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Cancer Research 67, 11263, December 1, 2007. doi: 10.1158/0008-5472.CAN-07-3016
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Runx2 Disruption Promotes Immortalization and Confers Resistance to Oncogene-Induced Senescence in Primary Murine Fibroblasts

Anna Kilbey1, Karen Blyth1, Sandy Wotton1, Anne Terry1, Alma Jenkins1, Margaret Bell1, Linda Hanlon2, Ewan R. Cameron1 and James C. Neil1

1 Molecular Oncology Laboratory, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Glasgow, United Kingdom and 2 J. Kissil Laboratory, The Wistar Institute, Philadelphia, Pennsylvania

Requests for reprints: Anna Kilbey, Institute of Comparative Medicine, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, United Kingdom. Phone: 44-141-330-3444; Fax: 44-141-330-2271; E-mail: A.Kilbey{at}vet.gla.ac.uk or James C. Neil, Institute of Comparative Medicine, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, United Kingdom. Phone: 44-141-330-2365; E-mail: j.c.neil{at}vet.gla.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Runx genes play paradoxical roles in cancer where they can function either as dominant oncogenes or tumor suppressors according to context. We now show that the ability to induce premature senescence in primary murine embryonic fibroblasts (MEF) is a common feature of all three Runx genes. However, ectopic Runx–induced senescence contrasts with Ras oncogene–induced senescence, as it occurs directly and lacks the hallmarks of proliferative stress. Moreover, a fundamental role for Runx function in the senescence program is indicated by the effects of Runx2 disruption, which renders MEFs prone to spontaneous immortalization and confers an early growth advantage that is resistant to stress-induced growth arrest. Runx2–/– cells are refractory to H-RasV12–induced premature senescence, despite the activation of a cascade of growth inhibitors and senescence markers, and are permissive for oncogenic transformation. The aberrant behavior of Runx2–/– cells is associated with signaling defects and elevated expression of S-G2-M cyclins and their associated cyclin dependent kinase activities that may override the effects of growth inhibitory signals. Coupling of stress responses to the cell cycle represents a novel facet of Runx tumor suppressor function and provides a rationale for the lineage-specific effects of loss of Runx function in cancer. [Cancer Res 2007;67(23):11263–71]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The three members of the Runx family of mammalian transcription factors are related to Runt, the Drosophila pair rule gene (1), sharing a highly conserved DNA-binding domain and a common DNA-binding cofactor. Their products regulate multiple cell fate decisions and have been implicated in a wide range of cancers where there is unequivocal evidence that members of this family can act as oncogenes or as tumor suppressors according to context (2).

The identification of somatic and germ-line loss-of-function mutations associated with acute myeloid leukemia (AML) strongly supports the view that RUNX1 operates as a tumor suppressor gene in this lineage. Further evidence that the RUNX genes can inhibit tumor development comes from a number of reports suggesting that methylation and down-regulation of RUNX3 is etiologically associated with the development and progression of a spectrum of epithelial cancers (ref. 2 and references therein). However, it is clear that the tumor suppressor role of the RUNX gene family is expressed in a lineage-restricted manner. Functional redundancy and partially overlapping expression patterns of the three family members have been proposed to account for these observations. Functional similarity between the Runx genes is also suggested by the finding that all three genes act as a single complementation group by retroviral activation in experimental models of hematologic malignancy (3–5).

The mechanisms underlying these contrasting manifestations in cancer are not fully understood, although it seems that ectopic Runx expression can inhibit apoptosis, particularly in transgenic mice overexpressing the Myc gene. Moreover, the growth suppressive features of Runx2 are evident in this T-cell transgenic model, as prelymphomatous mice display a proliferative defect in immature thymocytes, which is overcome by ectopic Myc expression (6). Loss of Runx1 seems to result in increased proliferation and failure of hematopoietic precursor cells to differentiate (7), suggesting that homeostatic control of hematopoiesis is a central function of this gene, whereas RUNX3 down-regulation and hypermethylation in epithelial cells has been linked to loss of growth inhibitory responses to transforming growth factor-β (TGF-β; refs. 8, 9). Together, these observations are consistent with growth inhibitory Runx functions that are selectively lost in cancers or overcome by complementing oncogenic mutations affecting collaborating genes, such as Myc or p53, which serve to reveal the oncogenic side of their dual nature (10, 11). However, it is not known whether these various inhibitory phenomena represent shared functions across family members and in different lineages, or whether they can fully account for the tumor suppressor activities of this gene family.

