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Overexpression of tuberous sclerosis complex 2 exerts antitumor effect on oral cancer cell lines
Oral Oncology (2003) 39 836–841
www.elsevier.com/locate/oraloncology
Overexpression of tuberous sclerosis complex 2 exerts antitumor effect on oral cancer cell lines Shin-ichi Kawaguchia, Koji Haradab,*, Supriatnob, Hideo Yoshidab, Mitsunobu Satob a
Department of Oral and Maxillofacial Radiology, University of Tokushima, School of Dentistry, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan b Second Department of Oral and Maxillofacial Surgery, University of Tokushima, School of Dentistry, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan
Received 15 April 2003; accepted 14 May 2003
KEYWORDS TSC2; Tuberin; Oral cancer; p27Kip1; Antitumor effect
Summary Tuberous sclerosis is caused by mutations in tuberous sclerosis complex (TSC) 2 on chromosome 16p13.3, encoding tuberin which is thought to be essential for p27Kip1 to regulate the cell cycle. In this study, we conducted to examine whether overexpression of TSC2 can affect the growth of oral cancer cells which have different expression level of p27Kip1 protein. We constructed an expression vector containing sense-oriented rat TSC2 cDNA with pcDNA3.1. We transfected oral cancer cells, B88t (high expression of p27Kip1 protein) and HI (low expression of p27Kip1 protein) with the sense expression vector to up-regulate the expression of TSC2 gene. Overexpression of TSC2 exerted the growth inhibitory effect of B88t and HI in vitro and in vivo. These findings suggest that overexpression of TSC2 may exert the antitumor effect on oral cancer cells whether they have high expression of p27Kip1 protein or not. # 2003 Elsevier Ltd. All rights reserved.
Introduction Tuberous sclerosis complex (TSC) is an inherited syndrome in which affected individuals are at increased risk for developing benign tumors included hamartomas, rhabdomyomas, angiofibromas and fibromas.1 Central nervous system manifestations include mental retardation, autism and seizures.2 Rarely, more malignant tumors develop. Linkage analysis of TSC resulted in the identification of two distinct genetic loci on chromosome 9 * Corresponding author. Tel.: +81-88-633-7354; fax: +81-88633-7462. E-mail address: [email protected] (K. Harada).
and 16.3 These genes are TSC1 and TSC2, respectively.4 TSC1 and TSC2 follow the classic retinoblastoma tumor suppressor gene model and appear to function as negative growth regulators.5 The TSC2 gene product, tuberin is a 180—200 kDa protein that contains a coiled-coil domain believed to mediate its interaction with hamartin and a carboxyl terminal GTPase activating protein.6 Tuberin may function as a tumor suppressor by induction of p27Kip1 protein. Interestingly, overexpression of tuberin results in reduce cell proliferation in vitro7 and increased amounts of the cell cycle regulator p27Kip1 in rat fibroblast.8 In addition, it is thought that tuberin is essential for p27Kip1 to regulate the cell cycle because tuberin can retain
1368-8375/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1368-8375(03)00106-4
Tuberous sclerosis complex 2 and oral cancer cell lines p27Kip1 protein in nuclei of cancer cells.8 In general, it has been thought that the prognosis of the oral squamous cell carcinoma (SCC) patients who have p27Kip1 proteins in nuclei of their cancer cells should be good.9 In short, tuberin may be closely associated with p27Kip1 to exert the function as tumor suppressor gene. In the present study, we conducted to examine whether overexpression of TSC2 can affect the growth of B88t and HI cells in vitro and in vivo by transfecting an expression vector containing sense-oriented rat TSC2 cDNA to B88t and HI cells.
Material and methods Establishment of a cell line and cell culture B88t cells was recovered from a subcutaneous tumor of formed by B88 cells on a nude mouse.10 B88t cells showed a high tumorigenesity on a nude mouse than B88 cells (data not shown). The original B88 cells were isolated from an oral SCC patient in our laboratory.11 The original tumor of B88 cells was moderately differentiated SCC of tongue, and was not invasive into muscle layer. B88 cells were established from a cervical lymph-node metastasis. We established a cell line in this experiment, designated as HI. HI cells were derived from a moderately differentiated SCC at tongue. The original tumor of HI cells was highly invasive to muscle layer and grew aggressively at the primary site. HI cells were established from a skin metastasis near the primary site. Cancer tissues were minced into 1 mm3 pieces and placed in 100 mm Petri dishes (Falcon; Becton Dickinson Labware, Lincoln Park, NJ, USA) containing a small amount of Dulbecco’s modified Eagle medium (DMEM; Sigma, St Louis, MO, USA) supplemented with 10% fetal calf serum (FCS; Moregate BioTech, Bulimba, Australia), and 100 mg/ml streptomycin, 100 units/ml penicillin (Gibco), and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 C. Epithelial cells were isolated and cloned. The cloned cells were cultured on plastic Petri dishes with the above medium and were passaged to new dishes before confluence. In addition, selected clones obtained after transfection were maintained in the same medium supplemented with Geneticin (800 mg/ml G418, Sigma).
