Oncogene (2002) 21, 3827 ± 3835 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc ORIGINAL PAPERS Tbx3 impinges on the p53 pathway to suppress apoptosis, facilitate cell transformation and block myogenic di€erentiation

Hanqian Carlson1,2, Sara Ota1, Youngsup Song1, Yanwen Chen1,2 and Peter J Hurlin*,1,2

1Shriners Hospitals for Children, Oregon Health Sciences University, 3101 Sam Jackson Park Road, Portland, Oregon 97201, USA; 2Department of Cell and Developmental Biology, Oregon Health Sciences University, 3101 Sam Jackson Park Road, Portland, Oregon 97201, USA

Tbx3 is a member of the T-box family of transcription not been linked to any human disease or birth defects, factors. Mutations in Tbx3 cause ulnar-mammary mutations in a number of other Tbx genes have. These syndrome, an autosomal dominant disorder characterized include Tbx1 (DiGeorge syndrome, see Schinke and by upper limb defects, apocrine-gland defects including Izumo (2001) for review), Tbx3 (ulnar-mammary mammary hypoplasia, and tooth, hair and genital syndrome, Bamshad et al., 1997), Tbx5 (Holt-Oram defects. In cell culture, Tbx3 and its close relative syndrome, Basson et al., 1997; Li et al., 1997) and Tbx2 are capable of immortalizing mouse embryo Tbx22 (X-linked cleft palate and ankyloglossia, Bray- ®broblasts. We show that expression of Tbx3 together brook et al., 2001). Consistent with known functions of with Myc or oncogenic Ras (H-RasVal17) leads to ecient Brachyury, a common theme amongst the human transformation of mouse embryo ®broblasts. Oncogene disease syndromes caused by mutations in Tbx family cooperation by Tbx3 correlates with an ability of Tbx3 genes is the disruption of regional epithelial/mesoder- to suppress the induction of p19ARF and p53 that is mal interactions and associated defects in mesoderm typically caused by overexpression Myc and Ras, and to induction and di€erentiation events. protect against Myc-induced apoptosis. Whereas Tbx3 is Ulnar-mammary syndrome (UMS), caused by het- capable of interfering with apoptosis caused by excessive erozygous mutations in Tbx3 (Bamshad et al., 1997, Myc levels, a Tbx3 mutant lacking its C-terminal 1999), is an autosomal dominant disorder characterized repression domain shows no anti-apoptotic activity and by de®ciencies and duplications of posterior elements fails to repress levels of p19ARF or p53. Consistent with of the forelimb, by mammary hypoplasia and apocrine an ability to suppress p53 pathway function, we ®nd that gland de®ciency/dysfunction and by tooth, hair and sex Tbx3, but not a Tbx3 C-terminal mutant, eciently organ defects. Tbx3 is related to Brachyury and other blocks myogenic di€erentiation of C2C12 myoblasts. Tbx family members by their shared DNA-binding Our results support the idea that deregulation and/or motif known as the T-domain. Some mutations in excessive levels of Tbx3 may have oncogenic potential in Tbx3 that cause UMS disrupt the T-domain and are vivo. thought to function as null alleles (Bamshad et al., Oncogene (2002) 21, 3827 ± 3835. DOI: 10.1038/sj/ 1997), but others occur downstream from the T- onc/1205476 domain and may a€ect the transcriptional repression activity or other functions of Tbx3 (Bamshad et al., Keywords: T-box; Tbx3, Myc, Ras, p19ARF; p53 1999; He et al., 1999; Carlson et al., 2001). The range and nature of defects associated with UMS, together with sites of Tbx3 expression (Chapman et al., 1996; Introduction Gibson-Brown et al., 1998) are consistent with a key role of Tbx3 as a mediator of regional mesoderm The Tbx family of transcription factors perform a induction and di€erentiation. However, very little is variety of functions critical to early embryonic known concerning the underlying molecular mechan- development (Smith, 1999). The prototypical Tbx isms responsible for such activity. protein brachyury is required for posterior, axial and Whereas the relationship between most Tbx family paraxial mesoderm formation as well as formation of members is limited to the conserved T-domain, Tbx2 the notochord (Smith, 1999). Whereas homozygous and Tbx3 show much more extensive sequence mutations in Brachyury cause early embryonic leth- relationship. Indeed the Tbx2/Tbx3 gene pair and the ality, heterozygous Brachyury mutations are respon- more distantly related Tbx4/Tbx5 gene pair is thought sible for the classical mouse mutant tailess (Herrmann to have evolved from an ancestral gene through a two- et al., 1990). Although mutations in Brachyury have step process involving an initial tandem duplication event followed by a parallel duplication event (Agulnik, 1996). Beyond the sequence similarities, it is now known that Tbx2 and Tbx3 both behave as *Correspondence: PJ Hurlin; E-mail: [email protected] Received 23 January 2002; revised 7 March 2002; accepted 12 transcriptional repressors and contain a conserved March 2002 repression domain in their C-terminus (He et al., Tbx3 effects on cell growth and differentiation H Carlson et al 3828 1999; Carlson et al., 2001). Tbx3 and Tbx2 also share to downregulate p19ARF, together with the ®nding similar, but not identical expression patterns during that it is ampli®ed in a subset of mammary embryonic development (Chapman et al., 1996; carcinomas, has raised the possibility that it may Gibson-Brown et al., 1998). However, mutations in function as an oncogene (Jacobs et al., 2000). In this Tbx2 have