A striking demonstration of RUNX-induced negative growth regulation is the senescence-like phenotype observed after ectopic expression of RUNX1 in primary murine embryonic fibroblasts (MEF; refs. 11, 12). Premature senescence or growth stasis shares many of the characteristics of replicative senescence but is independent of telomere shortening and occurs in response to activation of stress pathways that detect or heighten cellular responses to DNA damage (13). Thus, the aberrant expression of oncogenes, such as activated Ras, can result in a transient round of cell proliferation followed by permanent withdrawal from the cell cycle and other phenotypic features indicative of cellular senescence. Moreover, it is now clear that this is not merely a feature of in vitro conditions and that this phenomenon limits tumor development in vivo (14, 15). Although species and cell type differences exist, many of the essential components of the Ras-induced senescence pathway have been identified and there is considerable overlap between these components and genes with established tumor suppressor function. In accord with this hypothesis activation of the major tumor suppressor pathways mediated by p53 and/or pRb family members is a hallmark of premature senescence (16).

We now report that induction of premature senescence in MEFs is a common feature of all three Runx genes. However, this process differs markedly from Ras-induced senescence as it occurs without an initial aberrant proliferative phase. Moreover, MEFs lacking Runx2 display an early growth advantage that is resistant to in vitro culture stress and are susceptible to H-RasV12–induced cellular transformation. Our results suggest that Runx genes play a central role in regulating the cell cycle in response to stress signals, including ectopic oncogene expression, offering new insights into the mechanisms of Runx tumor suppression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, constructs, and retroviral transductions. Littermate wild-type and Runx2–/– MEFs were prepared from E14.5 embryos derived from mating Runx2+/– mice (17). The virus packaging line Phoenix (ecotropic) was obtained from G. Nolan (Stanford University, Palo Alto, CA). All cultures were maintained in DMEM (Invitrogen) with 10% heat-inactivated FCS, glutamine (2 mmol/L), and penicillin/streptomycin (200 units/mL; 100 µg/mL). The retroviral vectors were all based on the pBabe plasmid (18), carrying the puromycin-selectable marker. pBabe-PURO Runx2, kindly provided by M. Stewart (Glasgow University, Glasgow, United Kingdom), comprises a 2.3-kb cDNA fragment encoding the Cbfa1-G1 isoform (3) subcloned into the polylinker region of pBabe-PURO. pBabe-PURO Ras was kindly provided by S. Lowe (Cold Spring Harbor Laboratory, New York, NY; ref. 16). Retroviral transductions were performed as previously described (11).

Growth curves, senescence staining, and 3T3 passage culture. Early passage (p3–p4) MEFs were plated at 2.5 x 104 cells per well in 12-well plates in growth medium or in selection medium containing 2 µg/mL puromycin. Live cell counts were carried out in triplicate using trypan blue as a vital stain. Triplicate wells were pooled from each time point for analysis of protein expression. Medium changes were carried out every 3 to 4 days. For UVC irradiation, early passage MEFs were plated overnight at 5.0 x 105/60-mm dish. The medium was then removed, and the cells were irradiated using an XL-1500 UV cross-linker (Spectrolinker). The cultures were incubated in fresh medium, and live/dead cell counts were performed after 24 h by trypan blue exclusion. Graphs were plotted using Sigma plot, and significance values were determined by Student's t test. Error bars relate to SDs. Senescence staining was assayed on a parallel plate after 7 to 10 days using a solution of X-gal (Invitrogen) at pH 6.0 to detect senescence-associated β-galactosidase (SA-β-gal) activity as described previously (16). 3T3 passage culture was essentially performed according to the protocol of Todaro and Green (19).

Cell cycle analysis. MEFs plated at 3 x 105 cells/10-cm dish were sampled for flow cytometry in parallel with growth curves. The protocol for cell cycle analysis has been described previously (20). For simultaneous labeling with bromodeoxyuridine (BrdUrd), cells were incubated at 37°C with 10 µmol/L BrdUrd (Sigma) and then rinsed thrice in warmed PBS. After harvesting, cells were washed in 2 mL cold PBS, resuspended in 0.2 mL cold PBS, and fixed for at least 30 min in 2 mL of 70% ethanol at 4°C. Cells were denatured in 4 mol/L HCl for 20 min, rinsed twice in cold PBS, incubated for 10 min in PBS/0.5% bovine serum albumin/0.1% Tween 20, and labeled with FITC-conjugated anti-BrdUrd antibody (Roche). Samples were washed twice in PBS and resuspended in PBS containing 10 µg/mL propidium iodide. Analysis was carried out on a Beckman Coulter Epics XL using EXPO32 software.

