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Induction of apoptosis and inhibition of cell growth by developmental regulator hTBX5
BBRC Biochemical and Biophysical Research Communications 297 (2002) 185–192 www.academicpress.com
Induction of apoptosis and inhibition of cell growth by developmental regulator hTBX5 Ming-Liang He,a,* Ying Chen,a,b Ying Peng,a Dadao Jin,b,c Dan Du,a Jun Wu,a Ping Lu,b,c Marie C. Lin,a and Hsiang-Fu Kunga a
Institute of Molecular Biology, Open Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong, Hong Kong SAR, China b Department of Medicine, The University of Hong Kong, Hong Kong SAR, China c Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai 200080, China Received 13 August 2002
Abstract T box (Tbx) genes are a large family of transcription regulators that play critical roles in invertebrate and vertebrate development. Mutations in Tbx5 gene have been found to cause Holt–Oram syndrome (HOS) in humans. Partial dysfunction of TBX5 in mouse also causes HOS phenotype. Little is known about its molecular and cellular mechanism. Here, we report that ectopic expression of TBX5 inhibited colony formation, induced apoptosis, and decreased the growth rate of cells. The two point mutations in T domain and a truncated mutation in C-terminal found in human HOS patients produced TBX5 mutant proteins with a significantly reduction of colony suppression activity. Deletion of the DNA-binding domain, however, nearly completely abrogated its ability to suppress colony formation. These results reveal TBX5 as a new regulator of apoptosis and cell growth, suggesting a possible mechanism for Holt–Oram syndrome, and a potential reagent for controlling tumor growth. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Apoptosis; Growth inhibition; T box gene; Tbx5
Mouse T (Brachyury) gene, the founding member of the T box (Tbx) superfamily, was first discovered in 1927 [1]. Its cDNA was first cloned in 1990 [2]. There are now more than 20 known Tbx genes in the invertebrates and the vertebrates [3–5]. Each Tbx gene product contains a T domain, which is composed of approximately 200 amino acid residues and binds to the DNA [3,6]. Some TBX proteins are transcription activators, while others act as transcription repressors [6,7]. Functional studies of Tbx genes in Drosophila, Xenopus, zebrafish, and mice show that they all are important developmental regulators [3–5], as they play important roles in the development of the endoderm, mesoderm, and neural ectoderm in the vertebrate. TBX5 is one of the key molecules playing multiple functions in organogenesis during embryonic development. TBX5 is widely expressed in the developing heart, *
Corresponding author. Fax: +852-2817-1006. E-mail address: [email protected] (M.-L. He).
forelimb, eye, body wall, lung, and liver of vertebrate embryos during critical stages in morphogenesis and patterning [3,8–12]. It is essential in heart development [9,10,12]. Mutation in Tbx5 gene leads to multiple cardiac malformation, including frequent atrial and ventricular septal malformations and abnormalities of left-sided structures [14–18]. TBX5 heterozygous mice develop HOS [12], whereas over-expression in developing ventricles in mice results in heart looping defects or abnormalities in early chamber morphogenesis [11]. Ectopic expression of Tbx5 in the developing eye affects both the positional restricted patterns of gene expression and the growth of retina axon projection [20]. TBX5 also determines limb identity [20–27], as overexpression of Tbx5 in the hindlimbs of developing chickens results in arrested limb development and hypoplasia [20,21]. The differential expressions of the Tbx5 and Tbx4 genes have been implicated in determining the identities of the forelimbs and the hindlimbs, respectively [19–28]. Heterozygous mutations in the Tbx5 gene
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cause autosomal dominant Holt–Oram syndrome (HOS) with abnormalities in the development of the limbs and the heart [12,15–18]. Apoptosis and cell cycle arrest are fundamental processes in organogenesis. It has been found that ectopic expression of TBX5 in D17 canine osteosarcoma cells inhibits cell proliferation [11]. TBX5 is expressed in multiple developing organs and possibly functions differently. However, whether ectopic expression of TBX5 has different effects in other cell lines, such as apoptosis, has not been investigated. Here, we report that ectopic expression of human TBX5 inhibited colony formation, induced apoptosis, and suppressed cell growth. Both the DNA-binding Tdomain and the C-terminal transcriptional regulatory domain are important for these functions, suggesting that the intact activity of transcription regulation is essential for apoptosis and cell growth.
After incubation with the primary antibody, samples were washed with PBS four times, for 5 min each. The second antibody was conjugated with Cy3. After being viewed under the fluorescence microscope, cells were stained with Hoechst dye 33258. Cells expressing the proteins (TBX5 or SRP20) were counted by positive myc antibody staining. Apoptotic cells were indicated by the condensed and fragmented nuclei. Western blot analysis. Cells were harvested 48 h after transfection and lysed with the RIPA buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 1.0% NP-40, 0.1% SDS, 1 mM dithiothreitol, 2 ng/ml aprotinin, 0.1 mM phenymethylsulfonyl fluoride, and 2 ng/ml leupeptin). Assays for b-galactosidase were performed to estimate transfection efficiency and standardized amounts of cell extracts were separated by SDS–PAGE, transferred onto the nitrocellulose membrane, and incubated with a monoclonal antibody against the myc epitope (9E10). Cell proliferation assay. H1299 cells were transfected with either control empty vector or TBX5 expression plasmid, followed by selection of transfected cells, using 350 lg/ml of G418 for two weeks. Single cell colonies were isolated and amplified. Three random picked myc positive cell lines were used for cell proliferation assay. Cells were plated in 12-well dishes at 104 cells per well. The cell numbers were determined using a hematometer by visualization under phase microscope at 16 h after plating.
