly induced tuberin expression in the OSOM at 4 mo (Fig. 3B), it had no effect on tuberin levels in the cortex (Fig. 3C), consistent with the localization of TGHQ-induced stress and cell injury to the OSOM. In contrast, whereas short-term treatment of rats with TGHQ (4 mo) markedly increased tuberin expression within the OSOM, induction of tumors by TGHQ required the inactivation of the Tsc2 gene. Loss of Tsc2 occurs as an early, perhaps initiating, event in preneoplastic lesions (toxic tubular dysplasias) (29), leading to the development of renal tumors in TGHQ-treated Tsc2EK/+ rats. The early upregulation of tuberin expression observed at 4 mo in response to TGHQ suggests that tuberin is induced in response to acute cellular stress or injury and/or acts as a brake to dampen subsequent cell proliferation. Our findings from primary renal epithelial cells support this view. Establishment of primary cell culture is stressful to cells, and although tuberin expression is not detected in normal kidney, primary renal epithelial cells isolated from the OSOM exhibit high tuberin expression in culture (58). The precise function or role of the upregulation of tuberin expression in response to TGHQ is unclear. Tuberin is involved in cell cycle control (16, 44, 45). For example, loss of tuberin expression induces quiescent G0-arrested cells to reenter the cell cycle and also prevents them from reentering G0 (45). Reducing levels of tuberin with antisense oligonucleotides also increases cyclin D1 protein expression and causes a transition from the G0/G1 to the S phase (45). Therefore, perhaps the upregulation of tuberin expression in response to tissue injury functions to limit the mitogenic repair response, thereby counteracting increases in cell proliferation. The finding that tuberin induction in OSOM tissue obtained from rats treated with TGHQ for 4 mo (Fig. 3B) appears to be inversely correlated with the cell proliferation supports this view (Fig. 2A).

Tuberin is a structurally complex protein (46). In addition to possessing a Rap1GAP homology domain, tuberin also contains potentially important domains for gene expression and protein-protein interactions: two transcriptional activation domains (12), a zinc-finger-like region and a potential src-homology 3 binding domain (24). Although most studies have focused on the Rap1GAP function of tuberin, detailed functional analysis of the other regions has not been performed, and such domains may yet provide important functional roles for the tumor-suppressing activity of tuberin.

Four Rap1GAPs have been identified, including tuberin (4, 27, 40), but it is not known whether tuberin represents a major source of Rap1GAP activity in the kidney. However, with the exception of tuberin, there are no studies reported on Rap1GAP expression in the kidney. Rap1GAPs are likely expressed in a tissue- and cell-specific manner. Tuberin may therefore be the predominant Rap1GAP in the kidney. In this case, loss of tuberin would predispose cells to disregulation of the Rap1-mediated signaling pathway.

Interestingly, there was no significant difference in ERK activity between control (Tsc2EK/+ and Tsc2+/+) rats or rats treated with TGHQ for either 1 or 2 mo despite clear increases in cell proliferation at these times (Fig. 2A). Thus cell proliferation in response to TGHQ-induced tissue injury was not driven by the upregulation of ERK. However, after 4 mo of TGHQ treatment, Tsc2EK/+ rats exhibited higher ERK activity than either TGHQ-treated or control Tsc2+/+ rats (Fig. 5), and tuberin expression in these animals was lower than in their Tsc2+/+ counterparts. Renal tumors derived from TGHQ-treated Tsc2EK/+ rats also exhibited significantly higher ERK activity compared with normal kidneys. Thus, while changes in ERK activity did not correlate with changes in cell proliferation per se, the highest ERK activity was associated with decreased tuberin function under comparable exposure conditions. In support of this view, constitutive activation of ERKs has been shown to cause cell transformation (15, 34) and leads to reduced dependence of cellular growth on mitogens (2). In addition, inhibition of the ERK pathway reverts tumor cells to a nontransformed phenotype in vitro, arrests tumor growth in vivo (42), and inhibits the growth of Ras-transformed cells in vitro (31). Transient transfection of Tsc2 cDNA into tuberin-negative cells (Fig. 6) indicated that restoration of tuberin function is either directly or indirectly associated with downregulation of ERK activity, further suggesting that LOH at the Tsc2 locus in TGHQ-induced renal tumors may contribute to high ERK activity in developing renal tumors.

In conclusion, cell proliferation per se is insufficient to drive tumor formation after nephrotoxicant injury. Tuberin, the protein product of the Tsc2 tumor suppressor gene, suppresses cell proliferation and tumorigenesis and plays an important role in TGHQ-induced nephrocarcinogenicity. Tuberin is initially upregulated after TGHQ treatment, most likely in response to tissue injury and the compensatory cell proliferation. However, the Tsc2 gene is subsequently inactivated during TGHQ-induced tumor development and is accompanied by the loss of tuberin expression. The mechanism by which tuberin exerts its antiproliferative and antitumorigenic functions is unclear. However, our data indicate that tuberin is coupled to ERK activity and cyclin D1 expression, implying that the Tsc2 gene may exert its function as a tumor suppressor gene, either directly or indirectly, through regulation of the ERK cascade.


    ACKNOWLEDGEMENTS

The authors thank Dr. Raymond S. Yeung at the University of Washington, Seattle, WA, for kindly providing Tsc2 cDNA used in these studies and I. Badagnani for assistance with the Western blot analysis of tuberin.


    FOOTNOTES

This work was supported by National Institutes of Health Grants GM-39338 (to S. S. Lau), CA-63613 (to C. L. Walker), and ES-07784.

Address for reprint requests and other correspondence: S. S. Lau, Div. of Pharmacology and Toxicology, College of Pharmacy, Univ. of Texas at Austin, Austin, TX (E-mail: slau{at}mail.utexas.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

February 12, 2002;10.1152/ajprenal.00261.2001


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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