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Endocrinology |
Scott Department of Urology [L. L., G. Y., S. E., T. S., Y. N., T. L. T., C. R., J. W., S. A. T., T. C. T.] and Departments of Molecular and Cellular Biology [T. C. T.] and Radiology [T. C. T.], Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cav-1 is the principal component of caveolae, subinvaginations of the plasma membrane and trans-Golgi network that have been implicated in sphingolipid-cholesterol transport and signal transduction pathways (reviewed in Refs. 10, 11, 12, 13 ). Under some conditions, cav-1 has been shown to suppress growth of specific cell lines in vitro and in vivo (8 , 14, 15, 16, 17) , and it has been suggested that cav-1 functions as a tumor suppressor gene (18) . However, specific genetic analysis of cav-1 did not support this contention (19) .4 Recent studies have indicated that some genes can manifest seemingly opposing functional activities in a context-dependent fashion. One example is the bcl-2 gene that can demonstrate pro- or antiapoptotic activities depending on its level of expression (20) . These opposing functions may be related to separate bcl-2 protein domains that have been shown to independently mediate growth arrest or survival depending on cell context (21 , 22) . Additional examples are the Cox-1 and Cox-2 genes that have been shown to be up-regulated in numerous human malignancies, but overexpression of these genes can suppress growth and induce apoptosis in vitro (23 , 24) . Recent studies suggest that the growth-suppressive effects of Cox-1 are not related to its enzymatic activities within the prostaglandin synthesis pathway (24) . Overall, these results indicate the need to clearly define the regulation, biological activities, and mechanism(s) of action for these multipotential genes within the context of malignant progression. In this report, we demonstrate that cav-1 is a downstream effector of T-mediated survival activities and that modest but not high levels of cav-1 can promote both cell survival and metastatic activities in mouse and human prostate cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MTT Assay.
Subconfluent cells were trypsinized, collected by centrifugation, and washed once with SFM. A single-cell suspension was then seeded at low cell density (
200 cells/well of a 96-well plate) in SFM alone or with T (Sigma Chemical Co., St. Louis, MO; SFMT). After 3 days, viability of the cells was determined by incubation with 0.05 mg/ml MTT (Sigma Chemical Co.) at 37°C for a time period ranging from 2 h to overnight. The viability (viable cells:total cells) was determined by counting blue-stained (viable) cells and total cells microscopically and expressed as relative cell viability by normalization to control (control = 1; in cav-1 induction experiments, T = 0 nM was used as control; in virus infection experiments, uninfected was used as control). Previous experiments demonstrated that under conditions of growth/survival factor depletion and low cell density, proliferation was minimal, and therefore the activities monitored by this MTT assay represent predominately cell viability (5)
. The viability data are representative of at least three independent triplicate experiments. Error bars show SDs of a triplicate experiment.
ATPLite Assay.
ABAC3 (1 x 104 cell/well) and LNCaP (1 x 105 cells/well) cells were seeded in SFM or SFM plus various concentrations of T in 12-well plates. After 3 days, floating cells and trypsin-detached cells were combined and counted with a Coulter Particle Counter (Coulter Corp., Miami, FL). One thousand cells were seeded into each well of a 96-well black culture plate (Packard Instrument Co., Meriden, CT). Cell viability was determined with a luminescent ATP detection kit, Packard ATPLite-M, according to the manufacturer’s directions. Light units generated by ATP in each sample were normalized to control (T = 0 nM) and expressed as the relative ATP level. The ATPLite assay was also performed on LNCaP cells infected with an adenoviral vector expressing human sense cav-1 or with control RSV adenoviral vector in 6-well plates as described below. After 2 days in the complete medium postinfection, cells were subjected to growth/survival factor depletion for 3 days in SFM and then collected for ATP determination.
Clonogenic Assay.