Western blotting and antibodies. Preparation of whole cell protein extracts was performed as described previously (11). Samples equivalent to 30 µg total protein (Bio-Rad protein assay) were resolved on 8%, 10%, or 17% SDS-polyacrylamide gels and transferred to enhanced chemiluminescence (ECL; Amersham) nitrocellulose membranes. The antibodies used were {alpha}-p16INK4a, {alpha}-p21WAF1, {alpha}-actin (sc-1207, sc-471, and sc-1616, Santa Cruz Biotechnology), {alpha}-p19ARF (ab80, Abcam), {alpha}-p53, {alpha}–phospho-p38 mitogen-activated protein kinase (MAPK), {alpha}-p38 MAPK (IC12, 9211, and 9212, Cell Signaling Technology), {alpha}–c-H-ras (Oncogene Ab-1), and {alpha}-RUNX2 (D130-3, Stratech). Western blots were developed using ECL according to the manufacturer's protocol. Positive controls were as follows: p16INK4a and p19ARF (SV3T3 cell extract), p21WAF1 and p53 (UVC-treated wild-type MEF extract) and p38 MAPK (Jurkat cell extract). Negative controls were as follows: p16INK4a and p19ARF (NIH3T3 cell extract), p21WAF1 (RAT 1 fibroblast extract) and p53 (p53 null MEF extract).

In vivo transplantation studies. Runx2-null and wild-type MEFs were transduced with either the constitutive H-RasV12 or PURO-vector control and expanded in culture for 7 days. Cells (106) in 0.2 mL PBS were injected s.c. into the left flank of 5 to 6 MFI nu/nu mice (Harlan) for each cell group. Animals were housed in sterile filter-top cages, monitored thrice weekly, and humanely sacrificed when tumor masses reached a predetermined size or became ulcerated. All animal work was undertaken in line with the United Kingdom Animals Act of 1986 (Scientific Procedures).

Colony assays. For soft agar colony assays, cells were diluted into 0.3% agar (Noble Difco) in DMEM containing 20% FCS and seeded in triplicate onto solidified 0.6% agar containing culture medium at 103, 104, or 105 cells/6-cm plate (Bibby Sterilin). The colonies were fed every 3 to 4 days and evaluated after 5 weeks.

RNA extraction and cDNA preparation. Runx2–/– and wild-type MEFs were transduced with either constitutive H-RasV12 or the PURO-vector control and plated on six-well plates at 7.5 x 104 cells per well. The cells were harvested from triplicate wells at days 3, 7, 10, and 13 into 1-mL RNA-Bee (AMS Biotechnology), and RNA was prepared according to the manufacturer's protocol. RNA pellets were dissolved in diethyl pyrocarbonate (DEPC)-treated water (Ambion), and the concentration was determined using an Agilent Bioanalyzer. cDNA was prepared from 1-µg aliquots of RNA using a Quantitect Reverse Transcription kit (Qiagen) and diluted 1 in 20 in DEPC-treated water to give a working stock.

Quantitative real-time PCR. Five-microliter aliquots of cDNA were amplified in triplicate on an ABI 7500 real-time PCR system using Power SYBR Green PCR master Mix (Applera UK) and primers for murine cyclin A2, cyclin B1, cyclin E1 (Qiagen Quantitect Primer Assays QT00102151, QT00152040, and QT00103495), or endogenous control hprt (106F 5'-AGCGTCGTGATTAGCGATGAT-3' and 253R 5'-CCTTCATGACATCTCGAGCAAG-3'). Relative quantification was carried out and calibrated to day 0 wild-type sample.

In vitro kinase assays. Immunoprecipitation kinase assays for cyclin A–, cyclin B1–, and cyclin E–associated kinase activities with histone H1 as a substrate were performed as described previously (21). Essentially Runx2–/– and wild-type MEFs were plated at 1.5 x 106 cells/150-mm dish and grown to 80% to 90% confluence for 4 to 5 days. The contents of one dish were extracted into 0.5-mL lysis buffer, and the protein concentrations were determined as for Western extraction. Five hundred micrograms of total cellular extract were analyzed per reaction. Monoclonal antibodies against cyclin A (MAB3682, Chemicon) and cyclin B1 (sc-245, Santa Cruz Biotechnology) were used to immunoprecipitate cyclin A– and cyclin B1– associated kinase complexes directly. For cyclin E–associated kinase activity, cell lysates were sequentially immunoprecipitated with {alpha}-cyclin A and {alpha}-CDK2 (sc-163; Santa Cruz Biotechnology). Kinase reactions were transferred to nitrocellulose membranes for autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of premature senescence in primary mouse fibroblasts is a common feature of the Runx family but is distinct from H-RasV12–induced senescence. We previously showed that RUNX1 mediates senescence-like growth arrest in primary MEFs (11). To determine whether this response is a general property of the Runx family, we tested the effect of introducing ectopic Runx2 into wild-type cells by retroviral transduction. Western blot analysis revealed high intracellular levels of ectopic Runx2 and a corresponding dramatic reduction in cell numbers over a 7-day culture period (Fig. 1A ). In contrast, cells carrying the control vector (PURO control) showed at least a 10-fold increase in cell number over the same period. Moreover, cells expressing ectopic Runx2 displayed a senescent-like phenotype characterized by an enlarged flattened appearance and SA-β-gal staining. These results clearly show that ectopic Runx2 can induce a profound growth arrest in cultured cells and implicate Runx2 as a direct mediator of this response. Parallel experiments with Runx3 gave essentially identical results (Fig. 1B). However, this phenomenon differed from premature senescence induced by H-RasV12 in primary MEFs, which produced an early proliferative response followed by a sustained growth arrest with retention of fibroblastic and spindle shapes in a proportion of the culture (Fig. 1C). Although H-RasV12–induced senescence seems to be activated in response to aberrant cellular proliferation (ref. 22 and references therein), Runx-induced senescence is a more direct process reflecting negative growth regulation.