Construction of a mammalian expression vector The mammalian expression vectors pcDNA3.1TSC2 (containing sense-oriented rat TSC2 cDNA)
837 was constructed as follows: pcDNA3.1 (+) (Invitrogen, Carlsbad, CA) is mammalian expression vectors containing a CMV promoter. pcDNA3.1 (+) was digested with Xba I (Takara Biomedicals, Kusatsu, Japan) and Xho I (Takara Biomedicals), and dephosphorylated by calf intestinal alkaline phosphatase (Roche Diadnostics, Mannheim, Germany). The rat TSC2/pBluescript including the rat TSC2 cDNA fragment (5.4 kbp Xba I and Xho I fragment) was obtained as a generous gift from Professor Okio Hino (Experimental Pathology, The Cancer Institute, Tokyo, Japanese Foundation for Cancer Research). This fragment was ligated to the prepared cloning site of pcDNA3.1 (+) by T4 DNA ligase (Takara Biomedicals). The direction of the ligated fragment was confirmed by sequencing analysis.
Transfection Cells (5105 cells/dish) were seeded in 100 mm culture dishes (Falcon) in DMEM supplemented with 10% FCS. Twenty-four hours later, the cells were transfected with 5 mg of pcDNA3.1-TSC2 or pcDNA3.1 without insert (empty vector) by use of the Superfect reagent (Qiagen, Hilden, Germany). The cells were incubated for 24 h in DMEM containing 10% FCS, then trypsinized and seeded at a 1:5 ratio in 100 mm culture dishes in DMEM medium containing 10% FCS. Forty-eight hours later, the cells were switched to a selective medium containing Geneticin (800 mg/ml G418, Sigma). After 14 days of culture in the selective medium, ten representative G418-resistant clones were isolated and expanded in a 24-well cluster dish (Falcon).
Western blotting Cell lysates were prepared from the transfectants as follows: cells were cultured to subconfluence, washed three times with 100 mM phosphate-buffered-saline, and lysed with 50 mM N-2-hydroxyethyl piperazine-N0 -2-ethanesulfonic acid, HEPES (pH 7.5) containing 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM NaF, 100 mM p-nitrophenyl phosphate, 5 U/ml aprotinin, and 1 mM phenyl-methylsulfonyl fluoride. The protein concentration of the samples was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Fifty micrograms of protein samples were electrophoresed on SDS-polyacryl amide gel. Proteins from gels were transferred to PVDF membrane (Bio-Rad), and the membrane was incubated with a 1:500 dilution of the monoclonal or polyclonal antibody against tuberin (N-19, rabbit polyclonal antibody, Santa Cruz Biotechnology,
838 Santa Cruz, CA, USA), p27 protein (clone 1B4, monoclonal antibody, Novocastra Laboratories, New Castle, UK) as the primary antibody, and an Amersham ECL kit (Amersham Pharmacia Biotech). Also, anti-a-tubulin monoclonal antibody (Zymed Laboratories, SanFrancisco, CA, USA) was used for normalization of Western blot analysis.
MTT Assay Cells were seeded on 96-well plates (Falcon) at 5103 cells per well in DMEM containing 10% FCS with/without Geneticin. After 1, 2 and 3 days, the number of cells was quantitated by an assay in which MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) was used.
S. Kawaguchi et al.
Growth of cells in vitro Relative cell number was evaluated by comparing the absorbance in each cells at day 1, 2 and 3. In the case of B88t, no significant difference in the cell number was seen among the cells on day 1. However, the cell number of sense transfectants was significantly decreased when compared with that of control cells at day 2 and 3 (P < 0.01; oneway ANOVA). In the case of HI, no significant difference in the cell number was seen among the cells on day 1 and 2. However, the cell number of sense transfectants was significantly decreased when compared with that of control cells at day 3 (P < 0.01; one-way ANOVA) (Fig. 3).
In vivo tumorigenesis assay The tumorigenicity of tumor cells was examined in the nude mouse with Balb/cA Jcl-nu genetic background (CLEA Japan, Inc. Tokyo, Japan). Tumor cells (1106) were suspended in 0.1 ml of serum-free medium and injected into the subcutaneous tissue of 5-week-old nude mice using a 27-gauge needle. The size of the tumors were determined by first measuring length (L) and width (W) and then calculating the volume (0.4LW 2) every 2 days. The body weight of the mice also measured every 2 days. The mice were sacrificed 24 days after inoculation.