Materials and methods Plasmid constructs. To express hTBX5, hTBX5 mutatants, or the wild type SRP20 protein, cDNA tagged at the C terminus with the myc epitope followed by an internal ribosome entry site (IRES) was inserted into expression vector pCS2+. Human TBX5 cDNA was a gift from Dr. Brook [16]. The TBX5-G80R and -R237Q point mutations were generated by the two-step PCR strategy. The nucleotide sequences of all plasmid constructs were confirmed by automated sequencing on an ABI 377 sequencer. Cell culture. U2OS is a human osteosarcoma cell line in which the pRB protein was non-functional due to pRB hyperphosphorylation caused by a loss of p16 kinase inhibitor. H1299 is a human lung carcinoma cell line lack of tumor suppressor p53. U2OS and H1299 cells were cultured in DMEM, supplemented with 10% fetal calf serum and antibiotics (penicillin–streptomycin) at 37 °C/5% CO2 . Colony formation assay. U2OS cells and H1299 cells were transfected by the calcium phosphate method [31]. Cells were plated in 10cm dishes at 106 cells per dish. After overnight incubation, cells were transfected with 1 lg of CMV GFP plasmid and 10 lg vector or TBX5expression plasmid. The transfection efficiency was roughly estimated under a fluorescence microscope. Sixteen hours after transfection, the media were replaced with those containing 250 or 350 ng G418/ml, respectively. The media were changed every three days. Cells were selected with G418 for two weeks and stained with crystal violet [31]. For making stable cell lines, the individual colony was picked after G418 selection and amplified in 24-well dishes, following duplicated amplification in 6-well dishes. One set of cells was stained with an antimyc antibody. The myc positive cell lines were used for cell proliferation assay. Cell cycle analysis. U2OS cells were co-transfected with CD20-expression plasmid and either SRP20- or TBX5-expression plasmid. Cells were harvested and stained with FIFC-conjugated anti-CD20 antibody. The cells were then fixed in 70% ethanol before being treated with 50 ng/ml propidium iodide and 100 U/ml RNase A. At least 10,000 FITC-positive cells were analyzed by fluorescence activated cell sorting (FACS), using a FACScan flow cytometer. Cell distribution was analyzed with the CellQuest software [31]. The cells with DNA content below 2n were scored as dead cells. Antibody staining. Transfected cells were harvested, washed with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde for 20 min, followed by treatment with 1% Triton X-100 for 10 min.
Results Effect of TBX5 on colony formation To explore the potential effect of TBX5 on apoptosis and cell proliferation, we first examined the effect of TBX5 on colony formation. Colony formation assay has been extensively used to measure the growth suppression effect of a tested protein, such as tumor suppressor gene pRB and WT1 [31,32]. Osteosarcoma U2OS cells derived from osteroblasts, which play important roles in limb development, have been used extensively for this assay [31,32]. We constructed a TBX5-expression plasmid with the CMV promoter and the neomycin (neo) resistant gene as the selection marker, to express TBX5 protein tagged with a myc epitope. This plasmid was transfected into U2OS cells. Production of the myctagged TBX5-protein was confirmed by Western blot analysis using the anti-myc antibody (Fig. 1A). To test the effect of TBX5 on colony formation, we transfected U2OS cells (106 per 10-cm culture plate) with either a control vector expressing the neo gene alone or the TBX5 plasmid expressing both neo and TBX5. Cells were selected with 250 lg=ml G418 for two weeks, fixed with methanol, and stained with crystal violet. In a typical control vector transfected plate, more than 1000 colonies were formed (Fig. 1C). In contrast, plates transfected with TBX5 plasmid contained only a few colonies (Fig. 1D). Fig. 1B is the calculated average number of colonies per plate from the control vector and TBX5 plasmid transfected plates. Results are the combined data from eight experiments with a total of 18 control plates and 22 TBX5 transfected plates (12 plates had 0 colonies, 4 plates had 2 colonies; 2 plates had 3
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Fig. 1. Over-expression of TBX5 inhibited colony formation in U2OS cells. (A) Western blot analysis showing TBX5 expression in U2OS cells. Lane 1 is cell extracts from cells transfected with the control vector; lane 2 is cell extracts from cells transfected with myc-tagged TBX5 expression plasmid. (B) Cells per plate after transfection with control or TBX5 expressing plasmids. Cells per plate (106 ) were transfected and then selected with 250 lg/ml G418 for two weeks before fixation, staining, and counting. The numbers of colonies were from three plates. The numbers of colonies were very low after TBX5 transfection (see text for further explanation). (C) A typical plate of cells two weeks after transfection with the neo expressing vector and G418 selection. (D) A typical plate after cells were originally transfected with the plasmid expressing TBX5 and neo and selected with G418 for two weeks.
Fig. 2. Induction of apoptosis by TBX5. (A) Flow cytometry profile showing the number of cells (on the y axis) vs. DNA content (on the x axis). U2OS cells were transfected with a plasmid expressing CD20 and a vector. Cells were harvested 60 h post-transfection and stained with a FITCconjugated anti-CD20 antibody. All the cells (6.4%) were dead. Among the live cells, 32.5% was in the G0/G1, 26.0% in the S, and 41.5% in the G2/M phases. (B) U2OS cells were transfected with plasmids expressing CD20 and TBX5. All the cells (29.5%) were dead. Among the live cells, 37.3% was in the G0/G1, 19.6% in the S, and 43.1% in the G2/M phases. (C) The y axis shows the ratio of TBX5 or SRP20 expressing cells among all U2OS cells. The x axis indicates the day after the transfection. A significant reduction of TBX5 expressing cells occurred on day 3 (P ¼ 0:02), whereas in the control group, the ratio of SRP20 expressing was not significantly changed on day 3 (P ¼ 0:27). (D) Apoptosis scored by nuclear morphology. U2OS cells were transfected with expressing myc-tagged SRP20 or TBX5 proteins. They were double-stained with the anti-myc antibody and Hoechst 33258 dye. Condensed and fragmented nuclei were scored as apoptotic. Shown here is the ratio of apoptotic cells among myc-positive cells. A significant difference was found between TBX5 expressing cells ð9:5% 1:4%Þ and non-expressing cells (2% 0:3%; P ¼ 0:01). SRP20 expressing cells (3:4% 1:0%), which were not significantly different from non-expressing cells in the same plates (1:7% 0:5%; P ¼ 0:16). (E) Apoptosis assayed by TUNNEL staining. 15.7% of TBX5 expressing cells were TUNNEL positive, which is different from the percentage of TUNNEL positive cells among SRP20 expressing cells (7.2%; P ¼ 0:05).