Cells were suspended at low density in SFM or SFMT (T = 10 nM for ABAC3 cells and 5 nM for LNCaP cells) in 96-well plates as described for the MTT assay. After 3 days, the medium was removed carefully, and the cells were trypsinized and reseeded in 10-cm plates at a density of 103 cells/plate with complete medium. After 10–15 days, colonies were stained with 0.05 mg/ml MTT in the culture medium for 30 min, and the number of colonies was counted using Advanced Colony Counting software after capturing the image of each plate with a NucleoVision image analysis system (NucleoTech, Hayward, CA). Adenoviral vector-infected cells were grown in complete medium for 2 days after infection and then subjected to low cell density growth/survival factor depletion for 3 days. Cells were then seeded into 10-cm plates at a density of 103 cells/plate in complete medium. Colonies were counted as described above after 3 weeks.
Induction of cav-1 Protein by T.
Cells were seeded at a density similar to that used in the viability assay (2.0 x 105 cells/15-cm plate) in SFM or SFMT with varying concentrations of T. After 2 days, cells were scraped from plates and collected by centrifugation. The cell pellets were washed once with PBS and then lysed with TNES lysis buffer [50 mM Tris (pH 7.5), 2 mM EDTA, 100 mM NaCl, 1% NP40, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride] on ice for 45 min. Proteins were separated on a 12% SDS- polyacrylamide gel and then transferred electrophoretically onto a nitrocellulose membrane. cav-1 and AR were detected with purified polyclonal cav-1 antibody (SC894) and polyclonal AR antibody (SC-826; Santa Cruz Biotechnology, Santa Cruz, CA). A ß-actin monoclonal antibody (A5441; Sigma Chemical Co.) was used to detect ß-actin for loading control. All Western blots shown are representative of at least three independent experiments.
Luciferase Assay for the Mouse cav-1 Promoter Reporter.
A 721-bp mouse cav-1 promoter sequence was subcloned into the luciferase reporter vector, pGL3-basic (Promega Corp., Madison, WI), to generate a mouse cav-1 promoter-controlled luciferase reporter vector, pGL3-mcav-1-luc (9)
. One µg of pGL3-mcav-1-luc or pGL3-basic was cotransfected with 0.25 µg of pCMV-ß-gal into ABAC3 cells (per well of a 6-well plate) using LipofectAMINE Plus (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s protocol. Three h after lipofection, fresh medium was added, and the FCS concentration was brought to 10%. Twenty-four h later, the cells were trypsinized and washed once with SFM, and a single cell suspension was seeded in SFM or SFMT (T = 20 nM) at low density (2 x 105 cells/10-cm plate). Cells were collected after 24 h, lysed in 50 µl of LucLite substrate buffer (Packard) for 15 min at room temperature, and then diluted to the desired volume with PBS containing 1 mM Mg2+ and 1 mM Ca2+. Luciferase assays were performed using the Packard LucLite kit (Packard), and luciferase activities were measured on a TopCount luminescence counter (Packard). ß-Galactosidase activity was measured as an internal control for the transfection efficiency using a ß-galactosidase assay kit (Promega). Tfx-50 reagent (Promega) was used for the transfection of LNCaP cells. Two µg of pGL3-mcav-1-luc or its control vector, pGL3-basic, were cotransfected with 0.25 µg of pCMV-ß-gal into LNCaP cells using 2:1 charge ratio of Tfx reagent:DNA. One h after transfection, 2 ml of fresh SFM or SFMT were added to each well (final concentration of T = 10 nM). The androgen antagonist casodex (1 µM) was also added to the selected SFMT wells. Cells were harvested, and cell lysates were prepared 48 h after transfection. The reporter activity was expressed as relative luciferase activity (light units) by normalization to ß-galactosidase activity. The data reported are representative of at least three independent experiments.
Adenoviral Vector-mediated Sense and Antisense Human cav-1 Expression.