Figure 1
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  		Figure 1. Runx2 and Runx3 induce a senescence-like growth arrest in wild-type (WT) MEFs that is distinct from H-RasV12. A, cells were transduced with a vector containing Runx2 or a control vector containing the PURO-selectable gene. Growth curves showing viable cell numbers of Runx2 (dashed line) and vector control (solid line) cells are shown. Similar results were obtained in two independent experiments. SA-β-gal staining of matched day 8 cultures are displayed. Photographs are at the same magnification. B, parallel growth curves and SA-β-gal staining in wild-type MEFs transduced with a vector containing Runx3 (dashed line) and (C) mutant H-RasV12 (dashed line).

 
Runx2–/– fibroblasts are prone to spontaneous immortalization and display an early growth advantage in vitro. Analysis of Runx gene family expression in primary MEFs by Western blot and gene expression microarray revealed that Runx2 is the most abundantly expressed family member, and this observation led us to consider the possibility that this gene has a nonredundant role in their growth regulation. Under typical culture conditions, primary MEFs exit the cell cycle in increasing numbers during passage, resulting in eventual growth stasis (19). The use of a 3T3 passage protocol can occasionally permit the accumulation of secondary events that serve to immortalize primary cells in vitro. Using this approach, we observed cessation of growth in six out of six cultures of wild-type MEFs (Fig. 2A ). In contrast, five of six lines derived from Runx2–/– embryos exhibited an enhanced growth rate around passages 8 to 10, indicating the emergence of clones with an increased proportion of proliferating cells (Fig. 2A). Further study revealed that these cells were immortalized and, unlike their wild-type counterparts, could grow beyond passage 25 with little or no sign of reduced proliferation. However, these cells remained nontumorigenic in immunocompromised mice.


Figure 2
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  		Figure 2. Runx2–/– fibroblasts are prone to secondary events that lead to immortalization. A, 3T3 passage culture was performed on five wild-type and six Runx2–/– lines. Cells were plated at 3.0 x 105/T25 flask and passaged every 3rd day, reseeding 3.0 x 105 cells into a fresh flask. The increase in total viable cell numbers was calculated at every passage and added to a cumulative count over time. Secondary events that predispose to immortalization occurred between passage 8 and 10. B, Runx2–/– fibroblasts display an early growth advantage in vitro. Growth curve of two Runx2–/– lines compared with two wild-type lines seeded at passage 4. The experiment was repeated on six littermate-matched Runx2–/– and wild-type lines with essentially identical results. C, the percentage of BrdUrd incorporation in three Runx2–/– and three wild-type lines seeded at passage 5 and labeled for 3 h. A comparison is shown for NIH3T3 cells labeled for 1 h (hatched column).

 
To further investigate the nature of their growth deregulation, we examined the properties of early passage Runx2–/– MEFs. As shown in Fig. 2B, Runx2–/– cells expanded significantly faster than litter-matched controls over 7 days in culture. Flow cytometry analysis of BrdUrd accumulation indicated that the increase in total cell count was due, at least in part, to increased cell division in the Runx2–/– cultures (Fig. 2C). However, we noted that the overall rates of proliferation of early MEF cultures were very low compared with established 3T3 fibroblasts (Fig. 2C), with only 10% of control cells taking up BrdUrd over a 3-h period.

Primary rodent fibroblasts display a progressive loss in proliferative capacity in culture that has been attributed to culture stress and may account for the low proliferative rates of primary MEFs observed in this study (23, 24). To determine whether the growth advantage of Runx2–/– fibroblasts was a consequence of reduced sensitivity to "culture shock," cells of both genetic backgrounds were plated at passages 3, 5, and 7, and their growth was compared over 7 days in culture. As shown in Fig. 3A , Runx2–/– fibroblasts retain a greater proliferative capacity than their wild-type counterparts with successive passages but have not lost their sensitivity to replication-induced growth inhibition. In contrast, differential susceptibility was seen in the response to low-dose UVC, where Runx2–/– cells showed increased viability relative to their wild-type counterparts (Fig. 3B). In view of evidence that the Runx genes play important roles in negative growth regulation by TGF-β in a range of cell types (9, 25, 26), we also tested the effects of Runx2 disruption on this response. Surprisingly, we saw no impairment in TGF-β growth arrest over a range of concentrations (Fig. 3C).