not been found associated with UMS or any study, we demonstrate that Tbx3 downregulates other disease syndrome and the apparent haplo- p19ARF and p53 levels in cells, can suppress apoptosis insuciency basis for UMS supports the notion that and facilitate cell transformation by Myc and Ras Tbx2 and Tbx3 do not have absolutely redundant oncogenes and interfere with myogenic di€erentiation. functions during embryonic development. Nonetheless, Our results suggest that events leading to excessive the close sequence relationship between Tbx3 and Tbx2 expression levels of Tbx3 may contribute to tumor- suggests that these proteins may share redundant igenesis in vivo, and that defective regulation of p53 biological activities. caused by mutations in Tbx3 may be at least partly Recent cell culture assay systems demonstrate that responsible for ulnar-mammary syndrome. Tbx2 has functions that impinge on the replicative lifespan of mouse and human ®broblasts (Jacobs et al., 2000). Tbx2 was found capable of bypassing the Results premature senescence phenotype of mouse embryo ®broblasts lacking the polycomb group protein Bmi1 Tbx3 cooperates with Myc and Ras to transform cells (Jacobs et al., 2000). Further, Tbx2 and, more recently, Tbx3, was found capable of immortalizing primary We previously showed that ectopic expression of Tbx3 mouse embryo ®broblasts (Jacobs et al., 2000; Carlson was capable of immortalizing primary MEFs (Carlson et al., 2001; Brummelkamp et al., 2002). The under- et al., 2001). Since cell immortalizing events, such as lying mechanism for the ability of Tbx2 and Tbx3 to mutation or loss of p19ARF or p53 are required for prevent senescence and immortalize cells appears to be oncogenic transformation or progression caused by through transcriptional repression of the Cdkn2a oncoproteins such as Myc and oncogenic H-Ras (p19ARF) gene (Jacobs et al., 2000; Brummelkamp et (Serrano et al., 1997; Zindy et al., 1998; Palmero et al., 2002). Since cells lacking p19ARF fail to senesce in al., 1998), we tested whether expression of Tbx3 could culture and can be propagated inde®nitely (Kamijo et cooperate with Myc and H-RasV12 to transform cells. al., 1997), downregulation of p19ARF by Tbx2 and Primary MEFs at passage 1 were transfected with Tbx3 Tbx3 provides a molecular explanation for its ability to alone, Myc alone or H-RasV12 alone or transfected with immortalize cells. p19ARF regulates the abundance of equal amounts of Tbx3 and Myc or H-RasV12. Whereas p53 tumor suppressor protein through its physical Tbx3 alone, Myc alone and H-RasV12 alone produced interaction with Mdm2 oncoprotein, a ubiquitin ligase only rare or no foci of transformed cells, coexpression that mediates the degradation of p53 (Sherr and of Tbx3 with either Myc or H-RasV12 produced a large Weber, 2000; Sharpless and DePinho, 1999). Binding number of transformed foci (Figure 1a,b). Further, of p19ARF to Mdm2 blocks Mdm2 nucleo-cytoplas- coexpression of Tbx3 with Myc and H-RasV12 resulted mic shuttling and sequesters it to the nucleolus, thus in the production of far more foci than observed with preventing its targeting of p53 for destruction (Tao and Myc and H-RasV12 alone (Figure 1a,b). These results Levine, 1999). It is therefore predicted, but has not yet suggest that functions associated with the ability of been demonstrated, that downregulation of p19ARF Tbx3 to immortalize cells (Carlson et al., 2001), allows and immortalization by Tbx2 and Tbx3 should Tbx3 to facilitate cell transformation by Myc and H- correlate with decreased levels of p53 and suppression RasV12 oncoproteins. of its cell cycle arrest/senescence functions. In addition to focus-formation, cells infected with In addition to its functions in regulating cellular Tbx3 and Myc or Tbx3 and H-RasV12 were tested for lifespan, the p19ARF/Mdm2/p53 pathway plays an the ability to form colonies in soft agar. Transfected important role in regulating apoptosis (Sherr and MEFs were allowed to grow for 2 weeks, at which time Weber, 2000; Sharpless and DePinho, 1999). One way transformed foci became visible in Tbx3 plus Myc, that the apoptotic function of the p19ARF/MDM2/ Tbx3 plus H-RasV12 and H-RasV12 plus Myc popula- p53 pathway is activated, is in response to excessive tions. Cells were pooled from individual transfectant proliferation stimuli, such as that caused by oncogene populations, plated in soft agar and the number of activation/overexpression. For example, oncogenes colonies formed were counted 3 weeks later. Tbx3 such as Myc and Ras induce p19ARF expression and expression was found to have little or no ability to consequently allow stabilization and accumulation of promote soft agar colony formation in the presence of p53 (Serrano et al., 1997; Zindy et al., 1998; Palmero et H-RasV12, Myc or H-RasV12 plus Myc (data not al., 1998). Such increased p53 levels promote apoptosis shown). and/or cell cycle arrest and this function is hypothe- sized to serve as a default mechanism to promote the Tbx3 downregulates p19ARF and p53 and protects destruction, or interfere with the progression of cancer against Myc-induced apoptosis prone cells (Evan and Vousden, 2001). Furthermore, mutations that disrupt p53-mediated apoptosis appear Thus the ability of Tbx3 to immortalize MEFs and central to cancer development. Thus the ability of Tbx2 facilitate cell transformation by ectopic expression of