Results
Figure 1 TSC2 plasmid construction. pcDNA3.1-TSC2 contains the sense-oriented 5.4 kbp rat TSC2 cDNA under the transcriptional control of the cytomegalovirus (CMV) promoter. The rat TSC2 cDNA fragment (5.4 kbp Xba I and Xho I fragment), lacking exon 25 and 31, was cloned into the mammalian expression vector pcDNA3.1 (+) (Invitrogen) at the Xba I and Xho I restriction sites.
Expression of tuberin and p27Kip1 by Western blotting After transfection with pcDNA3.1-TSC2 (Fig. 1), or pcDNA3.1neo, we obtained more than 100 G418resistant colonies in sense transfectants and isolated 10 representative G418-resistant clones in sense transfectants. They were screened for the expression of tuberin by Western blot analysis. Approximately 90% of the representative G418resistant clones in sense transfectants (B88t-TSC2, HI-TSC2) expressed the up-regulation of tuberin (data not shown). As shown in Fig. 2A, we detected the up-regulation of tuberin in sense-TSC2 transfectants when compared with that in parental cells (B88t, HI) or control cells which were transfected with pcDNA3.1 without insert (B88t-neo, HI-neo). The expression of a-tubulin as an internal control was approximately the same in all of the cells. Also, the expression level of p27Kip1 protein of B88t was high than that of HI (Fig. 2B).
Figure 2 Expression of tuberin in the transfectants. A Western blot analysis. Cell lysates were prepared from parental cells (B88t, HI), control cells (B88t-neo, HI-neo) and the sense transfectants (B88t-TSC2, HI-TSC2). Protein samples (50 mg) were electrophoresed on SDS-polyacrylamide gel and transferred to PVDF membrane, and incubated with anti-tuberin polyclonal antibody, antip27 monoclonal antibody and anti-a-tubulin monoclonal antibody.
Tuberous sclerosis complex 2 and oral cancer cell lines
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Figure 3 Growth of transfectants in vitro. Relative cell number was evaluated by comparing the absorbance in each cells at day 1, 2 and 3. The values shown are the mean of six determinations; bars, S.D. *, P <0.01 when compared with that of control cells by one-way ANOVA.
In vivo tumorigenicity of the transfectants All five mice that received parental cells (B88t, HI) developed moderate size tumors, and all five mice that received control cells (B88t-neo, HI-neo) also developed moderate size tumors. All five mice
that received sense transfectants (B88t-TSC2, HITSC2) developed small tumors. As shown in Fig. 4, tumors induced by TSC2-up-regulated transfectants become much smaller than those of control cells and parental cells (Fig. 4). During the experimental period, no loss of body weight was observed in each group.
Figure 4 Growth of tumors formed by transfectants in nude mice. Tumor cells (1106) were suspended in 0.1 ml of serum-free medium and injected into the subcutaneous tissue of nude mice. Sizes of the tumors were determined by first measuring length (L) and width (W) and then calculating the volume (0.4LW 2). Each group had 5 mice. The values shown are the mean of four tumors (mm3); bars, S.D. *, P <0.01 when compared with that of control cells by one-way ANOVA.
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Discussion Recently, we have reported that low p27Kip1 expression is associated with poor prognosis, and that p27Kip1 is a useful predictor of survival in oral SCC.12 Similar results have been observed in most solid tumours, including in breast cancer,13 colorectal carcinoma,14 gastric carcinoma,15 prostate cancer16 and non-small cell lung cancer17 suggesting that down-regulation of p27Kip1 is a general phenomenon in malignancies associated with aggressive tumor growth. However, some studies on colorectal carcinoma reported that p27Kip1 expression showed no relationship to tumor progression or overall survival.18 We have also reported that up-regulation of p27Kip1 protein can exert the growth inhibitry effects through induction of G1 arrest and apoptosis on oral cancer cell line.11 In short, high p27Kip1 expression should be associated with good prognosis when p27Kip1 protein function normally. In the case of colorectal carcinoma, p27Kip1 protein may not function as a cell cycle regulator for some reason, or some factor may regulate the p27Kip1 protein at up-stream of p27Kip1. So, we paid attention to TSC2 as a regulator