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colonies; and 1 plate each had 5, 7, 13, and 18 colonies). TBX5 over-expression also inhibited colony formation in the human lung carcinoma H1299 cells (data not shown). Induction of apoptosis by TBX5 Suppression of colony formation can be the results of a reduced cell proliferation rate, increased apoptosis or cell cycle arrest. We first examined whether inhibition of colony formation was caused by cell cycle arrest. U2OS cells in duplicated plates were cotransfected with either CD20- and TBX5-expression plasmids (1:5) or CD20expression plasmid and the control empty vector. Cells were harvested at 60 h post-transfection stained with FITC-conjugated anti-CD20 antibody and propidium iodide. The FITC positive cells were scored and analyzed by fluorescence-activated cell sorting (FACS). We detected no difference in cell cycle profile between the TBX5 expressing cells (Fig. 2B) and the control cells (Fig. 2A). However, approximately 30% of the TBX5 expressing cells was dead as compared to 6% of the control cells (Figs. 3A and B). To understand how TBX5 induces cell death, U2OS cells were transfected with myc-tagged TBX5 or with myc-tagged SRP20, a splicing factor that does not affect cell growth or cell death [33]. We determined the number of non-transfected cells as well as the TBX5- or SRP20expressing cells per plate. In TBX5 transfected cells, the percentage of the TBX5-expression cells among the total cells steadily decreased over a period of 4 days (Fig. 2C). When the nuclei were stained with the Hoechst dye 33258, apoptotic cells were observed. The percentage of apoptotic cells among total TBX5-expressing cells was also determined. As shown in Fig. 2D, an estimated 9.8% of TBX5-expressing cells was apoptotic at 60 h post-transfection, while only 2% of untransfected cells in the same culture was apoptotic (P ¼ 0:01). In contrast,
in SRP20-expressing cells, only 3.4% was apoptotic at 60 h post-transfection, which is similar to that of the untransfected cells in the same cultures (Student’s t test, P ¼ 0:16). In addition, we evaluated TBX5-induced apoptosis by staining cells using the anti-myc antibody for TBX5-expression, the TUNNEL staining for nuclei fragmented DNA, and the Hoechst dye for nuclear morphology determinations. Results from c-myc staining showed that TBX5 is localized in the nuclei (Fig. 3A). The TUNNEL staining (Figs. 3C and E) showed that 15.7% of TBX5-expressing cells was apoptotic, three times that of the SRP20-expressing cells (6.2%). A correlation was observed between myc-staining, TUNNEL staining, and Hoechst staining (Figs. 3A–H), confirming that it is the TBX5-expression cells that are apoptotic with aberrant nuclei morphology. Taken together, these results all support the notion that TBX5 induces apoptosis. Inhibition of cell proliferation by TBX5 Next, we investigated whether TBX5 inhibits cell proliferation in U2Os and H1299 cells. Unfortunately, for reasons unknown, attempts to make U2OS cell lines stably expressing TBX5 have failed. We therefore chose to use the human lung carcinoma H1299 for this study, as H1299 has been used extensively to investigate cell proliferation due to its p53 deficiency. H1299 cell lines stably expressing a myc-tagged TBX5 were obtained. To determine whether the ectopic expression of TBX5 could alter cell proliferation, stable H1299 cells were plated in 12-well dishes at 104 cells per well. Cells were cultured for 1–4 days. Every 24 h, the cells were trypsinized and cell numbers were determined under microscope using a hematometer. Our results (Fig. 4) showed that TBX5expressing H1299 cells grew at a significantly lower rate. After 4 days in culture, the number of TBX5-expressing cells was 20% of that of the control wild type H1299 cells
Fig. 3. Apoptotic cells after transfection with plasmids expressing either TBX5 or SRP20. Top panels: cells in a plate transfected with the TBX5 expressing plasmid; lower panels: cells in a plate transfected with a plasmid expressing SRP20. (A and B) anti-myc antibody staining showing TBX5 or SRP20 expression and localization in the nuclei; (C and D) TUNNEL staining; (E and F) Hoechst dye staining (color converted from the original blue to white for better visualization); (G) composites of A and C; (H) composites of B and F.
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Inhibition of colony formation by TBX5 required the DNA binding domain (T domain) and transcription regulatory domain (C-terminal region)
Fig. 4. TBX5 over-expression inhibited cell growth in H1299 cells. TBX5 stable cell lines (H1299) were generated and used to measure the cell growth rate. Cells per well (104 ) were plated in 12-well dishes. The total cell numbers were obtained every 24 h.
(P < 0:01; n ¼ 3). In contrast, there is no significant difference in the cell number between the wild type H1299 cells and the mock transfected cells (P ¼ 0:16; n ¼ 3). Taken together, our results indicate that ectopic expression of TBX5 leads to an increased apoptosis rate and a decreased cell proliferation rate, which results a decreased colony formation.
To determine the functional domains, we made various mutant TBX5 constructs and determined the effects of these mutations on the ability of their protein products to inhibit colony formation in U2OS cells. Most mutations found in human HOS patients occur in the T domain region, leading to concentrated losses in the T domain and the C-terminal region [15–18]. Point mutations G80R ðGly80 ! ArgÞ and R237Q ðArg237 ! Gln237Þ are located in the DNA-binding domain (Fig. 5A). Mutant either G80R or R237Q disrupts the interactions between the T-domain and the target DNA [17,34]. A third mutation leads to a truncated protein that lacks more than half the C-terminal region, which performs important function on the activation of target gene transcription [29]. These three and additional mutations provide us with good models to investigate the functional domains of TBX5. As shown in Fig. 5B, the point mutations G80R and R237Q and the truncated mutation TBX5(1–384), which mimicked autosomal dominant mutations (Gly80 ! Arg80 and Arg237 ! Gln237) and truncated mutation found in HOS patients, reduced the number of colonies formed in U2OS cells to approximately 10–20%
Fig. 5. Dissection of the regulatory domain in the TBX5 protein. (A) Diagram of TBX5 mutations found in human HOS syndrome. X-ray crystallographic studies have predicted binding sites for target DNA major groove (gray) and minor groove (black) [7,19]. The Gly80Arg and Arg237Gln missense mutation was located at these sites, respectively. The C-terminal region is responsible for transcriptional activation [29]. (B) Diagrammatic comparison of the relative number of colony formation ðmean SDÞ. The numbers are: vector, 1447 129; TBX5, 2 1; TBX5G80R, 123 15; TBX5R237Q, 130 9; TBX5(1–384), 277 46; and TBX5d(80–237), 1177 266. (C) Results of Western blot analysis showing protein expression. Lane 1, vector; lane 2, wild type TBX5; lane 3, TBX5 mutant G80R; lane 4, R237Q; lane 5 TBX5(1–384); and lane 6, TBX5d(80–237).