Oncogene Tbx3 effects on cell growth and differentiation H Carlson et al 3829 A Myc and H-RasV12, suggested that Tbx3 might exert its activity by interfering with p53 pathway functions. Further, the closely related Tbx2 protein was previously demonstrated to downregulate the p19ARF gene (Jacobs et al., 2000). We examined the expression of p19ARF, Mdm2, p53 and levels of the p53 target gene product p21, in cells infected with Tbx3 and in cells sequentially transfected with Tbx3 and c-Myc or H-Ras (Figure 2a). Coinfection eciencies using this protocol were typically in the range of 25 ± 50%, based on the infection eciency of a control green-¯uorescent protein-expressing virus (not shown). Levels of p19ARF were found to be downregulated by Tbx3 alone and the elevated p19ARF levels caused by Myc or H-Ras over- expression were suppressed in the presence of Tbx3 (Figure 2a). Consistent with downregulation of p19ARF levels, the elevated levels of p53 and p21 in cells overexpressing Myc and Ras were down- regulated in the presence of Tbx3 (Figure 2a). In contrast to p19ARF, levels of p16 (Cdkn1a) were not a€ected by Tbx3, but were upregulated by H-Ras as previously reported (Serrano et al., 1997). Examina- tion of total pRb levels demonstrated the presence of a slower mobility species that appeared most abundant in Tbx3-expressing cells. The presence of a slower mobility form(s) of pRb is consistent with hyper-phosphorylation and inactivation of Rb. Using antibodies that speci®cally recognize phosphorylated forms of pRb, a form of pRb speci®cally phosphory- lated at Ser 807/810 was detected. Phosphorylation of pRb at 807/811 within the pRb `C-pocket' has been demonstrated to cause release of bound Abl tyrosine kinase (Welch and Wang, 1993, 1995). Unlike the 807/811-phosphorylated form of pRb, we were unable to consistently show the presence of other phos- phorylated forms, nor were we able to demonstrate increased activity of cyclin-dependent kinase 2, 4 or 6 (data not shown). Primary cells overexpressing Myc are prone to apoptosis under serum (survival factor) ± limiting condition (Evan and Vousden, 2001) and several studies indicate the disruption of p53 pathway functions associated with apoptosis is required, or greatly facilitates Myc-induced oncogenic transforma- tion (Zindy et al., 1998; Eischen et al., 1999; Jacobs et al., 1999; Maestro et al., 1999). Therefore we next examined the ability of Tbx3 to suppress apoptosis and its a€ects on the cell cycle. MEFs expressing Tbx3 alone or in combination with Myc or Ras were subjected to serum-limiting conditions (0.2% fetal bovine serum) for 48 h and apoptotic cells identi®ed by FACS analysis of propidium iodide-stained cells. As shown in Figure 2b, overexpression of Myc Figure 1 FocusformationassayshowingthatTbx3cooperateswith induced apoptosis as indicated by a dramatic increase Myc and H-RasV12 to transform primary cells. Mouse embryo in the percentage of the sub-G1 fraction of cells when ®broblasts (MEFs) at passage 1 were transfected with plasmids in 0.2% serum (see arrow). Coexpression of Tbx3 with expressing the indicated proteins. Plates on the right side were co- Myc led to a decrease in the level of apoptosis to near transfected with Tbx3 expression vector. Growth medium was replenished every 2 days for 14 ± 18 days and cells stained with that of control cells infected with empty virus or Tbx3 methylenebluetohighlightcoloniesthatformeddensefoci(a).Similar alone (Figure 2b, see arrow). Coexpression of Tbx3 results were achieved in each of two independent experiments (b) with H-Ras appeared to cause a decrease in the

