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Tbx3 expression is related to apoptosis and cell proliferation in rat bladder both hyperplastic epithelial cells and carcinoma cells
Cancer Letters 219 (2005) 105–112 www.elsevier.com/locate/canlet
Tbx3 expression is related to apoptosis and cell proliferation in rat bladder both hyperplastic epithelial cells and carcinoma cells Aya Ito, Makoto Asamoto*, Naomi Hokaiwado, Satoru Takahashi, Tomoyuki Shirai Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan Received 9 July 2004; accepted 18 July 2004
Abstract In the present study, Tbx3, a member of the T-box family of transcription factors, was identified as an up-regulated gene by mRNA differential display in the regression (apoptosis) stage after uracil-induction of papillomatosis in the rat urinary bladder. Immunohistochemical analysis revealed that apoptosis cells are negative and apoptosis resistant cells are positive for Tbx3 expression. That suggests that Tbx3 is an apoptosis resistant gene rather than an apoptosis induced gene. We have found the rat bladder carcinoma cell line, BC31, to feature Tbx3 expression detectable by RT-PCR. To investigate its role in such cancer cells, they were transfected with an anti-sense Tbx3 expression vector. The obtained stable transfectant clones showed reduced expression of Tbx3 and much slower cell growth, as compared with mock transfectants, and many apoptotic cells were observed under normal culture conditions. These results indicate that Tbx3 is a negative regulator of apoptosis in bladder normal epithelial cells and suppression of Tbx3 expression causes inhibition of cell proliferation and induction of apoptosis in BC31, a rat bladder carcinoma cells. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Tbx3; Apoptosis; Cell proliferation; Bladder
1. Introduction Marked hyperplasia (papillomatosis) of the bladder epithelium can be easily induced by feeding a diet containing 3% uracil to F344 rats [1–3], this being closely associated with urolithiasis produced by uracil calculi deposited in the urinary bladder [2]. * Corresponding author. Tel.: C81 52 853 8156; fax: C81 52 842 0817. E-mail address: [email protected] (M. Asamoto).
Since epithelial hyperplasia does not occur without the urolithiasis, mechanical irritation is considered to be responsible [4]. Withdrawal of uracil from the diet leads to rapid disappearance of calculi accompanied by a gradual diminution of the hyperplasia and return to a normal appearance within 2 weeks. Many apoptotic bodies become evident in the regressing epithelium, providing an excellent in vivo apoptosis model. We have focused on identifying genes changing their expression during this process using mRNA differential display and found Tbx3 to be
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.07.051
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up-regulated, as confirmed by the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). T-box genes are members of a highly conserved family of transcription factors [5–7]. Heritable abnormalities in several human T-box genes cause a variety of congenital human diseases [8–11]. In Tbx3, they may cause an autosomal dominant disorder, the ulnar-mammary syndrome, characterized by deficiencies and duplications of posterior elements of the forelimb, mammary hypoplasia and apocrine gland deficiency/dysfunction, as well as altered teeth, hair and genitalia [12]. In this paper, we describe sequential alternation of Tbx3 expression in the stages of papillomatosis and regression in rat urinary bladder epithelium. We also analyzed Tbx3 function in a p53 mutated rat urinary bladder cell line, BC31 [13–15], by transfection of anti-sense Tbx3. We thereby found that inhibition of Tbx3 induces apoptosis.
was converted to cDNA with avian myoblastosis virus reverse transcriptase (Takara, Otsu, Japan) in a 20 ml of reaction mixture. Aliquots of 2 ml of cDNA samples were subjected to quantitative RT-PCR in 20 ml reactions. RT-PCR reactions were performed using alpha-35S-dATP, AmpliTaq (Applied Biosystems Japan, Tokyo, Japan) and RNAmap KitA (GeneHunter Co., Brookline, MA, USA) according to the manufacturer’s instructions. Aliquots of the reaction mixtures were separated in 6% denaturing polyacrylamide gels in TBE buffer. Dried gels were used to expose X-ray films. Bands with increased intensity after uracil withdrawal were identified, and the DNA was reamplified by PCR to generate cDNA probes using RNAmap KitA (GeneHunter Co.) according to the manufacturer’s instructions.
2. Materials and methods
A cDNA library was prepared from mRNA of rat bladder epithelial mucosa of rats 2 days after withdrawal of uracil using the pEAK8 vector (EdgeBioSystems, Gaitherburg, MD, USA). Clones were screened and obtained using the cDNA probes obtained by mRNA differential display analysis. Sequences of the clones were determined by sequencing both strands using a genetic analyzer (ABI PRISM 310, Applied Biosystems Japan, Tokyo, Japan).