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of the control level. These levels of reduction, however, are significantly lower than those of the wild type TBX5 (which reduced the colony number to less than 1% of that of the control). More specifically, these TBX5 mutants produced about 60–100 times more colonies than those by wild type TBX5. On the other hand, deletion of the T domain in mutant d(80–237) nearly totally abolished the colony suppression activity (Fig. 5B, last column). To test whether the varied colony suppression activity is due to the difference in protein expression, we took advantage of the fact that all mutant proteins have been tagged with the myc-epitope and examined the expression of mutant TBX5 proteins by Western blot analysis using the anti-myc antibody. As shown in Fig. 5C, all mutant proteins were expressed and their steady-state levels could not explain their different activities in the colony suppression. For example, while the expression levels of wild-type TBX5 and mutants TBX5G80R and TBX5d(80–237) were similar, they have different colony suppression activities. As a transcription factor, TBX5 protein presumably functions in the nucleus. We have shown that the wild type TBX5 is localized in the nucleus (Fig. 3A). To determine whether the mutant proteins have aberrant subcellular localization, we used immuno-cytochemistry to examine their localization. Our results showed that although there was a small portion of the mutant TBX5(1–384) found in the cytoplasm (Fig. 6, G–I), all of the mutant proteins were localized in the nucleus. Thus, the loss of the colony suppression activity of the
TBX5d(80–237) and other mutants is due to the loss of the T domain and other protein motifs, not to the expression level or subcellular localization.
Discussion TBX5 plays critical roles in vertebrate morphogenesis. Little is known about its specific functions in the cells. In this study, we showed that ectopic expression of TBX5 causes apoptosis and inhibits cell growth, and the intact transcription activity is essential for its functions. The TBX5 activities on transcriptional regulation contribute to induction of apoptosis and/or inhibition of cell proliferation [30, and this study]. Mutations G80R, R237Q, and C-terminal truncation cause human Holt– Oram syndrome [16–18]. DNA-binding experiments revealed that mutations on Gly80 and Arg237 disrupt DNA-binding activity [34]. The C-terminal domain is responsible for activation of target gene transcription [29]. All these mutations disrupt the transcription–activation activity of TBX5 proteins [29,34]. Our results showed that G80R, R237Q, and C-terminal truncation mutation greatly reduced the efficiency of inhibition of colony formation, indicating that the intact transcriptional activity is essential for induction of apoptosis and inhibition of cell proliferation. It is possible that TBX5 have different specificities in different cell types. As TBX5 is widely expressed in different organs, mutations in TBX5 affect only heart and limb development. G80R mutant causes severe
Fig. 6. The nucleus localization of TBX5 and its mutations. Cells transfected with TBX5 mutants (tagged with the myc-epitope) were stained with an anti-myc antibody (in red) and with Hoechst 33258 (original color in blue, but converted into white for better visualization). The top panels show protein localization with anti-myc staining. The middle panels show nuclear staining with the Hoechst dye. The bottom panels are composites of the top and the middle panels. (A–C) TBX5 mutant Gly80Arg; (D–F) TBX5d(80–237); (G–I) TBX5(1–384). For wild type TBX5, see Fig. 3.
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cardiac malformations but only minor skeletal problems whereas R237Q mutant causes significantly limb abnormalities [17,18]. In COS-7 cell, G80R mutation abolished its transcriptional activation on Nppa promoter while R237Q mutation keeps its activity almost intact [29]. Our experiments showed that TBX5 could efficiently inhibit colony formation of different cancer cells, such as U2OS cells, HeLa cells, H1299 cells, and Saos-2 cells, with different sensitivities (data not shown). TBX5 is a potent inhibitor of colony formation on U2OS cells. Attempts to establish a U2OS cell line for constant expression of exogenous TBX5 proved unsuccessful, even with a low stringent selecting condition (reduced G418 concentration from 500 to 250 lg/ml), whereas we easily obtained H1299 stable cell lines. Both G80R and R237Q mutants reduce the inhibitory activity for colony formation on U2OS cell line. These observations suggest that TBX5 have either different downstream target genes or different pathways in different cells. The gradient distribution of TBX5 in developing hearts and limb buds indicates diverse roles in vivo [11,13]. We must note that Tbx5’s presence in the mature heart [9] and its potency in inhibiting cell growth and induction of apoptosis suggest a possible reason for the low frequency of primary tumoriogenesis in the heart and that TBX5 may potentially be used to suppress the growth of certain tumor cells.
Acknowledgments This work was supported by seed funds (to M.-L.H and H.-F. K) from the URC of the University of Hong Kong, RGC, and AoE grant from UGC of Hong Kong.