Oncogene Tbx3 effects on cell growth and differentiation H Carlson et al 3830 slightly elevated level of apoptosis seen in cells but when deprived of serum, are highly prone to overexpressing H-Ras in 0.2% serum and also caused apoptosis (Mateyak et al., 1999). We used retroviral a slight increase in the percentage cells in S phase infection to introduce Tbx3 and a mutant form of compared to cells overexpressing H-Ras alone (Figure Tbx3 lacking the entire C-terminus downstream from 2b). the T-domain (Tbx3-300, Carlson et al., 2001) into Since the sequential viral infection protocol used in HO-Myc cells and examined the apoptotic response the above experiments allowed only a fraction of the following reduction of serum levels to 0.2% (Figure target cells to be infected, a second approach was 3a,b). Retroviral infection eciencies using HO-Myc used to examine the extent to which Tbx3 could cells were typically 65 ± 80% (not shown). When protect cells against Myc-dependent apoptosis. For infected with either empty vector or Tbx3-300, HO- these experiments, we took advantage of an immortal Myc cells showed a strong apoptotic response. In ®broblast cell line developed by Mateyak et al. contrast, HO-Myc cells infected with Tbx3 virus (1999) called HO ± Myc, which lacks endogenous c- showed signi®cant resistance to apoptosis (Figure Myc, but expresses ectopic, deregulated c-Myc at 3a,b). However, by 96 h, many of the Tbx3-infected levels that are moderately elevated compared to its population had succumbed to apoptosis (Figure parental Rat1a cell line. Under normal serum levels 3a,b). In comparison, overexpression of Bcl2 in (10%) these cells grow similarly to wild-type cells, HO-Myc cells resulted in at least a 2-fold greater