2.1. Animal treatments A total of 45 male 7-week-old Fisher 344 rats (Charles River Japan, Inc., Atsugi, Japan) were housed five per plastic cage on hardwood chip bedding in an environment-controlled room, maintained at 23G2 8C at 50% relative humidity and a 12 h light/dark cycle. Diet (Oriental MF; Oriental Yeast Co., Tokyo, Japan) and water were given ad libitum. Five non-treated rats served as controls. Uracil obtained from Yamasa Shoyu Co. (Choshi, Japan) was mixed in the diet at a final concentration of 3% and given to 40 rats for 5 weeks. They were then maintained for a further 3 weeks on diet not containing uracil. Sub-groups of five rats each were killed at the end of weeks 1, 2 and 5, and 1, 2, and 4 days, 1 week and 2 weeks after withdrawal of uracil. Since uracil-induced mucosal lesions are diffuse, one randomly chosen part of each bladder mucosa was frozen for RNA extraction and the remainder was fixed in 4% buffered formalin for histopathological and immunohistochemical examination. 2.2. mRNA differential display Total RNAs were extracted from bladder mucosas treated with uracil at various time points. 1 mg of RNA
2.3. cDNA library preparation and isolation of Tbx3 clones
2.4. Quantitative RT-PCR cDNA fragments were synthesized as previously described and raveled with FastStart DNA Master SYBR Green I and a Light Cycler apparatus (Roch Diagnostics, Mannheim, Germany). Primers used were 5 0 -CTACGGGGGAGCAATGGATG-3 0 and 5 0 -AGTTTAGTATAGTAAATCCG-3 0 for Tbx3. Initial denaturation at 95 8C for 10 min was followed by 40 cycles with denaturation at 95 8C for 15 s, annealing at 55 8C for 5 s for Tbx3. The fluorescence intensity of the double-strand specific SYBR Green I, reflecting the amount of formed PCR-product, was monitored at the end of each elongation step. GAPD mRNA levels were used to normalize for sample cDNA content. Three samples per point were analyzed.
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2.5. Immunohistochemistry of Tbx3 Two antibodies, one against a synthetic peptide for amino acids 376–388 (ESDAEAESKEEHGPE), (Hokkaido System Science, Sapporo, Japan) and the other for amino acids 420–432 (EPSRARPTARLD) (Kitayama Labes, Ina, Nagano, Japan) were used to investigate localization of the Tbx3 protein in rat bladder tissues. Deparaffinized slide sections of formalin-fixed rat bladder tissues were heated in 0.05 M EDTA to 90 8C for 60 min for antigen retrieval then incubated with 1:1000 diluted Tbx3 antiserum. Antibody binding was visualized using a conventional immunostaining method using an autoimmunostaining apparatus (VENTANA HX SYSTEM, VENTANA Japan, Tokyo, Japan). 2.6. Preparation of anti-sense Tbx3 cDNA constructs A 392 bp cDNA fragment of anti-sense Tbx3 was amplified by PCR with synthetic primers. The PCR products were subcloned into the pIRESpuro2 vector that allows both a cloned gene and a puromycineresistant gene to be co-expressed (BD Bioscience Clontech, Palo Alto, CA, USA). The following primers were used for anti-sense Tbx3, the forward primer was 5 0 -ATGAGCCTCTCCATGAGAGA -3 0 , and the reverse primer was 5 0 -GCCTTCCTGACTTCGTAATG -3 0 . Sequences of the cloned fragments were confirmed by sequencing of both strands.
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became rounded, but stopped trypsinization by adding serum containing medium before detachment from the dish. The rounded colonies were transferred from the culture surface with cotton-tipped swabs which were then agitated briefly and then cultured in fresh medium of 24-well plates. 2.8. Apoptotic cell death detection in anti-sense Tbx3 transfected BC31 cells For detection of apoptotic cells, annexin assays were performed. These assays were followed by guava Nexin kit (Guava Technologies, Inc., Hayward, CA, USA) according to manufacture’s protocol. Briefly, anti-sense Tbx3 transfected BC31 cells for 48 h were tripsinized and washed three times by 1 ml of Nexin buffer. Cells were centrifuged to collect and resuspended in 50 ml Nexin buffer. 40 ml of the solution was added 5 ml of annexin V-PE and 5 ml of 7-aminoactinomycin D (7-AAD). Cells were incubated on ice for 20 min, shielded from light and analyzed by Guava PCA (Guava Technologies). In this assay, annexin V-negative and 7-AAD-positive indicates nuclear debris, annexin V-positive and 7-AAD-positive indicates late apoptotic cells, annexin V-negative and 7-AAD-negative indicates live health cells, and annexin V-positive and 7-AAD-negative indicates early apoptotic cells.