of p27Kip1 protein in the present study. The autosomal dominant disease tuberous sclerosis (TSC) is caused by mutations in either TSC1 on chromosome 9q34, encoding hamartin, or TSC2 on chromosome 16p13.3, encoding tuberin. Tuberin seemed to play an important role in exerting the function of p27Kip1 protein as a cell cycle regulator by retaining p27Kip1 protein in nuclei of cancer cells.8 However, the true biochemical functions of TSC2 as a tumor suppressor has not been clarified yet. Although overexpression of TSC2 resulted in inhibition of cancer cell proliferation and up-regulation of p27Kip1, TSC2 involved mutations also inhibited the cancer cell growth and increased amounts of the p27Kip1 in the same way.19 In addition, it is reported that TSC2 heterozygosity may provide a non-cell-autonomous growth advantage for astrocytes that may involve the reduction of p27Kip1 expression.20 Recently, it has been reported that the function of p27Kip1 protein as a cell cycle regulator inhibit by Akt/PKB (Protein Kinase B).2123 Interestingly, the Akt/PKB also is related with the degradation of tuberin.24 In short, the relationship between TSC2 and p27Kip1 protein is clarifying in these years. In this study, overexpression of TSC2 exerted the antitumor effect on oral cancer cells whether they have high expression of p27Kip1 protein or not. These finding may suggest that tuberin can regulate the p27Kip1 protein at up-stream of p27Kip1. In short, tuberin may be more important
S. Kawaguchi et al. as a prognostic factor in oral SCC rather than p27Kip1 protein. Next we should investigate the relationship between tuberin expression and prognosis in oral SCC. A significant suppression of tumor growth of sense transfectants in vivo would be worthy of note. In addition, transfectants did not affect the body weight of the nude mice. These findings may suggest that up-regulation of tuberin in tumor cells will be safe for the body, and TSC2 can be used as molecular target for gene therapy if the precise function of TSC2 can be clarified. Recently, extensive investigations have been performed to elucidate the role of TSC2 as a tumor suppressor in normal and neoplastic cells, but conclusions have not been drawn about the detailed function of TSC2. We should try to investigate the factors which regulate the TSC2 at up-stream of TSC2 to clarify the molecular mechanisms of the antitumor effect of TSC2. References 1. Roach ES, Gomez MR, Northrup H. Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. J Child Neurol 1998;13:624—628. 2. Gomez MR, Sampson JR, Whittemore VH. Tuberous sclerosis. 3rd ed. Oxford (UK): Oxford University Press; 1999. 3. Povey S, Burley MW, Attwood J, Benham F, Hunt D, Jeremiah SJ, et al. Two loci for tuberous: one on 9q34 and one on 16p13. Ann Hum Genet 1994;58:107—127. 4. Henske EP, Scheithauer BW, Short MP, Wollmann R, Nahmias J, Hornigold N. Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions. Am J Hum Genet 1996;59:400—406. 5. Uhlmann EJ, Apicelli AJ, Baidwin RL, Burke SP, Bajenaru ML, Onda H, et al. Heterozygosity for the tuberous sclerosis complex (TSC) gene products results in increased astrocyte number and decreased p27-Kip1 expression in TSC2+/ cells. Oncogene 2002;21:4050—4059. 6. Wienecke R, Konig A, DeClue JE. Identification of tuberin, the tuberous sclerosis 2 product. Tuberin possesses specific Rap1 GAP activity. J Biol Chem 1995;270:16409—16414. 7. Jin F, Wienecke R, Xiao GH, Maize JC, DeClue JE, Yeung RS. Suppression of tumorigenecity by the wild-type tuberous sclerosis 2 (TSC2) gene and its C-terminal region. Proc Natl Acad Sci USA 1996;93:9154—9159. 8. Soucek T, Yeung RS, Hengstschlager M. Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene 2. Proc Natl Acad Sci USA 1998;95:15653—15658. 9. Mineta H, Miura K, Suzuki I, Takebayashi S, Amano H, Araki K, et al. Low p27 expression correlates with poor prognosis for patients with oral tongue squamous cell carcinoma. Cancer 1999;85:1011—1017. 10. Supriatno, Harada K, Kawaguchi S, Yoshida H, Sato M. Effect of p27Kip 1 on the ability of invasion and metastasis of an oral cancer cell line. Oncol Rep 2003;10:527—532. 11. Supriatno, Harada K., Hoque M.O., Bando T., Yoshida H., Sato M. Overexpression of p27Kip 1 induces growth arrest and apoptosis in an oral cancer cell line. Oral Oncol 2002; 38:730—736.