Figure 2 E€ects of Tbx3 expression on levels of p53 pathway proteins and apoptosis. Primary MEFs at passage 1 were infected with empty virus, Tbx3, Myc, or Ras virus alone. For co-expression of Tbx3 with Myc and with Ras, Tbx3 infected cells were subsequently infected with either empty virus or by Myc or Ras viruses. For the singly expressing Tbx3, Myc and Ras cell populations, a second infection procedure was also performed, but with empty virus. Infected cells were harvested 2 days after the second infection for Western blot analysis (a) or for FACS analysis (b). For Western blots, equal amounts of cell extracts were used to determine the relative levels of the indicated proteins using antibodies listed in Materials and methods. Tbx3 and Tbx3-300 proteins contain Ha-antibody epitopes at their C-termini (Carlson et al., 2001). For FACS analysis, cells were ®xed in 70% ethanol and stained with propidium iodide. Relative DNA content of individual cells is shown in (b), with the growth phase 1 (G1, diploid DNA content) and growth phase 2/mitosis (G1/M, tetraploid DNA content) populations indicated, along with the percentage of apoptotic cells (exhibiting a sub-GI DNA content). Arrows point to cells infected with Myc and with Tbx3 plus Myc that show a sub-G1 DNA content characteristic of cells undergoing apoptosis. Note the decrease in the population of apoptotic cells that occurs when Tbx3 is coexpressed with Myc. The percentage of apoptosis present in each of the cell populations, based on sub-G1 DNA content is indicated

Oncogene Tbx3 effects on cell growth and differentiation H Carlson et al 3831

Figure 3 Tbx3 suppresses Myc-dependent apoptosis. HO ± Myc cells were infected with viruses expressing the indicated proteins and 2 days later growth medium (10% serum) was replaced with low serum (0.2%) medium. Cells were photographed at the indicated times following lowering the serum (a). The appearance of highly refractile, rounded and picnotic cells are characteristic of apoptosis. To quantitate apoptosis, all cells were collected from empty virus, Tbx3 ± 300 and Tbx3 infected populations at the indicated times, ®xed in 70% ethanol, stained with propidium iodide and DNA content measured by FACS (b). The percentage of apoptotic cells determined by the number of cells showing a sub-G1 (diploid) DNA content is indicated. Represented are the results of two separate experiments performed in duplicate (error bars indicate standard deviations). (c) Reduced expression levels of p53- pathway components caused by Tbx3 in HO ± Myc cells. HO ± Myc cells infected with empty virus, Tbx3 or Tbx3 ± 300 were harvested 2 days following viral infection, lysed and equal amounts of cell extracts used for Western blot analysis of the indicated proteins as described in Materials and methods number of cells protected from apoptosis under Tbx3-300 expression appeared to cause a slight identical conditions (not shown). These results upregulation of p19ARF, p53, Mdm2 and p21 (Figure suggest that Tbx3 signi®cantly delays the robust 3c). Thus overexpression of Tbx3 downregulates, but apoptotic response of these cells and may protect does not shut o€ expression of p19ARF and p53, and some HO-Myc cells from death, but does not such downregulation is consistent with the observation completely disrupt pathways required for apoptosis. that Tbx3 partially protects against apoptosis of HO- To examine the e€ects of Tbx3 on the p53 pathway Myc cells. in HO-Myc cells, levels of p19ARF, p53, Mdm2 and p21 were compared in uninfected cells and in cells Suppression of myogenic differentiation by Tbx3 infected with Tbx3 and Tbx3-300. Levels of p19ARF were consistently found to be signi®cantly lower in The p53 and pRb pathways play critical roles in the Tbx3 cells, as was p53, and the p53 target gene control of myogenic di€erentiation (Walsh and Perl- products Mdm2 and p21 (Figure 3c). In contrast, man, 1997; Cerone et al., 2000). The ability of Tbx3