3. Results 2.7. Transfection of BC31 cells 3.1. Identification of rat Tbx3 The rat BC31 cell line, established from a primary chemically-induced rat bladder transitional cell carcinoma [13–15] was maintained in culture in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum under 5% CO2/95% air at 37 8C in a humidified incubator. BC31 cells were transfected with anti-sense Tbx3 cDNA using LipofectAMINE (GIBCO BRL Life Technologies, Rockville, MD, USA). Stable transfectants were selected by growing cells in 5 mg/ml puromycin (SIGMA-ARDRICH Japan, Tokyo, Japan) for anti-sense Tbx3 cDNA. Cloning for anti-sense Tbx3 transfectants was performed using our original method. When independent colonies were appeared, treatment with trypsin was performed until cells
Tbx3 was identified by mRNA differential display analysis as a gene overexpressed during the apoptosis phase after induction of rat bladder epithelial papillomatosis by uracil administration. By screening the rat bladder cDNA library using a cDNA fragment obtained by the mRNA differential display analysis, six independent clones were obtained. On sequencing these clones, the full length coding sequence of rat Tbx3 could be determined (GeneBank accession no. AY289619). Three clones did not have exon 2a (60 bp), as a splicing variant reported previously for human Tbx3 [16]. The rat Tbx3 coding sequence was found to comprise 2232 bp with exon 2a (Fig. 1). The homology of rat Tbx3 nucleotide sequence to
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Fig. 1. Sequence of the rat Tbx3 cDNA, from the initiation codon to the stop codon (GeneBank accession no. AY289619).
the human Tbx3 was 85.8%, the T-box sequence being highly conserved. 3.2. Quantitative RT-PCR for Tbx3 The expression level of Tbx3 mRNA in rat bladder epithelium was relatively low during the period of uracil feeding, during which the mucosa demonstrated papillomatosis (Fig. 2). One day after withdrawal of uracil, expression of Tbx3 became elevated, and continued to increase to a peak at day 4 after the cessation of insult. At days 7 and 21, the expression levels were still relatively high (Fig. 2).
3.3. Immnohistochemistry of Tbx3 Immunohistochemistry of Tbx3 revealed localization in nuclei of almost all cells in the bladder except umbrella cells of the urothelial epithelium and apoptotic bodies (Fig. 3), indicating that senescent and apoptotic cells have no or little expression of the Tbx3 protein. 3.4. Stable transfectants of anti-sense Tbx3 We obtained 26 clones of stable transfectants of anti-sense Tbx3. We selected four (A2, B14,
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Fig. 2. Expression of rat Tbx3 in bladder tissues of rats treated with uracil and after withdrawal, revealed by quantitative RT-PCR. Note dramatic increase after withdrawal. The degree of hyperplasia (papillomatosis) is illustrated.
B19, B21) showing inhibition of Tbx3 expression on transfection of an anti-sense Tbx3 as assessed by quantitative RT-PCR. Levels of Tbx3 were much lower in the obtained stable transfectant clones compared to the three mock transfectants (C4, C5, C6) (Fig. 4). We found a dramatic inhibition of cell growth in the stable transfectants (Fig. 5), cell counts being markedly decreased. 3.5. Apoptosis in the transfectants Under normal conditions, we found dramatic morphologic changes in anti-Tbx3 transfectants (Fig. 6). Cell–cell intervals became wider and cell adhesion weaker as compared to the mock transfectant case. Many floating cells were also observed in the supernatant of the anti-sense Tbx3 transfectants, especially the A2 cell line which showed the slowest cell growth (Fig. 5). DNA collected from such floating demonstrated DNA ladders on gel electrophoresis (Fig. 6). To confirm these results, we performed annexin V and 7-AAF assay. We found most A2 cells (an antisense Tbx3 transfectant) show early apoptosis showing annexin V-positive and 7-AAF-negative (Fig. 7).
Other anti-sense transfectants showed similar tendency but less apoptotic cells.
4. Discussion In the present experiment, role of Tbx3 in the process of apoptosis was clarified using regression stage of urothelial papillomatosis by uracil [2]. The data clearly demonstrated that Tbx3 is a candidate for anti-apoptotic factor in rat bladder carcinoma cells. Although Tbx3 was initially isolated as an upregulated gene in the apoptosis phase of regression of uracil-induced rat urinary bladder papillomatosis, anti-sense Tbx3 transfected bladder carcinoma cells showed increased apoptosis and reduction of cell proliferation. Immunostaining revealed intense nuclear staining was evident in the urothelial cells, especially basal cells, but no or faint staining in apoptotic bodies and terminally differentiated umbrella cells. Therefore the observed up-regulated expression of Tbx3 in the apoptosis phase is considered to reflect levels in the normal urothelial cells, but not correctly represent the apoptotic cells. Thus down-regulation of Tbx3 appears to be associated with apoptosis or senescence of rat bladder epithelial
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down-regulation of Tbx3 in our rat bladder carcinoma cells with mutated p53 (data not shown). These results suggest that apoptosis can be involved by down-regulation of Tbx3. In conclusion, the data of the present experiments indicate a role for Tbx3 in the processes underlying apoptosis and growth in the urinary bladder normal epithelial cells and carcinomas.