Figure 3
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  		Figure 3. Loss of Runx2 conveys a growth advantage that is resistant to certain cellular stresses but not to TGF-β–mediated growth inhibition. A, total viable cell counts at day 7 from six wild-type and six Runx2–/– lines plated in triplicate at passages 3, 5, and 7. B, Runx2–/– fibroblasts are more resistant to UVC-induced cell death than wild-type controls. Two Runx2–/– and two wild-type cultures were plated in triplicate in 60-mm dishes and irradiated with 5, 10, and 20 J/m2 UVC irradiation. Live/dead cell counts were performed by trypan blue exclusion after 24 h in culture. Similar results were obtained in two independent experiments. C, the growth of Runx2–/– and wild-type fibroblasts was inhibited by TGF-β. 5 ng/mL, 0.5 ng/mL, or 0.05 ng/mL TGF-β was added to triplicate wells on days 1, 3, 5, and 6, and total viable cell counts were calculated on day 7 by trypan blue exclusion. Control cultures were fed fresh medium in parallel. The histogram is a representative of two independent experiments. Replicate experiments on three independent Runx2–/– and wild-type lines performed with 5 ng/mL TGF-β gave essentially identical results.

 
Loss of Runx2 permits escape from H-RasV12–induced senescence and oncogenic transformation. Because the Runx genes are potent agonists of premature senescence and Runx2 disruption confers differential sensitivity to certain stress signaling pathways in primary MEFs, we investigated the effect of Runx2 loss on the well-characterized phenomenon of Ras oncogene–induced senescence. As shown in Fig. 4A , expression of H-RasV12 in the wild-type cell background induced growth stasis after an initial proliferative burst that was comparable with the vector-transduced controls. In contrast, Runx2–/– lines expressing H-RasV12 displayed a 24-fold expansion in cell number over the same period and, unlike wild-type cells, were negative for SA-β-gal staining (Fig. 4A). The contrasting effects were not due to differential levels of H-RasV12 expression as wild-type and Runx2–/– lines expressed comparable levels of H-RasV12 by Western blotting. Also, Runx2–/– cells were not intrinsically resistant to growth stasis as reintroduction of Runx2 induced a senescent-like growth arrest and β-galactosidase staining in a manner indistinguishable from wild-type controls (Fig. 4B).


Figure 4
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  		Figure 4. Runx2–/– cells expressing H-RasV12 fail to undergo senescence. A, Runx2–/– and wild-type cells were transduced with a vector encoding H-RasV12 or a control vector containing only the PURO-selectable gene. Passage 4 growth curves over 14 d, showing viable cell numbers. Similar results were obtained in four independent experiments using littermate-matched Runx2–/– and wild-type lines. B, SA-β-gal activity of Runx2–/– cells transduced with a vector encoding murine Runx2. Transduction with the control vector containing only the PURO-selectable gene failed to generate positively stained cultures. Photographs are at the same magnification. C, colony formation of Runx2–/– fibroblasts retrovirally transduced with H-RasV12. Cells were transduced with a retrovirus encoding H-RasV12 or a control vector containing only the PURO-selectable gene and analyzed for oncogenic transformation. Numbers were determined for agar colonies (>0.1 mm) derived from plating 103 cells per 60-mm dish and fed every 3 to 4 d for 5 wk. Error bars, the average number of colonies derived from three independent dishes.

 
To determine whether Runx2 loss and H-RasV12 can collaborate in the oncogenic transformation of MEFs, H-RasV12 expressing Runx2–/– and wild-type cells were seeded in soft agar. As shown in Fig. 4C, anchorage-independent growth was significantly enhanced in H-RasV12/Runx2–/– cells compared with H-RasV12/ wild-type cells. The enhanced growth potential of H-RasV12/Runx2–/– cells was also seen in colony formation assays where these cells showed a much higher frequency of colony growth after low-density plating (data not shown). To determine whether loss of Runx2 renders primary MEFs susceptible to tumorigenesis in vivo, H-RasV12/Runx2–/–, H-RasV12/wild-type, and vector control cells were transplanted into athymic mice. While no tumor growth occurred with cells carrying the empty vector (control cells, 0 of 6; Runx2–/– cells, 0 of 6) or wild-type cells expressing H-RasV12 (0 of 6), those cells null for Runx2 and expressing H-RasV12 invariably produced tumors (6 of 6). Although most tumors appeared within 2 to 4 weeks, it was notable that these generally remained small and dormant before a later phase of expansion and vigorous growth. Examination of the rapidly growing tumors revealed a heterogeneous pattern with loss of expression of p16, p21, and/or p53 (data not shown). Although Runx2 loss is clearly sufficient to allow Ras-induced tumors to arise, it seems that the transformed cells are not immortalized and that additional events are necessary for tumor progression.