Recombinant adenoviral vectors containing sense (AdScav-1) or antisense (AdAScav-1) human cav-1 cDNA or control AdRSV without a cDNA were generated as described previously (5
, 9)
. LNCaP cells were seeded at a density of 5.0 x 105 cells/well in 6-well plates. After overnight incubation, the medium was replaced with 1 ml of SFM, and adenoviral vector at different MOIs was added. After 3 h, the medium was removed and replaced with complete culture medium. After 48 h, the cells were trypsinized for MTT assay and for the preparation of protein lysates. For ATPLite and clonogenic assays, the culture medium was replaced with SFM 48 h after infection, and the cells were subjected to growth/survival factor depletion for 3 days before each assay. Expression of cav-1 was also confirmed in adenoviral vector-infected cells by immunostaining with cav-1 antibody as described previously (4)
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A double infection with AdAScav-1 was adopted to minimize endogenous cav-1. ABAC3 cells were seeded at 1.0 x 105/well in a 6-well plate and grown overnight. The next day (day 1), cells were infected with the adenoviral-vector at the indicated MOI. A second infection was performed on day 3 (MOI calculations adjusted for increased cell number), followed by another 2-day growth period in complete medium. On day 5, the cells were trypsinized, washed with SFM, and seeded in SFM or SFMT (T = 10 nM) at low cell density as described above for examination of cav-1 protein expression and viability. For the clonogenic assay, SFM- or SFMT-treated cells were detached from a 96-well plate and seeded into 10-cm plates for colony counting as described above.
In Vivo Metastasis Analyses.
A panel of mouse stable antisense cav-1 clones (ABAC3, ABAC5, and BACS4) and control vector clones (ABH11, ABH14, and BHS3) established from high cav-1-expressing lung metastatic cell lines [148-1LMD or 151-2LMC (3)
] were used for orthotopic injection or tail vein injection into syngeneic 129/SV mice as described previously (26)
. Each cell clone was injected into eight or nine animals. Orthotopic tumors were established by injection of 5,000 cells, a cell number sufficient to establish a 100% tumor take (26)
, into the dorsolateral prostate. In some experiments, animals were surgically castrated or received sham surgery 3 days after orthotopic inoculation as described previously (5)
. Two weeks after orthotopic inoculation, animals were euthanized, the tumor was excised carefully, and the wet weight was recorded. The pelvic and retroperitoneal lymph nodes were excised, placed in formalin, embedded in paraffin, cut into 4–5-µm sections, and stained with H&E for histological examination. The extent of metastasis was assessed quantitatively on the stained slides via computer-assisted image analysis (5)
. An experimental metastasis assay consisted of the tail vein injection of 50,000 cells. Mice were euthanized after 14 days, and the lungs were weighed and fixed in Bouin’s fixative, and visible lung metastases were counted with the aid of a dissecting microscope at x10 magnification.
All mice were maintained in facilities accredited by the American Association for Accreditation of Laboratory Animal Care, and all experiments were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals.
Statistical Analysis.
Statistical analyses were performed with Statview 5.0 (SAS Institute, Inc, Cary, NC). Significance was determined by ANOVA with Fisher’s protected least significant difference.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For the human prostate cancer cell line LNCaP, maximum protection was observed at 5 nM T with a >60% (P < 0.0001) increase in viability (Fig. 1, A and B)
. To analyze the long-term effects of T-stimulated viability, we extended the viability assay to a modified clonogenic assay (see "Materials and Methods"). The results revealed that long-term T stimulation resulted in a 4-fold increase in colonies for ABAC3 and 6-fold increase in colonies for LNCaP (Fig. 1C)
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. LNCaP cells demonstrated slightly higher sensitivity to T with maximal induction of cav-1 at 5–10 nM T (Fig. 2)
. These results are in agreement with and extend the findings of a previous report demonstrating increased cav-1 protein levels after T treatment in vitro (27)
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. This activity could be blocked by the addition of 1 µM casodex, a direct AR antagonist (Fig. 3)
, indicating that the up-regulation of cav-1 by T is mediated by the AR.
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show that antisense cav-1 abrogated T-stimulated cav-1 up-regulation (compare AS25 with Un or R25 and AS50 with Un or R50). To demonstrate that the suppression of cav-1 up-regulation by T was a specific consequence of direct cav-1 gene antisense suppression rather than the result of a general toxic and/or nonspecific effect on cell viability, we also evaluated the expression of a known androgen-responsive gene, AR (28)
. The up-regulation of AR by T was not altered in the antisense cav-1-treated cells, confirming the cav-1 specificity of the antisense suppression (Fig. 4A)
. As a consequence of the suppression of cav-1 up-regulation by T, the increased viability stimulated by T was significantly reduced (Fig. 4B)
. At a MOI of 25 with the control AdRSV vector (R25), relative cell viability in SFMT was increased to 1.42 relative to SFM (P = 0.0007), whereas with antisense cav-1 (AS25), relative survival was only 1.1 in SFMT (P = 0.3214). Similarly, at a MOI = 50, T-stimulated survival was reduced from 1.5 (R50, P = 0.0003) to 1.07 (AS50, P = 0.5208). In clonogenic assays (Fig. 4C)
, T stimulated 2–3-fold increase colonies in uninfected and RSV controls (P < 0.0001), whereas in antisense cav-1-infected, SFMT-treated cells, the colony number was increased by 41% in AS25 (P = 0.09954) and 22% in AS50 (P = 0.3256). The results of these experiments, together with those described above, demonstrate that cav-1 is a downstream effector of T that is responsible in part for the survival/clonal growth stimulated by T in prostate cancer cells under these experimental conditions.