Fig. 4. Reduced Tbx3 expression in anti-sense Tbx3 transfectants revealed by RT-PCR. The RT-PCR was performed by the conventional methods using 1 mg of total RNA and 30 cycles of PCR reactions. (1) Mock-transfectant control 5; (2) mocktransfectant control 6; (3) anti-sense Tbx3 transfectant A2; (4) anti-sense Tbx3 transfectant B14; (5) anti-sense Tbx3 transfectant B19; (6)anti-sense Tbx3 transfectant B21. Left lane shows size makers of mass ladder (GIBCO BRL) indicating the product size is around 200 bp. Fig. 3. Expression and localization of the Tbx3 protein. The Tbx3 protein is localized in nuclei of epithelial cells of the urothelium, but not in umbrella cells and apoptotic bodies. (a) Hyperplasia induced by uracil administration for 7 days. Note umbrella cells (arrows) are not stained by Tbx3 anti-serum; (b) regressing hyperplasia containing many apoptotic bodies (arrow heads) not stained by Tbx3 anti-serum, 7 days after uracil withdrawal.
cells both in vivo and in vitro. This may be consistent with previous reports regarding the relationship between Tbx3 and apoptosis/senescence [17]. A previous study indicated that Tbx3 is a negative regulator of p19/ARF and inhibits senescence [17]. p19/ARF tumor-suppressor protein, encoded by the INK4A/ARF locus, up-regulates p53 activity to induce cell cycle arrest and sensitize cells to apoptosis by inactivating Mdm2 through the so-called ARF/ MDM2/p53 pathway [18–21]. In the present study, expression of p19/ARF, however, was not affected by
Fig. 5. Reduced cell proliferation in anti-sense Tbx3 transfectants. Closed circles, control 5; closed triangles, control 6; Open circles, B14; Open squares, B21; Open triangles, B19; X, A2.
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Fig. 6. Morphological changes and induction of apoptosis in anti-sense Tbx3 transfectants. (a and b) A2; (c and d) B21; (e and f) Control 5. A2 and B21 anti-sense transfectants demonstrate many floating cells undergoing apoptosis. In the A2 and B21 cases, cell–cell intervals are wider and cell adhesion appears weak. (g) DNA from floating A2 cells shows a DNA ladder indicating apoptosis. Left lane shows DNA size 100 bp makers (GIBCO BRL).
Fig. 7. Representative analysis for apoptosis in rat bladder carcinoma cells transfected by anti-sense Tbx3. In Control cell C5 (a, a mock transfectant), most cells showed annexin V negative 7-AAF negative (lower left) indicating living cells. However, in anti-sense Tbx3 transfected cell A2 (b), most cells annexin V-positive, 7-AAF-negative indicating early apoptotic cells (lower right).
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Acknowledgements This study was supported by research grants from the Ministry of Health, Labour and Welfare of Japan and the Society of Promotion for Pathology, Nagoya. We thank Dr Malcolm A. Moore for his kind linguistic advice during preparation of this manuscript.
References [1] T. Shirai, Y. Tagawa, S. Fukushima, K. Imaida, N. Ito, Strong promoting activity of reversible uracil-induced urolithiasis on urinary bladder carcinogenesis in rats initiated with N-butylN-(4-hydroxybutyl)nitrosamine, Cancer Res. 47 (1987) 6726– 6730. [2] T. Shirai, E. Ikawa, S. Fukushima, T. Masui, N. Ito, Uracilinduced urolithiasis and the development of reversible papillomatosis in the urinary bladder of F344 rats, Cancer Res. 46 (1986) 2062–2067. [3] T. Shirai, S. Fukushima, Y. Tagawa, M. Okumura, N. Ito, Cell proliferation induced by uracil-calculi and subsequent development of reversible papillomatosis in the rat urinary bladder, Cancer Res. 49 (1989) 378–383. [4] M. Kagawa, A. Yamamoto, K. Ogawa, T. Shirai, S. Fukushima, Uracil-induced urolithiasis in the urinary bladder of rats is irritation-dependent, Toxicol. Lett. 61 (1992) 21–26. [5] V.E. Papaioannou, T-box genes in development: from hydra to humans, Int. Rev. Cytol. 207 (2001) 1–70. [6] J. Smith, T-box genes: what they do and how they do it, Trends Genet. 15 (1999) 154–158. [7] A. Kispert, B. Koschorz, B.G. Herrmann, The T protein encoded by Brachyury is a tissue-specific transcription factor, Eur. Mol. Biol. Org. J. 14 (1995) 4763–4772. [8] C.T. Basson, D.R. Bachinsky, R.C. Lin, T. Levi, J.A. Elkins, J. Soults, et al., Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome, Nat. Genet. 15 (1997) 30–35. [9] Q.Y. Li, R.A. Newbury-Ecob, J.A. Terrett, D.I. Wilson, A.R. Curtis, C.H. Yi, et al., Holt–Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family, Nat. Genet. 15 (1997) 21–29.