Oncogene Tbx3 effects on cell growth and differentiation H Carlson et al 3832 to regulate p19ARF and p53 levels and to induce phosphorylation and possibly inactivate Rb (Figures 2a and 3c) led us to test whether ectopic expression of Tbx3 in the myogenic cell line C2C12 could prevent di€erentiation. For these experiments, pools of C2C12 cells expressing Tbx3, Tbx3-300 or no Tbx3 protein were generated by stable transfection using G418 selection. These di€erent populations were tested for their ability to di€erentiate into myotubes when the culture medium was changed from growth medium (15% bovine calf serum) to di€erentiation medium (2% horse serum). Whereas vector control cells and Tbx3-300 cells showed an abundance of myotubes following 5 days in di€er- entiation medium, very few myotubes were observed in the Tbx3 expressing population (Figure 4). Consistent with a disrupted di€erentiation response, Tbx3 expressing cells failed to upregulate the muscle di€erentiation marker myosin light chain (MLC) and the myogenic regulatory factor myogenin (Figure 5). Interestingly, the basal levels of both myogenin and myoD were reduced in Tbx3-expressing cells com- pared to vector control cells, as was MLC, and their expression remained suppressed following exposure to di€erentiation medium for 5 days (0 days, Figure 4). Figure 5 Northern blot comparing the induction of the muscle di€erentiation marker myosin light chain and expression levels of the muscle regulatory factors myogenin and MyoD in control, empty virus infected C2C12 cells and C2C12 cells stably expressing Tbx3. Glyceraldehyde phosphate dehydrogenase (GFAP) served as a loading control

It is important to note that Tbx3 expression appeared to be toxic to a sub-population of C2C12 cells and that another sub-population was capable of under- going di€erentiation into myotubes following more extended time in di€erentiation medium (not shown). One possibility is that these di€erent responses are caused by di€erent expression levels of Tbx3 in the pooled populations of transfected cells used in these experiments. Nonetheless, we conclude that Tbx3 expression provides a strong inhibitory signal for myogenic di€erentiation.

Discussion

A probable mechanism underlying the ability of Tbx3 to cooperate with Myc and Ras oncogenes to transform cells (Figure 1) and to inhibit myogenic di€erentiation (Figure 4) is reduction of p53 levels caused by downregulation of p19ARF by Tbx3 (Figures 2a and 3c). Based on results obtained with Tbx2 (Jacobs et al., 2000) and more recently with Figure 4 Tbx3 blocks myogenic di€erentiation. C2C12 myogenic Tbx3 (Brummelkamp et al., 2002), downregulation of cells were transfected with pFB, pFB ± Tbx3 or pFB ± Tbx3 ± 300 p19ARF by these factors is direct. The downregula- and stable transfectants selected by resistance to the G418 tion of p19ARF by Tbx3 is predicted to decrease resistance conferred by the virus. Pooled populations, growing cellular levels of p53 by freeing the p19ARF- in 15% fetal bovine serum, were established and examined for their ability to di€erentiate when placed in di€erentiation medium interacting protein Mdm2, leading to increased (2% horse serum). Cells were photographed (406) following Mdm2-mediated turnover of p53 (Sherr and Weber, incubation in di€erentiation medium for the indicated times 2000; Sharpless and DePinho, 1999). Indeed, we ®nd