Tuberous sclerosis complex 2 and oral cancer cell lines 12. Harada K, Supriatno, Yoshida H, Sato M. Low p27Kip 1 expression is associated with poor prognosis in oral squamous cell carcinoma. Anticancer Res 2002;22(5):2985—2989. 13. Catzavelos C, Bhattacharya N, Ung YC, et al. Decreased levels of the cell-cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer. Nature Med 1997;3:227—230. 14. Loda M, Cukor B, Tam SW, et al. Increased proteasomedependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinoma. Nature Med 1997;3:231—234. 15. Mori M, Mimori K, Shiraishi T, et al. p27 expression and gastric carcinoma. Nature Med 1997;3:593. 16. Tsihlias J, Kapusta LR, DeBoer G, et al. Loss of cyclindependent kinase inhibitor p27Kip1 is a novel prognostic factor in localized human prostate adenocarcinoma. Cancer Res 1998;58:542—548. 17. Esposito V, Baldi A, De Luca A, et al. Prognostic role of the cyclin-dependent kinase inhibitor p27 in non-small cell lung cancer. Cancer Res 1997;57:3381—3385. 18. Slebos RJ, Baas IO, Climent M, et al. Clinical and pathological associations with p53 tumor-suppressor gene mutations and expression of p21WAF1/CIP1 in colorectal carcinoma. Br J Cancer 1996;27:912—916. 19. Soucek T, Rosner M, Miloloza A, Kubista M, Cheadle JP,
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Sampson JR, et al. Tuberous sclerosis causing mutants of the TSC2 gene product affect proliferation and p27 expression. Oncogene 2001;20:4904—4909. Uhlmann EJ, Apicelli AJ, Baldwin RL, Burke SP, Bajenaru ML, Onda H, et al. Heterozygosity for the tuberous sclerosis complex (TSC) gene products results in increased astrocyte numbers and decreased p27-Kip1 expression in TSC2+/ cells. Oncogene 2002;21:4050—4059. Viglietto G, Motti ML, Bruni P, Melillo RM, D’Alessio A, Califano D, et al. Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/ Akt-mediated phosphorylation in breast cancer. Nat Med 2002;8:1136—1144. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, et al. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 2002;8:1153—1160. Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, Baselga J, et al. PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization. Nat Med 2002;8:1145—1152. Dan HC, Sun M, Yang L, Feldman RI, Sui XM, Ou CC, et al. Phosphatidylinositol 3-kinase/Akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin. J Biol Chem 2002;277:35364—35370.
www.elsevier.com/locate/oraloncology
Overexpression of tuberous sclerosis complex 2 exerts antitumor effect on oral cancer cell lines Shin-ichi Kawaguchia, Koji Haradab,*, Supriatnob, Hideo Yoshidab, Mitsunobu Satob a
Department of Oral and Maxillofacial Radiology, University of Tokushima, School of Dentistry, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan b Second Department of Oral and Maxillofacial Surgery, University of Tokushima, School of Dentistry, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan
Received 15 April 2003; accepted 14 May 2003
KEYWORDS TSC2; Tuberin; Oral cancer; p27Kip1; Antitumor effect
Summary Tuberous sclerosis is caused by mutations in tuberous sclerosis complex (TSC) 2 on chromosome 16p13.3, encoding tuberin which is thought to be essential for p27Kip1 to regulate the cell cycle. In this study, we conducted to examine whether overexpression of TSC2 can affect the growth of oral cancer cells which have different expression level of p27Kip1 protein. We constructed an expression vector containing sense-oriented rat TSC2 cDNA with pcDNA3.1. We transfected oral cancer cells, B88t (high expression of p27Kip1 protein) and HI (low expression of p27Kip1 protein) with the sense expression vector to up-regulate the expression of TSC2 gene. Overexpression of TSC2 exerted the growth inhibitory effect of B88t and HI in vitro and in vivo. These findings suggest that overexpression of TSC2 may exert the antitumor effect on oral cancer cells whether they have high expression of p27Kip1 protein or not. # 2003 Elsevier Ltd. All rights reserved.
Introduction Tuberous sclerosis complex (TSC) is an inherited syndrome in which affected individuals are at increased risk for developing benign tumors included hamartomas, rhabdomyomas, angiofibromas and fibromas.1 Central nervous system manifestations include mental retardation, autism and seizures.2 Rarely, more malignant tumors develop. Linkage analysis of TSC resulted in the identification of two distinct genetic loci on chromosome 9 * Corresponding author. Tel.: +81-88-633-7354; fax: +81-88633-7462. E-mail address: [email protected] (K. Harada).
and 16.3 These genes are TSC1 and TSC2, respectively.4 TSC1 and TSC2 follow the classic retinoblastoma tumor suppressor gene model and appear to function as negative growth regulators.5 The TSC2 gene product, tuberin is a 180—200 kDa protein that contains a coiled-coil domain believed to mediate its interaction with hamartin and a carboxyl terminal GTPase activating protein.6 Tuberin may function as a tumor suppressor by induction of p27Kip1 protein. Interestingly, overexpression of tuberin results in reduce cell proliferation in vitro7 and increased amounts of the cell cycle regulator p27Kip1 in rat fibroblast.8 In addition, it is thought that tuberin is essential for p27Kip1 to regulate the cell cycle because tuberin can retain
1368-8375/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1368-8375(03)00106-4
Tuberous sclerosis complex 2 and oral cancer cell lines p27Kip1 protein in nuclei of cancer cells.8 In general, it has been thought that the prognosis of the oral squamous cell carcinoma (SCC) patients who have p27Kip1 proteins in nuclei of their cancer cells should be good.9 In short, tuberin may be closely associated with p27Kip1 to exert the function as tumor suppressor gene. In the present study, we conducted to examine whether overexpression of TSC2 can affect the growth of B88t and HI cells in vitro and in vivo by transfecting an expression vector containing sense-oriented rat TSC2 cDNA to B88t and HI cells.