Loss of Runx2 does not block induction of cell cycle inhibitors but nevertheless disables cell cycle checkpoint controls in response to H-RasV12. H-RasV12–induced premature senescence is accompanied by activation of p38 MAPK (27, 28) and the induction of p53, p19ARF, p16INK4a, and p21WAF1 (16). To determine whether these responses are intact in Runx2–/– MEFs, we examined the levels of activated p38 MAPK using an antibody that specifically recognizes phospho Thr180 and Tyr182 p38 MAPK, previously shown to represent the active kinase (29). As shown in Fig. 5A , phospho-p38 MAPK was readily detectable in wild-type MEFs. The levels exceeded that of our positive control but could not be further induced by H-RasV12, indicating high endogenous activation of this stress signaling cascade. Activation of p38 MAPK was similarly high in Runx2–/– fibroblasts but, in contrast to wild-type controls, could be further activated by H-RasV12 (Fig. 5A). In vitro kinase assays using Activating Transcription Factor 2 as a substrate confirmed it to be fully functional (data not shown), indicating that signaling between H-RasV12 and p38 MAPK remained intact in Runx2–/– MEFs. An examination of the expression levels of p53, p19ARF, p16INK4a, and p21WAF1 revealed that despite lower basal levels in the Runx2–/– genetic background, H-RasV12 induced p53, p19ARF, p16INK4a, and p21WAF1 to a comparable level in both Runx2–/– and wild-type control cells (Fig. 5B). Therefore, although Runx2 is essential for H-RasV12 to exert its growth suppressing function, it does not seem to be required for the induction of a panel of genes that have been shown to contribute to the growth arrest.


Figure 5
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  		Figure 5. Loss of Runx2 does not block the induction of growth arrest pathways by Ras but facilitates transit through S-G2-M. A, total protein was extracted from early passage (p4) littermate-matched Runx2–/– and wild-type lines transduced with H-RasV12 (R) or the PURO (P) vector control and probed against antibodies to phospho-p38 MAPK or p38 MAPK as a loading control. The Western blot is representative of two independent experiments. B, Western blot analysis as in A probed against antibodies to p16INK4a, p19ARF, p21WAF1, and p53. Actin was used as a loading control. C, cell cycle analysis of H-RasV12–transduced wild-type and Runx2–/– MEFs is shown as the percentage of propidium iodide stained cells with 2N (top) and 4N (bottom) DNA content at day 13 after transduction. Two independent littermate-matched wild-type and Runx2–/– cultures are grouped (1 and 2). Error bars, triplicate samples. Similar results from two independent experiments. D, cytometry density plots of Runx2–/– (top) and wild-type (bottom) cells transduced with H-RasV12 that were labeled with BrdUrd for 15 h. Percentage of BrdUrd incorporation is given based on triplicate samples. Identical results were seen with two independent wild-type and Runx2–/– samples.

 
In contrast, cell cycle analysis confirmed and elucidated a profound difference in the growth response of the Runx2–/– cells to H-RasV12. Propidium iodide staining revealed a higher proportion of cells with a 2N (G1) DNA content and a significantly lower proportion of cells with a 4N (G2-M) DNA content in Runx2–/– cells compared with controls (Fig. 5C). Dual staining with propidium iodide and BrdUrd showed higher rates of BrdUrd incorporation in Runx2–/– H-RasV12 cultures, consistent with their observed expansion in cell numbers during the culture period (Fig. 5D). Of these cells, a significant proportion displayed a 2N DNA content, indicating successful transit through G2-M. A striking difference was seen in the wild-type cultures that displayed a much lower number of BrdUrd positive cells with a 2N DNA content. Together these results show that the H-RasV12 expressing Runx2–/– cells are actively cycling, while a significant proportion of wild-type cells are delayed or arrested during transit through S-G2-M.

Loss of Runx2 confers increased expression of S-G2-M cyclins and associated cyclin-dependent kinase activities. The continued cycling of Runx2–/– cells in the presence of a cascade of growth inhibitors suggested a compensatory mechanism possibly involving constitutive expression of positive growth mediators. We therefore compared the levels of cyclin gene expression in the wild-type and Runx2–/– genetic backgrounds. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that cyclin D1 expression was unaffected by Runx2 status, although it was up-regulated by H-RasV12 as previously reported (Fig. 6A ). However, the expression levels of cyclins A2 (Fig. 6B), B1 (Fig. 6C), and E1 (Fig. 6D) mRNA were all markedly up-regulated in Runx2–/– fibroblasts compared with wild-type controls (Fig. 6A). Moreover, unlike wild-type cells where expression of these cyclins declined steadily throughout the culture period, high levels of cyclin E1, cyclin A2, and cyclin B1 mRNA were sustained in Runx2–/– MEFs and persisted even as the cells reached confluence. Strikingly, deregulation of cyclin levels was observed in Runx2–/– cells whether or not they expressed H-RasV12.