Oncogene Tbx3 effects on cell growth and differentiation H Carlson et al 3833 that p53 levels decrease in response to ectopic bHLH protein Twist has properties similar to those expression of Tbx3, and that levels of the p53 target described here for Tbx3 and there is evidence that gene products Mdm2 (functioning as part of a it functions as an oncogenic factor in rhabdomyo- feedback mechanism regulating p53 abundance) and sarcoma (Maestro et al., 1999). Jacobs et al. p21 also decrease (Figures 2a and 3c). The ability of (2000), who made the original observation that Tbx3 to downregulate p19ARF, p53 and p53 target the Tbx2 protein can immortalize MEFs and can gene levels, is consistent with its ability to interfere repress p19ARF gene transcription, showed that with myogenic di€erentiation, which has been shown the Tbx2 gene is ampli®ed and overexpressed in to be dependent on p53 activity (Cerone et al., 2000). some breast cancers. Consistent with the notion Similarly, the ability of Tbx3 to negatively regulate that Tbx3 may be capable of promoting neoplastic p53 levels is consistent with its capacity to participate growth, null and/or inactivating mutations in Tbx3 in cell transformation. In contrast to Tbx3, both Myc in humans leads to hypoproliferation of a number and Ras stimulate increased p19ARF levels and of di€erent tissues, including breast tissue in ulnar- typically make cells prone to apoptosis and cell cycle mammary syndrome (Schinzel, 1987). Taken to- arrest respectively (Sherr and Weber, 2000; Sharpless gether with the Tbx2 results and work presented and DePinho, 1999). A likely manifestation of the here, a more comprehensive examination of Tbx2 decreased levels of p53 caused by Tbx3 expression is and Tbx3 involvement in tumorigenesis is war- partial suppression of apoptosis, as we show using ranted. primary MEFs overexpressing Myc (Figure 2b) and Although the present observations point to a the HO ± Myc cell line (Figure 3a,b). HO ± Myc cells potential role for Tbx3 in tumorigenesis, our results are derivatives of the immortal Rat1a cell line that may also shed some light on how mutations in Tbx3 express deregulated Myc on a myc-null background lead to ulnar-mammary syndrome. In experiments (Mateyak et al., 1999) and, like many cell types using a mutant Tbx3 lacking its C-terminus, a expressing elevated Myc levels, are highly sensitive to mutant form of Tbx3 that is similar to those apoptosis when serum-deprived. Although Myc-de- predicted to be produced by a number of Tbx3 pendent apoptosis was strongly suppressed by Tbx3 mutations found in ulnar ± mammary syndrome in primary MEFs (Figure 2b), suppression of (Bamshad et al., 1999; Carlson et al., 2001), we apoptosis in HO-Myc cells was less impressive found that it failed to interfere with apoptosis (Figure 3a,b). These results raise the possibility that (Figures 2b and 3a,b) and failed to block myogenic primary cells may be more responsive to Tbx3 e€ects di€erentiation (Figure 4). Thus, perhaps UMS on apoptosis than immortal cell lines. Alternatively, mutations lead to hypoplasia as a consequence of the HO ± Myc assay system may provide a more failure to adequately protect against apoptotic death accurate assessment of the protective e€ects of Tbx3 of key cell populations at critical times during expression. Nevertheless, we speculate that even a embryonic development. Alternatively or coinciden- partial disruption of p53-dependent apoptosis caused tally, mutant Tbx3 proteins and/or Tbx3 de®ciency, by downregulation of p19ARF in primary MEFs, is may lead to the premature or unscheduled di€er- sucient for Tbx3 to tip the balance in favor of entiation of key target tissues. However, even though growth promotion and cell transformation by Myc. It hypoplasia is evident in many tissues of UMS is important to recognize that Myc-induced apoptosis patients, UMS is a highly complex syndrome that is manifest much more readily in serum-limiting does not appear to be a disease of cell proliferation (survival factor-limiting) conditions and that the cell per se, but more a disease of defective epithelial/ transformation experiments using Tbx3 in combina- mesenchymal interactions (Bamshad et al., 1997; tion with Myc and Ras were performed in normal Smith, 1999). Furthermore, regulation of p19ARF growth conditions (as these types of experiments are levels and p53 pathway activity and possibly pRb typically performed). Thus, partial disruption of p53- pathway activity (Figures 2a, 3c) is likely to be but mediated apoptosis by Tbx3, combined with the one aspect of Tbx3 function and it will be important presence of survival factors above some threshold to determine other Tbx3 target genes and biological that bu€ers cells from the apoptotic and cell cycle activities. However, it is notable that mutations in arrest activities of excessive Myc and Ras levels, the p53 family member p63, cause EEC syndrome likely work together to promote cell transformation (ectrodactyly, ectodermal dysplasia, and cleft lip/ by Myc, as well as Ras oncogenes. Indeed, the ability palate) (Celli et al., 1999; van Bokhoven et al., of Tbx3 to cooperate with Myc to transform cells, 2001), the related Hays ± Wells syndrome (McGrath et yet only partially block Myc-dependent apoptosis, al., 2001), and are found in a subset of individuals suggests that additional activities of Tbx3, or with non-syndromic split hand ± split foot malforma- additional activities associated with downregulation tion (SHFM) (Ianakiev et al., 2000; van Bokhoven et of p19ARF, promote cell transformation. al., 2001) and limb-mammary syndrome (van Bokho- The ability of Tbx3 to impinge on p53 pathway ven et al., 2001). All of these diseases share some function, facilitate cell immortalization and trans- overlapping features with UMS, supporting the formation, and inhibit myogenic di€erentiation, is possibility that disrupted regulation of p53 and consistent with the idea that Tbx3 may participate perhaps p63 in some a€ected tissues may indeed be in tumorigenic progression in vivo. Interestingly, the an important underlying mechanism in this syndrome.