[10] L.A. Jerome, V.E. Papaioannou, DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1 Nat. Genet. 27 (2001) 286–291. [11] E.A. Lindsay, F. Vitelli, H. Su, M. Morishima, T. Huynh, T. Pramparo, et al., Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice, Nature 410 (2001) 97–101. [12] M. Bamshad, R.C. Lin, D.J. Law, W.C. Watkins, P.A. Krakowiak, M.E. Moore, et al., Mutations in human TBX3 alter limb, apocrine and genital development in ulnarmammary syndrome, Nat. Genet. 16 (1997) 311–315. [13] A. Ito, M. Asamoto, N. Hokaiwado, T. Shirai, Regulation of cell proliferation by induction of p21/WAF1 in rat bladder carcinoma cells using the Cre-loxP system, Cancer Lett. 193 (2003) 183–188. [14] M. Asamoto, S. Takahashi, K. Imaida, T. Shirai, S. Fukushima, Increased gap junctional intercellular communication capacity and connexin 43 and 26 expression in rat bladder carcinogenesis, Carcinogenesis 15 (1994) 1263–1266. [15] H. Hojo, A. Kaneko, N. Kayagaki, M. Saki, Y. Hashimoto, Subcellular localization and characterization of interleukin-1 alpha produced by rat bladder cancer cells, Immunol. Lett. 43 (1994) 215–220. [16] M. Bamshad, T. Le, W.S. Watkins, M.E. Dixon, B.E. Kramer, A.D. Roeder, et al., The spectrum of mutations in TBX3: Genotype/Phenotype relationship in ulnar-mammary syndrome, Am. J. Hum. Genet. 64 (1999) 1550–1562. [17] T.R. Brummelkamp, R.M. Kortlever, M. Lingbeek, F. Trettel, M.E. MacDonald, M. van Lohuizen, R. Bernards, TBX-3, the gene mutated in Ulnar-Mammary Syndrome, is a negative regulator of p19ARF and inhibits senescence, J. Biol. Chem. 277 (2002) 6567–6572. [18] D.E. Quelle, F. Zindy, R.A. Ashmun, C.J. Sherr, Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest, Cell 83 (1995) 993–1000. [19] S. Bates, A.C. Phillips, P.A. Clark, F. Stott, G. Peters, R.L. Ludwig, K.H. Vousden, p14ARF links the tumour suppressors RB and p53, Nature 395 (1998) 124–125. [20] Y. Zhang, Y. Xiong, W.G. Yarbrough, ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways, Cell 92 (1998) 725–734. [21] F.J. Stott, S. Bates, M.C. James, B.B. McConnell, M. Starborg, S. Brookes, et al., The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2, Eur. Mol. Biol. Org. J. 17 (1998) 5001–5014.
Tbx3 expression is related to apoptosis and cell proliferation in rat bladder both hyperplastic epithelial cells and carcinoma cells Aya Ito, Makoto Asamoto*, Naomi Hokaiwado, Satoru Takahashi, Tomoyuki Shirai Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan Received 9 July 2004; accepted 18 July 2004
Abstract In the present study, Tbx3, a member of the T-box family of transcription factors, was identified as an up-regulated gene by mRNA differential display in the regression (apoptosis) stage after uracil-induction of papillomatosis in the rat urinary bladder. Immunohistochemical analysis revealed that apoptosis cells are negative and apoptosis resistant cells are positive for Tbx3 expression. That suggests that Tbx3 is an apoptosis resistant gene rather than an apoptosis induced gene. We have found the rat bladder carcinoma cell line, BC31, to feature Tbx3 expression detectable by RT-PCR. To investigate its role in such cancer cells, they were transfected with an anti-sense Tbx3 expression vector. The obtained stable transfectant clones showed reduced expression of Tbx3 and much slower cell growth, as compared with mock transfectants, and many apoptotic cells were observed under normal culture conditions. These results indicate that Tbx3 is a negative regulator of apoptosis in bladder normal epithelial cells and suppression of Tbx3 expression causes inhibition of cell proliferation and induction of apoptosis in BC31, a rat bladder carcinoma cells. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Tbx3; Apoptosis; Cell proliferation; Bladder
1. Introduction Marked hyperplasia (papillomatosis) of the bladder epithelium can be easily induced by feeding a diet containing 3% uracil to F344 rats [1–3], this being closely associated with urolithiasis produced by uracil calculi deposited in the urinary bladder [2]. * Corresponding author. Tel.: C81 52 853 8156; fax: C81 52 842 0817. E-mail address: [email protected] (M. Asamoto).