Figure 6
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  		Figure 6. Loss of Runx2 confers increased expression of S-G2-M cyclins and associated cyclin-dependent kinase activities. A, cyclin D1 expression is refractory to loss of Runx2 but remains sensitive to induction by H-RasV12. cDNA was prepared from littermate-matched Runx2–/– and wild-type cultures transduced with H-RasV12 or the PURO control vector and plated for 0 to 13 d in culture. Cyclin D1 expression was assayed at each time point by relative quantification to endogenous control hprt and calibrated to the day 0 wild-type sample. B to D, loss of Runx2 confers increased expression of cyclin A2 (B), cyclin B1 (C), and cyclin E1 (D) and sustains downstream cyclin dependent kinase activity. cDNA was analyzed from the same data set as described in A. The data are displayed as raw RQ values and is representative of two independent experiments on two littermate-matched Runx2–/– and wild-type lines. Parallel cultures were harvested for immunoprecipitation kinase assays of total cyclin A–, cyclin B1–, and cyclin E–associated kinase activities. Cell extracts were immunoprecipitated with antibodies specific for the relevant cyclin, and the precipitates were analyzed for kinase activity using histone H1 (32 kDa) as a substrate. *, 25 and 37 kDa molecular weight markers. Data are representative of two independent experiments.

 
To determine whether cyclin-dependent kinase activity was also deregulated in the absence of Runx2, in vitro kinase assays were performed using histone H1 as a substrate. As shown in the parallel panels, elevated levels of A-, B1-, and E-dependent kinase activities were observed in Runx2–/– cells relative to wild-type controls after coimmunoprecipitation with the corresponding antibody. Comparable results were also obtained upon retroviral transduction with H-RasV12. A direct analysis of the kinase complex composition was problematic due to comigration of cyclin proteins with the IgG heavy chain moiety, but examination of cyclin protein expression by Western blotting confirmed the qRT-PCR data, thereby supporting a direct relationship between cyclin gene expression levels and S-G2-M cyclin-dependent kinase activities in Runx2–/– cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown that Runx2 can induce premature senescence in primary MEFs, revealing a further functional attribute shared by the Runx gene family. This process is markedly different from Ras-induced senescence that occurs in response to aberrant proliferation and DNA damage signaling (ref. 22 and references therein), and it seems that Runx signaling induces a senescence cascade downstream of these responses. However, the process remains dependent on p53, and our finding that p53 is required for Runx2-induced senescence recapitulates previous results for RUNX1 (11). More surprising is our finding that Runx2 has a nonredundant role in MEFs where gene disruption promotes immortalization, disables Ras-induced senescence, and facilitates oncogenic transformation. These findings offer a new perspective on the lineage-specific tumor suppressor activities of the Runx gene family.

Runx2–/– MEFs display enhanced proliferation, but it is important to note that this phenomenon was observed on a background of primary cultures that display low proliferative rates compared with established cell lines. The early growth advantage was refractory to the nonphysiologic conditions of in vitro culture (23, 24) but was amplified by low-dose UVC irradiation, suggesting that loss of Runx2 alters sensitivity to a subset of stress-signaling pathways. The early growth advantage of Runx2–/– cells may be sufficient to account for their increased susceptibility to become immortalized at much higher rates than wild-type cultures, although a more direct role in regulating cellular life span or replicative senescence in response to telomere shortening cannot be formally excluded. However, it is clear that immortalized Runx2–/– cells remain nontumorigenic, unlike their p53–/– counterparts, suggesting that this tumor suppressor–like property of Runx2 is likely to be less potently manifested in vivo. Another interesting contrast is that Runx2–/– cells retain full sensitivity to the growth inhibitory effects of TGF-β, whereas p53–/– cells are refractory (30). Whether Runx function is dispensable for TGF-β inhibition in primary MEFs or this process is mediated by low-level expression of another family member remains to be determined.