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[30] C.J. Hatcher, M.S. Kim, C.S. Mah, M.M. Goldstein, B. Wong, T. Mikawa, C.T. Basson, TBX5 transcription factor regulates cell proliferation during cardiogenesis, Dev. Biol. 230 (2001) 177–188. [31] H.S. Zhang, A.A. Postigo, D.C. Dean, Active transcriptional repression by the Rb-E2F complex mediates G1 arrest triggered by p16INK4a, TGFb, and contact inhibition, Cell 97 (1999) 53– 61. [32] L.S. Guan, J.J. Liu, Y.H. Xu, Z.Y. Wang, A point mutation within exon 5 of the WT1 gene of a sporadic unilateral Wilms’ tumor alters gene function, Cancer Res. 58 (1998) 4180–4184. [33] Z.H. Jiang, W.J. Zhang, Y. Rao, J.Y. Wu, Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors, Proc. Natl. Acad. Sci. USA 95 (1998) 9155–9160. [34] T.K. Ghosh, E.A. Packham, A.J. Bonser, T.E. Robinson, S.J. Cross, J.D. Brook, Characterization of the TBX5 binding site and analysis of mutations that cause Holt–Oram syndrome, Hum. Mol. Genet. 10 (2001) 1983–1994.
Induction of apoptosis and inhibition of cell growth by developmental regulator hTBX5 Ming-Liang He,a,* Ying Chen,a,b Ying Peng,a Dadao Jin,b,c Dan Du,a Jun Wu,a Ping Lu,b,c Marie C. Lin,a and Hsiang-Fu Kunga a
Institute of Molecular Biology, Open Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong, Hong Kong SAR, China b Department of Medicine, The University of Hong Kong, Hong Kong SAR, China c Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai 200080, China Received 13 August 2002
Abstract T box (Tbx) genes are a large family of transcription regulators that play critical roles in invertebrate and vertebrate development. Mutations in Tbx5 gene have been found to cause Holt–Oram syndrome (HOS) in humans. Partial dysfunction of TBX5 in mouse also causes HOS phenotype. Little is known about its molecular and cellular mechanism. Here, we report that ectopic expression of TBX5 inhibited colony formation, induced apoptosis, and decreased the growth rate of cells. The two point mutations in T domain and a truncated mutation in C-terminal found in human HOS patients produced TBX5 mutant proteins with a significantly reduction of colony suppression activity. Deletion of the DNA-binding domain, however, nearly completely abrogated its ability to suppress colony formation. These results reveal TBX5 as a new regulator of apoptosis and cell growth, suggesting a possible mechanism for Holt–Oram syndrome, and a potential reagent for controlling tumor growth. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Apoptosis; Growth inhibition; T box gene; Tbx5
Mouse T (Brachyury) gene, the founding member of the T box (Tbx) superfamily, was first discovered in 1927 [1]. Its cDNA was first cloned in 1990 [2]. There are now more than 20 known Tbx genes in the invertebrates and the vertebrates [3–5]. Each Tbx gene product contains a T domain, which is composed of approximately 200 amino acid residues and binds to the DNA [3,6]. Some TBX proteins are transcription activators, while others act as transcription repressors [6,7]. Functional studies of Tbx genes in Drosophila, Xenopus, zebrafish, and mice show that they all are important developmental regulators [3–5], as they play important roles in the development of the endoderm, mesoderm, and neural ectoderm in the vertebrate. TBX5 is one of the key molecules playing multiple functions in organogenesis during embryonic development. TBX5 is widely expressed in the developing heart, *
Corresponding author. Fax: +852-2817-1006. E-mail address: [email protected] (M.-L. He).
forelimb, eye, body wall, lung, and liver of vertebrate embryos during critical stages in morphogenesis and patterning [3,8–12]. It is essential in heart development [9,10,12]. Mutation in Tbx5 gene leads to multiple cardiac malformation, including frequent atrial and ventricular septal malformations and abnormalities of left-sided structures [14–18]. TBX5 heterozygous mice develop HOS [12], whereas over-expression in developing ventricles in mice results in heart looping defects or abnormalities in early chamber morphogenesis [11]. Ectopic expression of Tbx5 in the developing eye affects both the positional restricted patterns of gene expression and the growth of retina axon projection [20]. TBX5 also determines limb identity [20–27], as overexpression of Tbx5 in the hindlimbs of developing chickens results in arrested limb development and hypoplasia [20,21]. The differential expressions of the Tbx5 and Tbx4 genes have been implicated in determining the identities of the forelimbs and the hindlimbs, respectively [19–28]. Heterozygous mutations in the Tbx5 gene
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cause autosomal dominant Holt–Oram syndrome (HOS) with abnormalities in the development of the limbs and the heart [12,15–18]. Apoptosis and cell cycle arrest are fundamental processes in organogenesis. It has been found that ectopic expression of TBX5 in D17 canine osteosarcoma cells inhibits cell proliferation [11]. TBX5 is expressed in multiple developing organs and possibly functions differently. However, whether ectopic expression of TBX5 has different effects in other cell lines, such as apoptosis, has not been investigated. Here, we report that ectopic expression of human TBX5 inhibited colony formation, induced apoptosis, and suppressed cell growth. Both the DNA-binding Tdomain and the C-terminal transcriptional regulatory domain are important for these functions, suggesting that the intact activity of transcription regulation is essential for apoptosis and cell growth.
After incubation with the primary antibody, samples were washed with PBS four times, for 5 min each. The second antibody was conjugated with Cy3. After being viewed under the fluorescence microscope, cells were stained with Hoechst dye 33258. Cells expressing the proteins (TBX5 or SRP20) were counted by positive myc antibody staining. Apoptotic cells were indicated by the condensed and fragmented nuclei. Western blot analysis. Cells were harvested 48 h after transfection and lysed with the RIPA buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 1.0% NP-40, 0.1% SDS, 1 mM dithiothreitol, 2 ng/ml aprotinin, 0.1 mM phenymethylsulfonyl fluoride, and 2 ng/ml leupeptin). Assays for b-galactosidase were performed to estimate transfection efficiency and standardized amounts of cell extracts were separated by SDS–PAGE, transferred onto the nitrocellulose membrane, and incubated with a monoclonal antibody against the myc epitope (9E10). Cell proliferation assay. H1299 cells were transfected with either control empty vector or TBX5 expression plasmid, followed by selection of transfected cells, using 350 lg/ml of G418 for two weeks. Single cell colonies were isolated and amplified. Three random picked myc positive cell lines were used for cell proliferation assay. Cells were plated in 12-well dishes at 104 cells per well. The cell numbers were determined using a hematometer by visualization under phase microscope at 16 h after plating.