Oncogene Tbx3 effects on cell growth and differentiation H Carlson et al 3834 Materials and methods Myogenic differentiation assays and Northern blotting C2C12 cells (ATCC) were maintained in DMEM supple- Focus formation assays mented with 15% fetal calf serum (FCS) and grown in Primary mouse embryo ®broblasts (MEFs) were isolated humidi®ed incubators at 378C and 10% CO2. To induce from embryonic day 13.5 mouse (C57Bl/6) embryos as di€erentiation, cells were grown to 90% con¯uence and previously described (Spector et al., 1998) and grown in growth medium switched to 2% horse serum (di€erentiation DMEM supplemented with 10% FBS. Analogous to medium) for 5 days. Cells were photographed daily. Northern experiments pioneered by Land et al. (1983), focus formation blots were carried out using total RNA by standard methods (transformation) assays were performed by transfecting and hybridized with radiolabelled probes for myoD, MEFs at passage 1 or 2 with combinations of pBabe ± Myc myogenin and myosin light chain (gifts from M Thayer) (a kind gift from C Grandori) pEJ ± Hras and pCS2 ± Tbx3 and GFAP. plasmids (Carlson et al., 2001) using the calcium phosphate precipitation method (Chen and Okayama, 1987). Trans- Western blot analysis fected MEFs were fed every 2 days with growth medium and stained with methylene blue after 2 ± 3 weeks. Focus For retrovirus-infected cells, cells were harvested 48 h after formation experiments were performed in duplicate and infection and lysed with NP-40 bu€er (150 mM NaCl, 20 mM repeated with di€erent batches of MEFs at least two times Tris HCl pH 8, 1 mM EDTA, 1% NP-40, 10% glycerol, and with essentially identical results. For soft agar assays, instead 10 mg/ml Aprotinin, 1 mM PMSF, and 25 mg/ml leupeptin of staining cells used in focus formation assays, they were added fresh). Equal amounts of protein were resolved by pooled and plated in duplicate at 5000 cells/plate in soft agar SDS-polyacrylamide gel electrophoresis and transferred to as previously described (Hurlin et al., 1987). Soft agar assays polyvinylidene di¯uoride membranes (Millipore Corp., Bed- were performed twice with reproducible results. ford, MA, USA). Membranes were incubated in TTBS (10 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat dry milk overnight at 48C. Mem- Retroviral infection and apoptosis assays branes were probed using the following antibodies: p19ARF Retroviral infections of MEFs and HO-Myc (a kind gift of J (Abcam), p53, p21, p16, c-Myc, tubulin (Santa Cruz Sedivy) cells were carried out for 24 h in DMEM/10% FBS Biotechnology), Mdm2 (A Levine, Princeton), pRb, and using viral supernatants produced in transiently transfected phospho-speci®c Rb (New England Biolabs), Ha epitope Ecopak 293 cells (Clonetech). Infections were performed (Covance). Antibody incubations and washes were performed using pFB retrovirus (Stratagene), pFB ± Tbx3 ± Ha, or pFB ± according to conditions provided by the supplier. Tbx3 ± 300 (Carlson et al., 2001), pBabe ± Myc and pBabe ± H-Ras (a kind gift from Scott Lowe). Virus titers were determined by infecting NIH3T3 cells with a pFB ± GFP (green ¯uorescent protein) retrovirus and positive (green ¯uorescent) cells counted. Typical infection eciencies were 50 ± 80%. For apoptosis assays, infected cells were plated at 50 000 cells per 30 mm dish 24 h after infection (or sequential infections) and serum levels were lowered 12 h later from Acknowledgments 10% to 0.2% serum. Cells were photographed and collected We would like to thank Scott Lowe, Arnold Levine, John daily, ®xed in 70% ethanol, stained with propidium iodide Sedivy, Carla Grandori and Robert Eisenman for reagents. and sorted for DNA content by ¯uorescence-activated cell This work was supported by a grant from Shriners sorting (FACS). Assays were performed at least three times. Hospitals for Children to PJ Hurlin.

References

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