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. cav-1 expression increased the relative viability of LNCaP cells after growth/survival factor depletion. Interestingly, the maximum survival protection of cav-1 (>40%; P < 0.0001) was observed at a MOI = 10 (Fig. 5, B and C)
, which corresponded with a moderate level of cav-1 protein (Fig. 5A)
. Viability protection was observed with AdScav-1 at MOI 5–50, but not at MOI = 100 or 200 (Fig. 5, B and C)
. These data clearly suggest that cav-1 can induce survival activities when it is expressed at moderate levels; however, when it is expressed at high levels, cav-1 may be toxic to the cells. To determine the long-term effect of cav-1 expression on survival/clonal growth, we also performed a clonogenic assay on infected cells. A significant difference between the effects of AdScav-1 and control vector AdRSV was observed in this extended assay, with 8–10-fold more colonies for the cav-1 group in a 3-week period (Fig. 5D)
. To confirm expression of cav-1 in the LNCaP cells, we performed immunohistochemical staining that revealed an absence of cav-1 in uninfected LNCaP cells (Fig. 5E)
but readily detectable expression of cav-1 in LNCaP cells infected with AdScav-1 (Fig. 5F)
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. The antisense clones were about 10% smaller than the vector clones in the sham-operated animals, but this was not a significant difference (P = 0.226). However, a significant (39%; P < 0.001) decrease in tumor weight was observed in the antisense clones in castrated animals, but not in the vector clones. In these same animals, the extent of spontaneous lymph node metastasis was evaluated in terms of the number of animals with metastases (incidence) and the relative volume of the metastases as determined by computer-assisted microscopic quantitation (Fig. 6B)
. The antisense clones had less metastatic activity compared with the vector control clones in the sham-operated animals with a 17% decrease in incidence (P = 0.003) and a 52% reduction in relative volume (P < 0.0001). In castrated animals, there was no difference between the vector control clones and the sham-operated animals; however, the antisense clones had a significantly greater decrease in both incidence and volume of lymph node metastasis than the antisense clones in sham-operated animals (18% and 28%, respectively; P < 0.001). To further evaluate metastatic activity, we injected cell clones directly into the tail vein and counted the number of lung metastatic deposits that formed at 2 weeks (Fig. 6C)
. The antisense cav-1 clones had 40% fewer lung metastases as compared with vector control clones (P < 0.001).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the first series of experiments we demonstrated that T induces cav-1 expression in part at the level of transcriptional regulation. We demonstrated previously that cav-1 is expressed at very low to nondetectable levels in normal prostate epithelium but is expressed focally in prostate cancer, and further increased expression is associated with prostate cancer metastases (3, 4, 5) . Together, these results suggest that T is responsible, in part, for inducing cav-1 in prostate cancer cells during progression, but it is not yet clear how the cav-1 gene, which is relatively inactive in normal prostate epithelial cells, becomes responsive to T induction. Conceivably, demethylation could play a role, but previous reports have been inconclusive regarding the role of methylation in cav-1 expression in prostate cancer, and additional studies are needed (19) .