Materials and methods Plasmid constructs. To express hTBX5, hTBX5 mutatants, or the wild type SRP20 protein, cDNA tagged at the C terminus with the myc epitope followed by an internal ribosome entry site (IRES) was inserted into expression vector pCS2+. Human TBX5 cDNA was a gift from Dr. Brook [16]. The TBX5-G80R and -R237Q point mutations were generated by the two-step PCR strategy. The nucleotide sequences of all plasmid constructs were confirmed by automated sequencing on an ABI 377 sequencer. Cell culture. U2OS is a human osteosarcoma cell line in which the pRB protein was non-functional due to pRB hyperphosphorylation caused by a loss of p16 kinase inhibitor. H1299 is a human lung carcinoma cell line lack of tumor suppressor p53. U2OS and H1299 cells were cultured in DMEM, supplemented with 10% fetal calf serum and antibiotics (penicillin–streptomycin) at 37 °C/5% CO2 . Colony formation assay. U2OS cells and H1299 cells were transfected by the calcium phosphate method [31]. Cells were plated in 10cm dishes at 106 cells per dish. After overnight incubation, cells were transfected with 1 lg of CMV GFP plasmid and 10 lg vector or TBX5expression plasmid. The transfection efficiency was roughly estimated under a fluorescence microscope. Sixteen hours after transfection, the media were replaced with those containing 250 or 350 ng G418/ml, respectively. The media were changed every three days. Cells were selected with G418 for two weeks and stained with crystal violet [31]. For making stable cell lines, the individual colony was picked after G418 selection and amplified in 24-well dishes, following duplicated amplification in 6-well dishes. One set of cells was stained with an antimyc antibody. The myc positive cell lines were used for cell proliferation assay. Cell cycle analysis. U2OS cells were co-transfected with CD20-expression plasmid and either SRP20- or TBX5-expression plasmid. Cells were harvested and stained with FIFC-conjugated anti-CD20 antibody. The cells were then fixed in 70% ethanol before being treated with 50 ng/ml propidium iodide and 100 U/ml RNase A. At least 10,000 FITC-positive cells were analyzed by fluorescence activated cell sorting (FACS), using a FACScan flow cytometer. Cell distribution was analyzed with the CellQuest software [31]. The cells with DNA content below 2n were scored as dead cells. Antibody staining. Transfected cells were harvested, washed with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde for 20 min, followed by treatment with 1% Triton X-100 for 10 min.
Results Effect of TBX5 on colony formation To explore the potential effect of TBX5 on apoptosis and cell proliferation, we first examined the effect of TBX5 on colony formation. Colony formation assay has been extensively used to measure the growth suppression effect of a tested protein, such as tumor suppressor gene pRB and WT1 [31,32]. Osteosarcoma U2OS cells derived from osteroblasts, which play important roles in limb development, have been used extensively for this assay [31,32]. We constructed a TBX5-expression plasmid with the CMV promoter and the neomycin (neo) resistant gene as the selection marker, to express TBX5 protein tagged with a myc epitope. This plasmid was transfected into U2OS cells. Production of the myctagged TBX5-protein was confirmed by Western blot analysis using the anti-myc antibody (Fig. 1A). To test the effect of TBX5 on colony formation, we transfected U2OS cells (106 per 10-cm culture plate) with either a control vector expressing the neo gene alone or the TBX5 plasmid expressing both neo and TBX5. Cells were selected with 250 lg=ml G418 for two weeks, fixed with methanol, and stained with crystal violet. In a typical control vector transfected plate, more than 1000 colonies were formed (Fig. 1C). In contrast, plates transfected with TBX5 plasmid contained only a few colonies (Fig. 1D). Fig. 1B is the calculated average number of colonies per plate from the control vector and TBX5 plasmid transfected plates. Results are the combined data from eight experiments with a total of 18 control plates and 22 TBX5 transfected plates (12 plates had 0 colonies, 4 plates had 2 colonies; 2 plates had 3
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Fig. 1. Over-expression of TBX5 inhibited colony formation in U2OS cells. (A) Western blot analysis showing TBX5 expression in U2OS cells. Lane 1 is cell extracts from cells transfected with the control vector; lane 2 is cell extracts from cells transfected with myc-tagged TBX5 expression plasmid. (B) Cells per plate after transfection with control or TBX5 expressing plasmids. Cells per plate (106 ) were transfected and then selected with 250 lg/ml G418 for two weeks before fixation, staining, and counting. The numbers of colonies were from three plates. The numbers of colonies were very low after TBX5 transfection (see text for further explanation). (C) A typical plate of cells two weeks after transfection with the neo expressing vector and G418 selection. (D) A typical plate after cells were originally transfected with the plasmid expressing TBX5 and neo and selected with G418 for two weeks.
Fig. 2. Induction of apoptosis by TBX5. (A) Flow cytometry profile showing the number of cells (on the y axis) vs. DNA content (on the x axis). U2OS cells were transfected with a plasmid expressing CD20 and a vector. Cells were harvested 60 h post-transfection and stained with a FITCconjugated anti-CD20 antibody. All the cells (6.4%) were dead. Among the live cells, 32.5% was in the G0/G1, 26.0% in the S, and 41.5% in the G2/M phases. (B) U2OS cells were transfected with plasmids expressing CD20 and TBX5. All the cells (29.5%) were dead. Among the live cells, 37.3% was in the G0/G1, 19.6% in the S, and 43.1% in the G2/M phases. (C) The y axis shows the ratio of TBX5 or SRP20 expressing cells among all U2OS cells. The x axis indicates the day after the transfection. A significant reduction of TBX5 expressing cells occurred on day 3 (P ¼ 0:02), whereas in the control group, the ratio of SRP20 expressing was not significantly changed on day 3 (P ¼ 0:27). (D) Apoptosis scored by nuclear morphology. U2OS cells were transfected with expressing myc-tagged SRP20 or TBX5 proteins. They were double-stained with the anti-myc antibody and Hoechst 33258 dye. Condensed and fragmented nuclei were scored as apoptotic. Shown here is the ratio of apoptotic cells among myc-positive cells. A significant difference was found between TBX5 expressing cells ð9:5% 1:4%Þ and non-expressing cells (2% 0:3%; P ¼ 0:01). SRP20 expressing cells (3:4% 1:0%), which were not significantly different from non-expressing cells in the same plates (1:7% 0:5%; P ¼ 0:16). (E) Apoptosis assayed by TUNNEL staining. 15.7% of TBX5 expressing cells were TUNNEL positive, which is different from the percentage of TUNNEL positive cells among SRP20 expressing cells (7.2%; P ¼ 0:05).