Material and methods Establishment of a cell line and cell culture B88t cells was recovered from a subcutaneous tumor of formed by B88 cells on a nude mouse.10 B88t cells showed a high tumorigenesity on a nude mouse than B88 cells (data not shown). The original B88 cells were isolated from an oral SCC patient in our laboratory.11 The original tumor of B88 cells was moderately differentiated SCC of tongue, and was not invasive into muscle layer. B88 cells were established from a cervical lymph-node metastasis. We established a cell line in this experiment, designated as HI. HI cells were derived from a moderately differentiated SCC at tongue. The original tumor of HI cells was highly invasive to muscle layer and grew aggressively at the primary site. HI cells were established from a skin metastasis near the primary site. Cancer tissues were minced into 1 mm3 pieces and placed in 100 mm Petri dishes (Falcon; Becton Dickinson Labware, Lincoln Park, NJ, USA) containing a small amount of Dulbecco’s modified Eagle medium (DMEM; Sigma, St Louis, MO, USA) supplemented with 10% fetal calf serum (FCS; Moregate BioTech, Bulimba, Australia), and 100 mg/ml streptomycin, 100 units/ml penicillin (Gibco), and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 C. Epithelial cells were isolated and cloned. The cloned cells were cultured on plastic Petri dishes with the above medium and were passaged to new dishes before confluence. In addition, selected clones obtained after transfection were maintained in the same medium supplemented with Geneticin (800 mg/ml G418, Sigma).
Construction of a mammalian expression vector The mammalian expression vectors pcDNA3.1TSC2 (containing sense-oriented rat TSC2 cDNA)
837 was constructed as follows: pcDNA3.1 (+) (Invitrogen, Carlsbad, CA) is mammalian expression vectors containing a CMV promoter. pcDNA3.1 (+) was digested with Xba I (Takara Biomedicals, Kusatsu, Japan) and Xho I (Takara Biomedicals), and dephosphorylated by calf intestinal alkaline phosphatase (Roche Diadnostics, Mannheim, Germany). The rat TSC2/pBluescript including the rat TSC2 cDNA fragment (5.4 kbp Xba I and Xho I fragment) was obtained as a generous gift from Professor Okio Hino (Experimental Pathology, The Cancer Institute, Tokyo, Japanese Foundation for Cancer Research). This fragment was ligated to the prepared cloning site of pcDNA3.1 (+) by T4 DNA ligase (Takara Biomedicals). The direction of the ligated fragment was confirmed by sequencing analysis.
Transfection Cells (5105 cells/dish) were seeded in 100 mm culture dishes (Falcon) in DMEM supplemented with 10% FCS. Twenty-four hours later, the cells were transfected with 5 mg of pcDNA3.1-TSC2 or pcDNA3.1 without insert (empty vector) by use of the Superfect reagent (Qiagen, Hilden, Germany). The cells were incubated for 24 h in DMEM containing 10% FCS, then trypsinized and seeded at a 1:5 ratio in 100 mm culture dishes in DMEM medium containing 10% FCS. Forty-eight hours later, the cells were switched to a selective medium containing Geneticin (800 mg/ml G418, Sigma). After 14 days of culture in the selective medium, ten representative G418-resistant clones were isolated and expanded in a 24-well cluster dish (Falcon).
Western blotting Cell lysates were prepared from the transfectants as follows: cells were cultured to subconfluence, washed three times with 100 mM phosphate-buffered-saline, and lysed with 50 mM N-2-hydroxyethyl piperazine-N0 -2-ethanesulfonic acid, HEPES (pH 7.5) containing 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM NaF, 100 mM p-nitrophenyl phosphate, 5 U/ml aprotinin, and 1 mM phenyl-methylsulfonyl fluoride. The protein concentration of the samples was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Fifty micrograms of protein samples were electrophoresed on SDS-polyacryl amide gel. Proteins from gels were transferred to PVDF membrane (Bio-Rad), and the membrane was incubated with a 1:500 dilution of the monoclonal or polyclonal antibody against tuberin (N-19, rabbit polyclonal antibody, Santa Cruz Biotechnology,
838 Santa Cruz, CA, USA), p27 protein (clone 1B4, monoclonal antibody, Novocastra Laboratories, New Castle, UK) as the primary antibody, and an Amersham ECL kit (Amersham Pharmacia Biotech). Also, anti-a-tubulin monoclonal antibody (Zymed Laboratories, SanFrancisco, CA, USA) was used for normalization of Western blot analysis.
MTT Assay Cells were seeded on 96-well plates (Falcon) at 5103 cells per well in DMEM containing 10% FCS with/without Geneticin. After 1, 2 and 3 days, the number of cells was quantitated by an assay in which MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) was used.