To study the effects of T and cav-1 expression on prostate cancer cell survival and clonal growth activities in vitro, we developed a two-step assay system that mimics specific steps of metastasis in vivo. In the first step of this analysis, prostate cancer cells are maintained for 3 days at low density under serum-free conditions, mimicking the reduced growth factor and low density conditions encountered during vascular transit. After this 3-day period, cell viability was analyzed using two independent methods of analysis (the MTT and ATPLite assays). Cells were subsequently seeded into a clonogenic assay, which involved a 2–3-week growth period in vitro followed by analysis of colony number. This second step approximates growth at a distal metastatic site and is dependent on continued cell survival. The initial experiments using this assay system demonstrated that T can stimulate cell survival and clonal growth in both mouse and human prostate cancer cells.
Additional experiments in mouse prostate cancer cell lines using adenoviral vector-mediated antisense cav-1 demonstrated that cav-1 induction was responsible, in part, for T-stimulated cell survival/clonal growth in vitro. These results are consistent, in general, with the results of our previous studies that demonstrated that elevated cav-1 levels are associated with androgen insensitivity (5) . In the absence of T, it is conceivable that other growth factors stimulate cav-1 expression in prostate cancer. Others have shown that polypeptide growth factors can regulate cav-1 expression in NIH-3T3 cells (29) . However, to establish a clear correlation between cav-1 expression and androgen-insensitive human prostate cancer, it will be necessary to demonstrate that cav-1 expression is increased in androgen-insensitive disease and to generate experimental support for androgen-independent regulation of cav-1 expression in androgen-insensitive prostate cancer cells. Additional studies in this area are needed.
The substitution of increased cav-1 expression via infection with AdScav-1 demonstrated that modest levels of cav-1 could also maintain viability in the assay systems described above. The results of the clonogenic assay supported and extended the results of the survival analyses, indicating a severalfold increase in the number of colonies in AdScav-1-infected cells compared with that in control Ad-RSV-infected cells in human (LNCaP) prostate cancer cells.
The data presented in this report, together with our previous studies (9) , indicate that relatively modest but not high levels of cav-1 expression can lead to increased cell viability consistent with malignant progression. Overall, these results further reconcile previous reports that have shown that high levels of cav-1 can suppress growth in various cell types (8 , 14, 15, 16, 17) . A recent study indicates that although cav-1 is initially down-regulated in colon cancer cells, reexpression of cav-1 is selected for during the development of drug resistance and metastasis (8) . At the molecular level, this dichotomy between the role of cav-1 in tumorigenesis and metastasis may be explained in part by specific interactions between phosphorylated cav-1 and downstream signaling molecules (30) . Additional studies are required to define the molecular mechanism(s) through which cav-1 specifically promotes survival/clonal growth in prostate cancer cells.
Finally, we generated in vivo data that support our in vitro studies and demonstrate that experimental reduction of cav-1 expression results in suppression of metastatic activities in vivo. Using stable antisense mouse prostate cancer cell clones, our results indicate that both spontaneous and experimental metastatic activities can be significantly reduced by the suppression of cav-1 expression. The results of our castration studies further suggest that the presence of circulating T together with cav-1 can produce synergistic effects that increase metastatic activities. Interestingly, although a reduction of cav-1 levels suppressed metastatic activities it did not suppress primary tumor growth, demonstrating that the effects of cav-1 in vivo are metastasis specific in this prostate cancer model.
Our results have demonstrated that T can induce cav-1 expression in part through transcriptional regulation and that cav-1 overexpression is, in part, responsible for T-stimulated survival of mouse prostate cancer cells in vitro. Additional studies documented that modest but not high levels of cav-1 can support survival of prostate cancer cells under proapoptotic conditions, i.e., growth/survival factor depletion and low cell density, and promote clonal growth in vitro. Finally, analysis of spontaneous and experimental metastasis using stably transfected antisense cav-1 prostate cancer cells confirmed that elevated cav-1 levels contribute to metastasis of prostate cancer cells in vivo. Additional studies will be needed to define the molecular mechanism(s) through which cav-1 contributes to prostate cancer metastasis.
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FOOTNOTES |
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1 Supported by National Cancer Institute Grants CA 50588, CA 68814, and SPORE P50–58204 and the Department of Defense Grant DAMD17-98-1-8575. 