Escape from senescence in response to exogenous H-RasV12 is the most striking attribute of Runx2–/– cells. We have established that this phenotype is not due to an intrinsic loss of capacity to undergo senescence as Runx2–/– lines remain susceptible to senescence induced by ectopic expression of Runx2. However, attempts to identify the basis of their resistance to H-RasV12 revealed some unanticipated results. In wild-type MEFs, H-RasV12–induced senescence is accompanied by activation of the p38 MAPK signaling cascade (28) and the induction of p16INK4a, p19ARF, p21WAF1, and p53 (16). We found no obvious defect in these responses in Runx2–/– MEFs that express high levels of the active p38 MAPK and accumulate all four proteins to similar levels in response to H-RasV12. In fact, there was evidence of hyperactivation of p38 MAPK in Runx2–/– cells, suggestive of the loss of a feedback control mechanism. These findings were somewhat surprising as at least two of the genes involved have been implicated as direct targets for Runx regulation (12, 31, 32), and Runx2–/– MEFs expressed slightly lower basal levels of all of these markers. It seems that Runx2 is dispensable for the induction of this cascade of inhibitory signals by H-RasV12, and that the explanation for the continued proliferation of Runx2–/– cells must lie elsewhere downstream.

Analysis of other components of the cell cycle machinery provided us with a clear rationale for the aberrant growth properties of Runx2–/– cultures, as these cells were found to express high levels of cyclin A2, B1, and E1 that were refractory to down-regulation by Ras. Moreover, the corresponding cyclin-associated cyclin-dependent kinase activities were markedly elevated on the Runx2–/– background. Although earlier studies of H-RasV12–induced senescence have described a G1 arrest as the predominant feature (16, 33), cell cycle analysis of our MEF cultures revealed stronger evidence of a block at G2-M in wild-type cells, with accumulation of cells with a 4N DNA content, whereas Runx2–/– cells continued to cycle. Our observations do not exclude the existence of multiple blocks to cell cycle progression in H-RasV12–transduced cells but are in accord with recent studies showing that oncogene-induced senescence and associated DNA damage can result in activation of S-G2-M checkpoints (34, 35).

A precedent for the ability of deregulated cyclin expression to overcome the Ras-induced inhibitory cascade has been provided by two genes discovered in screens for mediators of escape from Ras-induced senescence. Thus, hDRIL binds E2F1 and activates the cyclin E1 promoter (36), whereas repression of BTG2 leads to up-regulation of cyclins D1 and E1 (37). Similarly, it now seems that the constitutive expression and/or lack of silencing of several cyclin genes underlie the resistance of Runx2–/– cells to culture stress and Ras-induced growth arrest. It is interesting to note that cyclin A2 is among the E2F-regulated genes that are sequestered in senescence-associated heterochromatic foci (38, 39), which appear in the course of a multistep process, leading to irreversible loss of replicative capacity in human fibroblasts. The possibility that Runx functions are required in this critical program merits further investigation, particularly in light of evidence implicating Runx2 in SWI/SNF chromatin remodeling in osteoblast differentiation (40). It is conceivable that other defects in Runx2 null cells also contribute to their resistance to Ras-induced senescence. A recent study in human fibroblasts has shown that Ras-induced senescence requires attenuation of MAPK and PI3K signaling in addition to induction of cell cycle inhibitors (41), and it is intriguing to note that we observed elevated p38 MAPK signaling in Runx2 null cells in the presence of Ras.

This study also has wider implications for the role of the Runx genes in cancer. Because all three genes induce premature senescence when expressed ectopically, the prospect of a similar redundant role for the endogenous Runx genes in the induction of the senescence program in response to oncogene-induced growth arrest merits further investigation. It may be expected that manifestations of loss of Runx expression or function will be limited to those tissues where a single family member plays a decisive role, as we have found for Runx2 in MEFs. Whether Runx2 loss or silencing is associated with specific cancers in vivo is an interesting question. As this gene plays a critical role in vivo in the development of chondrogenic and osteogenic tissues, its tumor protective action might be manifested in mesenchymal tissues. With regard to other family members, it is notable that RUNX1 loss of function mutations have been associated with activated Ras signaling in AML and myelodysplastic syndrome/AML (42, 43). In addition, epithelial cancers that have been associated with RUNX3 down-regulation (ref. 2 and references therein) display Ras pathway mutations at significant frequencies, suggesting the possibility of parallel roles for the other Runx family members. It will also be interesting to explore the Runx2-dependent growth arrest mechanism uncovered here for its relevance to the normal physiologic roles of the Runx genes in cell fate determination, particularly where these entail checks to the proliferation of stem cells or early progenitors to permit lineage-specific differentiation (44).


   Acknowledgments
 
Grant support: Major support for this work was provided by a joint programme grant from Cancer Research UK and the Leukaemia Research Fund, while some aspects were supported by a project grant from the Association for International Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Ming Hu for preliminary observations, Sharon Mackay and Nancy Mackay for their excellent technical assistance, and Professor David Gillespie for reagents and helpful discussions.

Received 8/ 7/07. Revised 9/19/07. Accepted 9/26/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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