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colonies; and 1 plate each had 5, 7, 13, and 18 colonies). TBX5 over-expression also inhibited colony formation in the human lung carcinoma H1299 cells (data not shown). Induction of apoptosis by TBX5 Suppression of colony formation can be the results of a reduced cell proliferation rate, increased apoptosis or cell cycle arrest. We first examined whether inhibition of colony formation was caused by cell cycle arrest. U2OS cells in duplicated plates were cotransfected with either CD20- and TBX5-expression plasmids (1:5) or CD20expression plasmid and the control empty vector. Cells were harvested at 60 h post-transfection stained with FITC-conjugated anti-CD20 antibody and propidium iodide. The FITC positive cells were scored and analyzed by fluorescence-activated cell sorting (FACS). We detected no difference in cell cycle profile between the TBX5 expressing cells (Fig. 2B) and the control cells (Fig. 2A). However, approximately 30% of the TBX5 expressing cells was dead as compared to 6% of the control cells (Figs. 3A and B). To understand how TBX5 induces cell death, U2OS cells were transfected with myc-tagged TBX5 or with myc-tagged SRP20, a splicing factor that does not affect cell growth or cell death [33]. We determined the number of non-transfected cells as well as the TBX5- or SRP20expressing cells per plate. In TBX5 transfected cells, the percentage of the TBX5-expression cells among the total cells steadily decreased over a period of 4 days (Fig. 2C). When the nuclei were stained with the Hoechst dye 33258, apoptotic cells were observed. The percentage of apoptotic cells among total TBX5-expressing cells was also determined. As shown in Fig. 2D, an estimated 9.8% of TBX5-expressing cells was apoptotic at 60 h post-transfection, while only 2% of untransfected cells in the same culture was apoptotic (P ¼ 0:01). In contrast,
in SRP20-expressing cells, only 3.4% was apoptotic at 60 h post-transfection, which is similar to that of the untransfected cells in the same cultures (Student’s t test, P ¼ 0:16). In addition, we evaluated TBX5-induced apoptosis by staining cells using the anti-myc antibody for TBX5-expression, the TUNNEL staining for nuclei fragmented DNA, and the Hoechst dye for nuclear morphology determinations. Results from c-myc staining showed that TBX5 is localized in the nuclei (Fig. 3A). The TUNNEL staining (Figs. 3C and E) showed that 15.7% of TBX5-expressing cells was apoptotic, three times that of the SRP20-expressing cells (6.2%). A correlation was observed between myc-staining, TUNNEL staining, and Hoechst staining (Figs. 3A–H), confirming that it is the TBX5-expression cells that are apoptotic with aberrant nuclei morphology. Taken together, these results all support the notion that TBX5 induces apoptosis. Inhibition of cell proliferation by TBX5 Next, we investigated whether TBX5 inhibits cell proliferation in U2Os and H1299 cells. Unfortunately, for reasons unknown, attempts to make U2OS cell lines stably expressing TBX5 have failed. We therefore chose to use the human lung carcinoma H1299 for this study, as H1299 has been used extensively to investigate cell proliferation due to its p53 deficiency. H1299 cell lines stably expressing a myc-tagged TBX5 were obtained. To determine whether the ectopic expression of TBX5 could alter cell proliferation, stable H1299 cells were plated in 12-well dishes at 104 cells per well. Cells were cultured for 1–4 days. Every 24 h, the cells were trypsinized and cell numbers were determined under microscope using a hematometer. Our results (Fig. 4) showed that TBX5expressing H1299 cells grew at a significantly lower rate. After 4 days in culture, the number of TBX5-expressing cells was 20% of that of the control wild type H1299 cells
Fig. 3. Apoptotic cells after transfection with plasmids expressing either TBX5 or SRP20. Top panels: cells in a plate transfected with the TBX5 expressing plasmid; lower panels: cells in a plate transfected with a plasmid expressing SRP20. (A and B) anti-myc antibody staining showing TBX5 or SRP20 expression and localization in the nuclei; (C and D) TUNNEL staining; (E and F) Hoechst dye staining (color converted from the original blue to white for better visualization); (G) composites of A and C; (H) composites of B and F.
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Inhibition of colony formation by TBX5 required the DNA binding domain (T domain) and transcription regulatory domain (C-terminal region)
Fig. 4. TBX5 over-expression inhibited cell growth in H1299 cells. TBX5 stable cell lines (H1299) were generated and used to measure the cell growth rate. Cells per well (104 ) were plated in 12-well dishes. The total cell numbers were obtained every 24 h.
(P < 0:01; n ¼ 3). In contrast, there is no significant difference in the cell number between the wild type H1299 cells and the mock transfected cells (P ¼ 0:16; n ¼ 3). Taken together, our results indicate that ectopic expression of TBX5 leads to an increased apoptosis rate and a decreased cell proliferation rate, which results a decreased colony formation.