S. Kawaguchi et al.
Growth of cells in vitro Relative cell number was evaluated by comparing the absorbance in each cells at day 1, 2 and 3. In the case of B88t, no significant difference in the cell number was seen among the cells on day 1. However, the cell number of sense transfectants was significantly decreased when compared with that of control cells at day 2 and 3 (P < 0.01; oneway ANOVA). In the case of HI, no significant difference in the cell number was seen among the cells on day 1 and 2. However, the cell number of sense transfectants was significantly decreased when compared with that of control cells at day 3 (P < 0.01; one-way ANOVA) (Fig. 3).
In vivo tumorigenesis assay The tumorigenicity of tumor cells was examined in the nude mouse with Balb/cA Jcl-nu genetic background (CLEA Japan, Inc. Tokyo, Japan). Tumor cells (1106) were suspended in 0.1 ml of serum-free medium and injected into the subcutaneous tissue of 5-week-old nude mice using a 27-gauge needle. The size of the tumors were determined by first measuring length (L) and width (W) and then calculating the volume (0.4LW 2) every 2 days. The body weight of the mice also measured every 2 days. The mice were sacrificed 24 days after inoculation.
Results
Figure 1 TSC2 plasmid construction. pcDNA3.1-TSC2 contains the sense-oriented 5.4 kbp rat TSC2 cDNA under the transcriptional control of the cytomegalovirus (CMV) promoter. The rat TSC2 cDNA fragment (5.4 kbp Xba I and Xho I fragment), lacking exon 25 and 31, was cloned into the mammalian expression vector pcDNA3.1 (+) (Invitrogen) at the Xba I and Xho I restriction sites.
Expression of tuberin and p27Kip1 by Western blotting After transfection with pcDNA3.1-TSC2 (Fig. 1), or pcDNA3.1neo, we obtained more than 100 G418resistant colonies in sense transfectants and isolated 10 representative G418-resistant clones in sense transfectants. They were screened for the expression of tuberin by Western blot analysis. Approximately 90% of the representative G418resistant clones in sense transfectants (B88t-TSC2, HI-TSC2) expressed the up-regulation of tuberin (data not shown). As shown in Fig. 2A, we detected the up-regulation of tuberin in sense-TSC2 transfectants when compared with that in parental cells (B88t, HI) or control cells which were transfected with pcDNA3.1 without insert (B88t-neo, HI-neo). The expression of a-tubulin as an internal control was approximately the same in all of the cells. Also, the expression level of p27Kip1 protein of B88t was high than that of HI (Fig. 2B).
Figure 2 Expression of tuberin in the transfectants. A Western blot analysis. Cell lysates were prepared from parental cells (B88t, HI), control cells (B88t-neo, HI-neo) and the sense transfectants (B88t-TSC2, HI-TSC2). Protein samples (50 mg) were electrophoresed on SDS-polyacrylamide gel and transferred to PVDF membrane, and incubated with anti-tuberin polyclonal antibody, antip27 monoclonal antibody and anti-a-tubulin monoclonal antibody.
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Figure 3 Growth of transfectants in vitro. Relative cell number was evaluated by comparing the absorbance in each cells at day 1, 2 and 3. The values shown are the mean of six determinations; bars, S.D. *, P <0.01 when compared with that of control cells by one-way ANOVA.
In vivo tumorigenicity of the transfectants All five mice that received parental cells (B88t, HI) developed moderate size tumors, and all five mice that received control cells (B88t-neo, HI-neo) also developed moderate size tumors. All five mice
that received sense transfectants (B88t-TSC2, HITSC2) developed small tumors. As shown in Fig. 4, tumors induced by TSC2-up-regulated transfectants become much smaller than those of control cells and parental cells (Fig. 4). During the experimental period, no loss of body weight was observed in each group.
Figure 4 Growth of tumors formed by transfectants in nude mice. Tumor cells (1106) were suspended in 0.1 ml of serum-free medium and injected into the subcutaneous tissue of nude mice. Sizes of the tumors were determined by first measuring length (L) and width (W) and then calculating the volume (0.4LW 2). Each group had 5 mice. The values shown are the mean of four tumors (mm3); bars, S.D. *, P <0.01 when compared with that of control cells by one-way ANOVA.