Since epithelial hyperplasia does not occur without the urolithiasis, mechanical irritation is considered to be responsible [4]. Withdrawal of uracil from the diet leads to rapid disappearance of calculi accompanied by a gradual diminution of the hyperplasia and return to a normal appearance within 2 weeks. Many apoptotic bodies become evident in the regressing epithelium, providing an excellent in vivo apoptosis model. We have focused on identifying genes changing their expression during this process using mRNA differential display and found Tbx3 to be
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.07.051
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up-regulated, as confirmed by the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). T-box genes are members of a highly conserved family of transcription factors [5–7]. Heritable abnormalities in several human T-box genes cause a variety of congenital human diseases [8–11]. In Tbx3, they may cause an autosomal dominant disorder, the ulnar-mammary syndrome, characterized by deficiencies and duplications of posterior elements of the forelimb, mammary hypoplasia and apocrine gland deficiency/dysfunction, as well as altered teeth, hair and genitalia [12]. In this paper, we describe sequential alternation of Tbx3 expression in the stages of papillomatosis and regression in rat urinary bladder epithelium. We also analyzed Tbx3 function in a p53 mutated rat urinary bladder cell line, BC31 [13–15], by transfection of anti-sense Tbx3. We thereby found that inhibition of Tbx3 induces apoptosis.
was converted to cDNA with avian myoblastosis virus reverse transcriptase (Takara, Otsu, Japan) in a 20 ml of reaction mixture. Aliquots of 2 ml of cDNA samples were subjected to quantitative RT-PCR in 20 ml reactions. RT-PCR reactions were performed using alpha-35S-dATP, AmpliTaq (Applied Biosystems Japan, Tokyo, Japan) and RNAmap KitA (GeneHunter Co., Brookline, MA, USA) according to the manufacturer’s instructions. Aliquots of the reaction mixtures were separated in 6% denaturing polyacrylamide gels in TBE buffer. Dried gels were used to expose X-ray films. Bands with increased intensity after uracil withdrawal were identified, and the DNA was reamplified by PCR to generate cDNA probes using RNAmap KitA (GeneHunter Co.) according to the manufacturer’s instructions.
2. Materials and methods
A cDNA library was prepared from mRNA of rat bladder epithelial mucosa of rats 2 days after withdrawal of uracil using the pEAK8 vector (EdgeBioSystems, Gaitherburg, MD, USA). Clones were screened and obtained using the cDNA probes obtained by mRNA differential display analysis. Sequences of the clones were determined by sequencing both strands using a genetic analyzer (ABI PRISM 310, Applied Biosystems Japan, Tokyo, Japan).
2.1. Animal treatments A total of 45 male 7-week-old Fisher 344 rats (Charles River Japan, Inc., Atsugi, Japan) were housed five per plastic cage on hardwood chip bedding in an environment-controlled room, maintained at 23G2 8C at 50% relative humidity and a 12 h light/dark cycle. Diet (Oriental MF; Oriental Yeast Co., Tokyo, Japan) and water were given ad libitum. Five non-treated rats served as controls. Uracil obtained from Yamasa Shoyu Co. (Choshi, Japan) was mixed in the diet at a final concentration of 3% and given to 40 rats for 5 weeks. They were then maintained for a further 3 weeks on diet not containing uracil. Sub-groups of five rats each were killed at the end of weeks 1, 2 and 5, and 1, 2, and 4 days, 1 week and 2 weeks after withdrawal of uracil. Since uracil-induced mucosal lesions are diffuse, one randomly chosen part of each bladder mucosa was frozen for RNA extraction and the remainder was fixed in 4% buffered formalin for histopathological and immunohistochemical examination. 2.2. mRNA differential display Total RNAs were extracted from bladder mucosas treated with uracil at various time points. 1 mg of RNA
2.3. cDNA library preparation and isolation of Tbx3 clones
2.4. Quantitative RT-PCR cDNA fragments were synthesized as previously described and raveled with FastStart DNA Master SYBR Green I and a Light Cycler apparatus (Roch Diagnostics, Mannheim, Germany). Primers used were 5 0 -CTACGGGGGAGCAATGGATG-3 0 and 5 0 -AGTTTAGTATAGTAAATCCG-3 0 for Tbx3. Initial denaturation at 95 8C for 10 min was followed by 40 cycles with denaturation at 95 8C for 15 s, annealing at 55 8C for 5 s for Tbx3. The fluorescence intensity of the double-strand specific SYBR Green I, reflecting the amount of formed PCR-product, was monitored at the end of each elongation step. GAPD mRNA levels were used to normalize for sample cDNA content. Three samples per point were analyzed.
A. Ito et al. / Cancer Letters 219 (2005) 105–112
2.5. Immunohistochemistry of Tbx3 Two antibodies, one against a synthetic peptide for amino acids 376–388 (ESDAEAESKEEHGPE), (Hokkaido System Science, Sapporo, Japan) and the other for amino acids 420–432 (EPSRARPTARLD) (Kitayama Labes, Ina, Nagano, Japan) were used to investigate localization of the Tbx3 protein in rat bladder tissues. Deparaffinized slide sections of formalin-fixed rat bladder tissues were heated in 0.05 M EDTA to 90 8C for 60 min for antigen retrieval then incubated with 1:1000 diluted Tbx3 antiserum. Antibody binding was visualized using a conventional immunostaining method using an autoimmunostaining apparatus (VENTANA HX SYSTEM, VENTANA Japan, Tokyo, Japan). 2.6. Preparation of anti-sense Tbx3 cDNA constructs A 392 bp cDNA fragment of anti-sense Tbx3 was amplified by PCR with synthetic primers. The PCR products were subcloned into the pIRESpuro2 vector that allows both a cloned gene and a puromycineresistant gene to be co-expressed (BD Bioscience Clontech, Palo Alto, CA, USA). The following primers were used for anti-sense Tbx3, the forward primer was 5 0 -ATGAGCCTCTCCATGAGAGA -3 0 , and the reverse primer was 5 0 -GCCTTCCTGACTTCGTAATG -3 0 . Sequences of the cloned fragments were confirmed by sequencing of both strands.