To determine the functional domains, we made various mutant TBX5 constructs and determined the effects of these mutations on the ability of their protein products to inhibit colony formation in U2OS cells. Most mutations found in human HOS patients occur in the T domain region, leading to concentrated losses in the T domain and the C-terminal region [15–18]. Point mutations G80R ðGly80 ! ArgÞ and R237Q ðArg237 ! Gln237Þ are located in the DNA-binding domain (Fig. 5A). Mutant either G80R or R237Q disrupts the interactions between the T-domain and the target DNA [17,34]. A third mutation leads to a truncated protein that lacks more than half the C-terminal region, which performs important function on the activation of target gene transcription [29]. These three and additional mutations provide us with good models to investigate the functional domains of TBX5. As shown in Fig. 5B, the point mutations G80R and R237Q and the truncated mutation TBX5(1–384), which mimicked autosomal dominant mutations (Gly80 ! Arg80 and Arg237 ! Gln237) and truncated mutation found in HOS patients, reduced the number of colonies formed in U2OS cells to approximately 10–20%
Fig. 5. Dissection of the regulatory domain in the TBX5 protein. (A) Diagram of TBX5 mutations found in human HOS syndrome. X-ray crystallographic studies have predicted binding sites for target DNA major groove (gray) and minor groove (black) [7,19]. The Gly80Arg and Arg237Gln missense mutation was located at these sites, respectively. The C-terminal region is responsible for transcriptional activation [29]. (B) Diagrammatic comparison of the relative number of colony formation ðmean SDÞ. The numbers are: vector, 1447 129; TBX5, 2 1; TBX5G80R, 123 15; TBX5R237Q, 130 9; TBX5(1–384), 277 46; and TBX5d(80–237), 1177 266. (C) Results of Western blot analysis showing protein expression. Lane 1, vector; lane 2, wild type TBX5; lane 3, TBX5 mutant G80R; lane 4, R237Q; lane 5 TBX5(1–384); and lane 6, TBX5d(80–237).
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of the control level. These levels of reduction, however, are significantly lower than those of the wild type TBX5 (which reduced the colony number to less than 1% of that of the control). More specifically, these TBX5 mutants produced about 60–100 times more colonies than those by wild type TBX5. On the other hand, deletion of the T domain in mutant d(80–237) nearly totally abolished the colony suppression activity (Fig. 5B, last column). To test whether the varied colony suppression activity is due to the difference in protein expression, we took advantage of the fact that all mutant proteins have been tagged with the myc-epitope and examined the expression of mutant TBX5 proteins by Western blot analysis using the anti-myc antibody. As shown in Fig. 5C, all mutant proteins were expressed and their steady-state levels could not explain their different activities in the colony suppression. For example, while the expression levels of wild-type TBX5 and mutants TBX5G80R and TBX5d(80–237) were similar, they have different colony suppression activities. As a transcription factor, TBX5 protein presumably functions in the nucleus. We have shown that the wild type TBX5 is localized in the nucleus (Fig. 3A). To determine whether the mutant proteins have aberrant subcellular localization, we used immuno-cytochemistry to examine their localization. Our results showed that although there was a small portion of the mutant TBX5(1–384) found in the cytoplasm (Fig. 6, G–I), all of the mutant proteins were localized in the nucleus. Thus, the loss of the colony suppression activity of the
TBX5d(80–237) and other mutants is due to the loss of the T domain and other protein motifs, not to the expression level or subcellular localization.
Discussion TBX5 plays critical roles in vertebrate morphogenesis. Little is known about its specific functions in the cells. In this study, we showed that ectopic expression of TBX5 causes apoptosis and inhibits cell growth, and the intact transcription activity is essential for its functions. The TBX5 activities on transcriptional regulation contribute to induction of apoptosis and/or inhibition of cell proliferation [30, and this study]. Mutations G80R, R237Q, and C-terminal truncation cause human Holt– Oram syndrome [16–18]. DNA-binding experiments revealed that mutations on Gly80 and Arg237 disrupt DNA-binding activity [34]. The C-terminal domain is responsible for activation of target gene transcription [29]. All these mutations disrupt the transcription–activation activity of TBX5 proteins [29,34]. Our results showed that G80R, R237Q, and C-terminal truncation mutation greatly reduced the efficiency of inhibition of colony formation, indicating that the intact transcriptional activity is essential for induction of apoptosis and inhibition of cell proliferation. It is possible that TBX5 have different specificities in different cell types. As TBX5 is widely expressed in different organs, mutations in TBX5 affect only heart and limb development. G80R mutant causes severe
Fig. 6. The nucleus localization of TBX5 and its mutations. Cells transfected with TBX5 mutants (tagged with the myc-epitope) were stained with an anti-myc antibody (in red) and with Hoechst 33258 (original color in blue, but converted into white for better visualization). The top panels show protein localization with anti-myc staining. The middle panels show nuclear staining with the Hoechst dye. The bottom panels are composites of the top and the middle panels. (A–C) TBX5 mutant Gly80Arg; (D–F) TBX5d(80–237); (G–I) TBX5(1–384). For wild type TBX5, see Fig. 3.
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cardiac malformations but only minor skeletal problems whereas R237Q mutant causes significantly limb abnormalities [17,18]. In COS-7 cell, G80R mutation abolished its transcriptional activation on Nppa promoter while R237Q mutation keeps its activity almost intact [29]. Our experiments showed that TBX5 could efficiently inhibit colony formation of different cancer cells, such as U2OS cells, HeLa cells, H1299 cells, and Saos-2 cells, with different sensitivities (data not shown). TBX5 is a potent inhibitor of colony formation on U2OS cells. Attempts to establish a U2OS cell line for constant expression of exogenous TBX5 proved unsuccessful, even with a low stringent selecting condition (reduced G418 concentration from 500 to 250 lg/ml), whereas we easily obtained H1299 stable cell lines. Both G80R and R237Q mutants reduce the inhibitory activity for colony formation on U2OS cell line. These observations suggest that TBX5 have either different downstream target genes or different pathways in different cells. The gradient distribution of TBX5 in developing hearts and limb buds indicates diverse roles in vivo [11,13]. We must note that Tbx5’s presence in the mature heart [9] and its potency in inhibiting cell growth and induction of apoptosis suggest a possible reason for the low frequency of primary tumoriogenesis in the heart and that TBX5 may potentially be used to suppress the growth of certain tumor cells.
Acknowledgments This work was supported by seed funds (to M.-L.H and H.-F. K) from the URC of the University of Hong Kong, RGC, and AoE grant from UGC of Hong Kong.
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