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Discussion Recently, we have reported that low p27Kip1 expression is associated with poor prognosis, and that p27Kip1 is a useful predictor of survival in oral SCC.12 Similar results have been observed in most solid tumours, including in breast cancer,13 colorectal carcinoma,14 gastric carcinoma,15 prostate cancer16 and non-small cell lung cancer17 suggesting that down-regulation of p27Kip1 is a general phenomenon in malignancies associated with aggressive tumor growth. However, some studies on colorectal carcinoma reported that p27Kip1 expression showed no relationship to tumor progression or overall survival.18 We have also reported that up-regulation of p27Kip1 protein can exert the growth inhibitry effects through induction of G1 arrest and apoptosis on oral cancer cell line.11 In short, high p27Kip1 expression should be associated with good prognosis when p27Kip1 protein function normally. In the case of colorectal carcinoma, p27Kip1 protein may not function as a cell cycle regulator for some reason, or some factor may regulate the p27Kip1 protein at up-stream of p27Kip1. So, we paid attention to TSC2 as a regulator of p27Kip1 protein in the present study. The autosomal dominant disease tuberous sclerosis (TSC) is caused by mutations in either TSC1 on chromosome 9q34, encoding hamartin, or TSC2 on chromosome 16p13.3, encoding tuberin. Tuberin seemed to play an important role in exerting the function of p27Kip1 protein as a cell cycle regulator by retaining p27Kip1 protein in nuclei of cancer cells.8 However, the true biochemical functions of TSC2 as a tumor suppressor has not been clarified yet. Although overexpression of TSC2 resulted in inhibition of cancer cell proliferation and up-regulation of p27Kip1, TSC2 involved mutations also inhibited the cancer cell growth and increased amounts of the p27Kip1 in the same way.19 In addition, it is reported that TSC2 heterozygosity may provide a non-cell-autonomous growth advantage for astrocytes that may involve the reduction of p27Kip1 expression.20 Recently, it has been reported that the function of p27Kip1 protein as a cell cycle regulator inhibit by Akt/PKB (Protein Kinase B).2123 Interestingly, the Akt/PKB also is related with the degradation of tuberin.24 In short, the relationship between TSC2 and p27Kip1 protein is clarifying in these years. In this study, overexpression of TSC2 exerted the antitumor effect on oral cancer cells whether they have high expression of p27Kip1 protein or not. These finding may suggest that tuberin can regulate the p27Kip1 protein at up-stream of p27Kip1. In short, tuberin may be more important
S. Kawaguchi et al. as a prognostic factor in oral SCC rather than p27Kip1 protein. Next we should investigate the relationship between tuberin expression and prognosis in oral SCC. A significant suppression of tumor growth of sense transfectants in vivo would be worthy of note. In addition, transfectants did not affect the body weight of the nude mice. These findings may suggest that up-regulation of tuberin in tumor cells will be safe for the body, and TSC2 can be used as molecular target for gene therapy if the precise function of TSC2 can be clarified. Recently, extensive investigations have been performed to elucidate the role of TSC2 as a tumor suppressor in normal and neoplastic cells, but conclusions have not been drawn about the detailed function of TSC2. We should try to investigate the factors which regulate the TSC2 at up-stream of TSC2 to clarify the molecular mechanisms of the antitumor effect of TSC2. References 1. Roach ES, Gomez MR, Northrup H. Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. J Child Neurol 1998;13:624—628. 2. Gomez MR, Sampson JR, Whittemore VH. Tuberous sclerosis. 3rd ed. Oxford (UK): Oxford University Press; 1999. 3. Povey S, Burley MW, Attwood J, Benham F, Hunt D, Jeremiah SJ, et al. Two loci for tuberous: one on 9q34 and one on 16p13. Ann Hum Genet 1994;58:107—127. 4. Henske EP, Scheithauer BW, Short MP, Wollmann R, Nahmias J, Hornigold N. Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions. Am J Hum Genet 1996;59:400—406. 5. Uhlmann EJ, Apicelli AJ, Baidwin RL, Burke SP, Bajenaru ML, Onda H, et al. Heterozygosity for the tuberous sclerosis complex (TSC) gene products results in increased astrocyte number and decreased p27-Kip1 expression in TSC2+/ cells. Oncogene 2002;21:4050—4059. 6. Wienecke R, Konig A, DeClue JE. Identification of tuberin, the tuberous sclerosis 2 product. Tuberin possesses specific Rap1 GAP activity. J Biol Chem 1995;270:16409—16414. 7. Jin F, Wienecke R, Xiao GH, Maize JC, DeClue JE, Yeung RS. Suppression of tumorigenecity by the wild-type tuberous sclerosis 2 (TSC2) gene and its C-terminal region. Proc Natl Acad Sci USA 1996;93:9154—9159. 8. Soucek T, Yeung RS, Hengstschlager M. Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene 2. Proc Natl Acad Sci USA 1998;95:15653—15658. 9. Mineta H, Miura K, Suzuki I, Takebayashi S, Amano H, Araki K, et al. Low p27 expression correlates with poor prognosis for patients with oral tongue squamous cell carcinoma. Cancer 1999;85:1011—1017. 10. Supriatno, Harada K, Kawaguchi S, Yoshida H, Sato M. Effect of p27Kip 1 on the ability of invasion and metastasis of an oral cancer cell line. Oncol Rep 2003;10:527—532. 11. Supriatno, Harada K., Hoque M.O., Bando T., Yoshida H., Sato M. Overexpression of p27Kip 1 induces growth arrest and apoptosis in an oral cancer cell line. Oral Oncol 2002; 38:730—736.
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