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became rounded, but stopped trypsinization by adding serum containing medium before detachment from the dish. The rounded colonies were transferred from the culture surface with cotton-tipped swabs which were then agitated briefly and then cultured in fresh medium of 24-well plates. 2.8. Apoptotic cell death detection in anti-sense Tbx3 transfected BC31 cells For detection of apoptotic cells, annexin assays were performed. These assays were followed by guava Nexin kit (Guava Technologies, Inc., Hayward, CA, USA) according to manufacture’s protocol. Briefly, anti-sense Tbx3 transfected BC31 cells for 48 h were tripsinized and washed three times by 1 ml of Nexin buffer. Cells were centrifuged to collect and resuspended in 50 ml Nexin buffer. 40 ml of the solution was added 5 ml of annexin V-PE and 5 ml of 7-aminoactinomycin D (7-AAD). Cells were incubated on ice for 20 min, shielded from light and analyzed by Guava PCA (Guava Technologies). In this assay, annexin V-negative and 7-AAD-positive indicates nuclear debris, annexin V-positive and 7-AAD-positive indicates late apoptotic cells, annexin V-negative and 7-AAD-negative indicates live health cells, and annexin V-positive and 7-AAD-negative indicates early apoptotic cells.
3. Results 2.7. Transfection of BC31 cells 3.1. Identification of rat Tbx3 The rat BC31 cell line, established from a primary chemically-induced rat bladder transitional cell carcinoma [13–15] was maintained in culture in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum under 5% CO2/95% air at 37 8C in a humidified incubator. BC31 cells were transfected with anti-sense Tbx3 cDNA using LipofectAMINE (GIBCO BRL Life Technologies, Rockville, MD, USA). Stable transfectants were selected by growing cells in 5 mg/ml puromycin (SIGMA-ARDRICH Japan, Tokyo, Japan) for anti-sense Tbx3 cDNA. Cloning for anti-sense Tbx3 transfectants was performed using our original method. When independent colonies were appeared, treatment with trypsin was performed until cells
Tbx3 was identified by mRNA differential display analysis as a gene overexpressed during the apoptosis phase after induction of rat bladder epithelial papillomatosis by uracil administration. By screening the rat bladder cDNA library using a cDNA fragment obtained by the mRNA differential display analysis, six independent clones were obtained. On sequencing these clones, the full length coding sequence of rat Tbx3 could be determined (GeneBank accession no. AY289619). Three clones did not have exon 2a (60 bp), as a splicing variant reported previously for human Tbx3 [16]. The rat Tbx3 coding sequence was found to comprise 2232 bp with exon 2a (Fig. 1). The homology of rat Tbx3 nucleotide sequence to
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Fig. 1. Sequence of the rat Tbx3 cDNA, from the initiation codon to the stop codon (GeneBank accession no. AY289619).
the human Tbx3 was 85.8%, the T-box sequence being highly conserved. 3.2. Quantitative RT-PCR for Tbx3 The expression level of Tbx3 mRNA in rat bladder epithelium was relatively low during the period of uracil feeding, during which the mucosa demonstrated papillomatosis (Fig. 2). One day after withdrawal of uracil, expression of Tbx3 became elevated, and continued to increase to a peak at day 4 after the cessation of insult. At days 7 and 21, the expression levels were still relatively high (Fig. 2).
3.3. Immnohistochemistry of Tbx3 Immunohistochemistry of Tbx3 revealed localization in nuclei of almost all cells in the bladder except umbrella cells of the urothelial epithelium and apoptotic bodies (Fig. 3), indicating that senescent and apoptotic cells have no or little expression of the Tbx3 protein. 3.4. Stable transfectants of anti-sense Tbx3 We obtained 26 clones of stable transfectants of anti-sense Tbx3. We selected four (A2, B14,
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Fig. 2. Expression of rat Tbx3 in bladder tissues of rats treated with uracil and after withdrawal, revealed by quantitative RT-PCR. Note dramatic increase after withdrawal. The degree of hyperplasia (papillomatosis) is illustrated.