2 To whom requests for reprints should be addressed, at Baylor College of Medicine, 6560 Fannin, Suite 2100, Houston, TX 77030. Phone: (713) 799-8718; Fax: (713) 799-8712; E-mail: timothyt{at}www.urol.bcm.tmc.edu
3 The abbreviations used are: cav-1, caveolin-1; T, testosterone; SFM, serum-free medium; AR, androgen receptor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SFMT, serum-free medium plus testosterone; RSV, Rous sarcoma virus; MOI, multiplicity of infection.
4 C. Ren, L. Garza, Y. Yuan, W. Tian, and T. C. Thompson, unpublished data.
Received 12/ 5/00. Accepted 3/29/01.
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 S. A. Tahir, C. Ren, T. L. Timme, Y. Gdor, R. Hoogeveen, J. D. Morrisett, A. Frolov, G. Ayala, T. M. Wheeler, and T. C. Thompson Development of an Immunoassay for Serum Caveolin-1: A Novel Biomarker for Prostate Cancer Clin. Cancer Res., September 1, 2003; 9(10): 3653 - 3659. [Abstract] [Full Text] [PDF]
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D. Wu and D. M. Terrian Regulation of Caveolin-1 Expression and Secretion by a Protein Kinase Cepsilon Signaling Pathway in Human Prostate Cancer Cells J. Biol. Chem., October 18, 2002; 277(43): 40449 - 40455. [Abstract] [Full Text] [PDF]
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C. Ren, L. Li, A. A. Goltsov, T. L. Timme, S. A. Tahir, J. Wang, L. Garza, A. C. Chinault, and T. C. Thompson mRTVP-1, a Novel p53 Target Gene with Proapoptotic Activities Mol. Cell. Biol., May 15, 2002; 22(10): 3345 - 3357. [Abstract] [Full Text] [PDF]
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B. Terris, E. Blaveri, T. Crnogorac-Jurcevic, M. Jones, E. Missiaglia, P. Ruszniewski, A. Sauvanet, and N. R. Lemoine Characterization of Gene Expression Profiles in Intraductal Papillary-Mucinous Tumors of the Pancreas Am. J. Pathol., May 1, 2002; 160(5): 1745 - 1754. [Abstract] [Full Text] [PDF]
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L. Zhuang, J. Lin, M. L. Lu, K. R. Solomon, and M. R. Freeman Cholesterol-rich Lipid Rafts Mediate Akt-regulated Survival in Prostate Cancer Cells Cancer Res., April 1, 2002; 62(8): 2227 - 2231. [Abstract] [Full Text] [PDF]
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C. Pramudji, S. Shimura, S. Ebara, G. Yang, J. Wang, C. Ren, Y. Yuan, S. A. Tahir, T. L. Timme, and T. C. Thompson In Situ Prostate Cancer Gene Therapy Using a Novel Adenoviral Vector Regulated by the Caveolin-1 Promoter Clin. Cancer Res., December 1, 2001; 7(12): 4272 - 4279. [Abstract] [Full Text] [PDF]
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C. D. Wolfgang, M. Essand, B. Lee, and I. Pastan T-Cell Receptor {gamma} Chain Alternate Reading Frame Protein (TARP) Expression in Prostate Cancer Cells Leads to an Increased Growth Rate and Induction of Caveolins and Amphiregulin Cancer Res., November 1, 2001; 61(22): 8122 - 8126. [Abstract] [Full Text] [PDF]
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K. Wiechen, L. Diatchenko, A. Agoulnik, K. M. Scharff, H. Schober, K. Arlt, B. Zhumabayeva, P. D. Siebert, M. Dietel, R. Schafer, et al. Caveolin-1 Is Down-Regulated in Human Ovarian Carcinoma and Acts as a Candidate Tumor Suppressor Gene Am. J. Pathol., November 1, 2001; 159(5): 1635 - 1643. [Abstract] [Full Text] [PDF]
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) and LNCaP (
) cells were seeded at low density (200 cells/well in a 96-well plate) and incubated for 3 days in SFM or SFMT with various concentration of T. Cells were stained with MTT, and then living and dead cells were counted microscopically. Viability was expressed as a fraction of viable cells (viable cells:total cells) and normalized to control (control = 1; T = 0 nM was used as control). B, ABAC3 (
) cell lysates were prepared, and reporter activities were determined. Error bar, SD. **, P < 0.0001 compared with SFM.