B19, B21) showing inhibition of Tbx3 expression on transfection of an anti-sense Tbx3 as assessed by quantitative RT-PCR. Levels of Tbx3 were much lower in the obtained stable transfectant clones compared to the three mock transfectants (C4, C5, C6) (Fig. 4). We found a dramatic inhibition of cell growth in the stable transfectants (Fig. 5), cell counts being markedly decreased. 3.5. Apoptosis in the transfectants Under normal conditions, we found dramatic morphologic changes in anti-Tbx3 transfectants (Fig. 6). Cell–cell intervals became wider and cell adhesion weaker as compared to the mock transfectant case. Many floating cells were also observed in the supernatant of the anti-sense Tbx3 transfectants, especially the A2 cell line which showed the slowest cell growth (Fig. 5). DNA collected from such floating demonstrated DNA ladders on gel electrophoresis (Fig. 6). To confirm these results, we performed annexin V and 7-AAF assay. We found most A2 cells (an antisense Tbx3 transfectant) show early apoptosis showing annexin V-positive and 7-AAF-negative (Fig. 7).
Other anti-sense transfectants showed similar tendency but less apoptotic cells.
4. Discussion In the present experiment, role of Tbx3 in the process of apoptosis was clarified using regression stage of urothelial papillomatosis by uracil [2]. The data clearly demonstrated that Tbx3 is a candidate for anti-apoptotic factor in rat bladder carcinoma cells. Although Tbx3 was initially isolated as an upregulated gene in the apoptosis phase of regression of uracil-induced rat urinary bladder papillomatosis, anti-sense Tbx3 transfected bladder carcinoma cells showed increased apoptosis and reduction of cell proliferation. Immunostaining revealed intense nuclear staining was evident in the urothelial cells, especially basal cells, but no or faint staining in apoptotic bodies and terminally differentiated umbrella cells. Therefore the observed up-regulated expression of Tbx3 in the apoptosis phase is considered to reflect levels in the normal urothelial cells, but not correctly represent the apoptotic cells. Thus down-regulation of Tbx3 appears to be associated with apoptosis or senescence of rat bladder epithelial
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down-regulation of Tbx3 in our rat bladder carcinoma cells with mutated p53 (data not shown). These results suggest that apoptosis can be involved by down-regulation of Tbx3. In conclusion, the data of the present experiments indicate a role for Tbx3 in the processes underlying apoptosis and growth in the urinary bladder normal epithelial cells and carcinomas.
Fig. 4. Reduced Tbx3 expression in anti-sense Tbx3 transfectants revealed by RT-PCR. The RT-PCR was performed by the conventional methods using 1 mg of total RNA and 30 cycles of PCR reactions. (1) Mock-transfectant control 5; (2) mocktransfectant control 6; (3) anti-sense Tbx3 transfectant A2; (4) anti-sense Tbx3 transfectant B14; (5) anti-sense Tbx3 transfectant B19; (6)anti-sense Tbx3 transfectant B21. Left lane shows size makers of mass ladder (GIBCO BRL) indicating the product size is around 200 bp. Fig. 3. Expression and localization of the Tbx3 protein. The Tbx3 protein is localized in nuclei of epithelial cells of the urothelium, but not in umbrella cells and apoptotic bodies. (a) Hyperplasia induced by uracil administration for 7 days. Note umbrella cells (arrows) are not stained by Tbx3 anti-serum; (b) regressing hyperplasia containing many apoptotic bodies (arrow heads) not stained by Tbx3 anti-serum, 7 days after uracil withdrawal.
cells both in vivo and in vitro. This may be consistent with previous reports regarding the relationship between Tbx3 and apoptosis/senescence [17]. A previous study indicated that Tbx3 is a negative regulator of p19/ARF and inhibits senescence [17]. p19/ARF tumor-suppressor protein, encoded by the INK4A/ARF locus, up-regulates p53 activity to induce cell cycle arrest and sensitize cells to apoptosis by inactivating Mdm2 through the so-called ARF/ MDM2/p53 pathway [18–21]. In the present study, expression of p19/ARF, however, was not affected by
Fig. 5. Reduced cell proliferation in anti-sense Tbx3 transfectants. Closed circles, control 5; closed triangles, control 6; Open circles, B14; Open squares, B21; Open triangles, B19; X, A2.
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Fig. 6. Morphological changes and induction of apoptosis in anti-sense Tbx3 transfectants. (a and b) A2; (c and d) B21; (e and f) Control 5. A2 and B21 anti-sense transfectants demonstrate many floating cells undergoing apoptosis. In the A2 and B21 cases, cell–cell intervals are wider and cell adhesion appears weak. (g) DNA from floating A2 cells shows a DNA ladder indicating apoptosis. Left lane shows DNA size 100 bp makers (GIBCO BRL).
Fig. 7. Representative analysis for apoptosis in rat bladder carcinoma cells transfected by anti-sense Tbx3. In Control cell C5 (a, a mock transfectant), most cells showed annexin V negative 7-AAF negative (lower left) indicating living cells. However, in anti-sense Tbx3 transfected cell A2 (b), most cells annexin V-positive, 7-AAF-negative indicating early apoptotic cells (lower right).
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Acknowledgements This study was supported by research grants from the Ministry of Health, Labour and Welfare of Japan and the Society of Promotion for Pathology, Nagoya. We thank Dr Malcolm A. Moore for his kind linguistic advice during preparation of this manuscript.
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