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Decreased Mdm2 expression inhibits tumor development induced by loss of ARF

Abstract

The tumor suppressor p14/p19ARF regulates Mdm2, which is known for controlling the p53 tumor suppressor. Here we report that loss of one allele of Mdm2 in cells that lack ARF resulted in a decreased rate of proliferation, fewer chromosomal aberrations, and suppression of Ras-induced transformation. Moreover, a haploinsufficiency of Mdm2 inhibited spontaneous tumor development in ARF-null mice. Remarkably, Mdm2+/−ARF−/− mice survived an average of 6 months longer than Mdm2+/+ARF−/− mice. The spectrum of tumors that arose in Mdm2+/−ARF−/− mice did not significantly differ from those that developed in mice lacking only ARF. However, the extended tumor latency allowed for the emergence of multiple primary tumors in a third of the Mdm2+/−ARF−/− mice, as compared to the single tumor type that arose in ARF-null only mice. Therefore, a decrease in Mdm2 levels restored regulation of critical cellular processes that are altered during transformation and that occur in the absence of ARF. Our findings also indicate that Mdm2 can function independently from ARF and imply that targeting Mdm2 in tumors that lack ARF expression should be an effective therapeutic approach.

Introduction

The p14ARF (human)/p19ARF (mouse) tumor suppressor is the second most commonly inactivated gene behind p53 in human and murine malignancies (Kamb et al., 1994; Sherr, 2001). ARF expression is lost in tumors by biallelic deletion of the entire ARF/Ink4a locus or by methylation of a CpG island in the ARF promoter (reviewed in Lowe and Sherr, 2003). ARF was first recognized as a cell cycle checkpoint regulator (Quelle et al., 1995). Loss of ARF leads to unregulated cell growth and immortalization (Zindy et al., 1998; Randle et al., 2001), whereas overexpression of ARF results in cell cycle arrest (Quelle et al., 1995). ARF-null mice develop tumors and have an average survival of 10 months (Kamijo et al., 1997). A single tumor type (sarcoma, lymphoma, or to a lesser extent carcinoma) spontaneously develops in mice lacking ARF (Kamijo et al., 1997, 1999). Deletion of ARF cooperates with oncogenes to induce and accelerate transformation. For example, primary ARF−/− mouse embryo fibroblasts (MEFs) are transformed by the expression of a single oncogene, such as c-Myc or RasV12, in contrast to wild-type MEFs that require both oncogenes for transformation (Kamijo et al., 1997). Moreover, loss of ARF dramatically accelerates B-cell lymphoma development in mice overexpressing the c-Myc oncogene in B-cells (Eμ-myc transgenic mice) (Eischen et al., 1999). Gamma irradiation or exposure to carcinogens, such as DMBA, will also accelerate tumorigenesis in ARF−/− mice (Kamijo et al., 1999). Conversely, mice that overexpress the ARF locus are resistant to the development of multiple types of carcinogen-induced tumors (Matheu et al., 2004). Therefore, ARF is clearly a tumor suppressor that regulates important processes necessary to prevent transformation; however, a means to inhibit or prevent tumorigenesis induced by ARF inactivation has not been previously investigated.

ARF was originally shown to regulate the p53 tumor suppressor by controlling p53's negative regulator murine double minute 2 (Mdm2) (Kamijo et al., 1998; Pomerantz et al., 1998; Stott et al., 1998; Zhang et al., 1998; Honda and Yasuda, 1999). Mdm2 binds to and regulates p53 function. Mdm2 controls p53 transcriptional activity and is responsible for ubiquitinating p53, leading to its degradation (Honda et al., 1997; Freedman and Levine, 1998; Roth et al., 1998). When ARF is present, it binds to Mdm2 and blocks Mdm2 from inhibiting p53. Although Mdm2 can also be sequestered in the nucleolus by ARF (Kamijo et al., 1998; Pomerantz et al., 1998; Zhang et al., 1998), preventing Mdm2 from interacting with p53, this is not necessary for inhibition of Mdm2 (Llanos et al., 2001; Korgaonkar et al., 2002). In support of Mdm2's regulation of p53, deletion of Mdm2 in mice results in an increase in p53 activity and an early embryonic lethality, and loss of p53 rescues this lethality (Jones et al., 1995; Montes de Oca Luna et al., 1995; de Rozieres et al., 2000). In contrast to Mdm2−/− mice, Mdm2+/− mice are born at Mendelian ratios and appear to have a normal life span (Montes de Oca Luna et al., 1995). However, the decreased Mdm2 levels in Mdm2+/− mice result in a reduced ability of these mice to regulate p53 under stressful conditions, such as irradiation and oncogene overexpression. Specifically, Mdm2+/− mice and the cells therein have an increased sensitivity to gamma irradiation due to increased p53 activation (Eischen et al., 2004; O'Leary et al., 2004). Similar observations were made with mice with a hypomorphic Mdm2 allele, resulting in severely reduced levels of Mdm2 (Mendrysa et al., 2003). The sensitivity to gamma irradiation of the Mdm2 hypomorphic mice was rescued with loss of p53, but not deletion of ARF (O'Leary et al., 2004). Heterozygosity of Mdm2 has also been shown to be insufficient at regulating p53 induced by oncogenes. Mdm2+/−Eμ-myc transgenic mice that overexpress the c-Myc oncogene in B-cells had increased p53-dependent B-cell apoptosis, resulting in a significant inhibition of B-cell lymphoma development when compared to Eμ-myc transgenics with both alleles of Mdm2 (Alt et al., 2003). Loss of only one allele of ARF fully restored p53 regulation in Mdm2+/−Eμ-myc transgenics, but deletion of ARF was not tested (Eischen et al., 2004). Therefore, decreased levels of Mdm2 protein lower the threshold of p53 activation and increase sensitivity to stimuli that activate p53, and loss of ARF expression does not appear to have an effect on these p53-dependent Mdm2 functions in vivo, particularly in the absence of oncogene overexpression. Based on these observations, we postulated that since decreased levels of Mdm2 appear to have cellular effects independent of ARF regulation, there should be an Mdm2 haploinsufficiency effect on tumor development in ARF-null mice. The results presented here reveal that loss of one allele of Mdm2 inhibited proliferation, accumulation of chromosomal aberrations, transformation, and tumor development induced by deletion of ARF. This study demonstrates the importance of Mdm2's influence on critical cellular processes that are altered during transformation and that occur in the absence of ARF. These results also provide a means by which tumorigenesis initiated by loss of ARF expression can be inhibited.

Results

Mdm2 haploinsufficiency inhibits the proliferation of ARF-null cells

Cells lacking ARF are immortal and proliferate at a faster rate than cells containing ARF (Kamijo et al., 1997). Consequently, expression of oncogenic Ras or Myc alone in ARF−/− MEFs is transforming. To determine whether a decrease in Mdm2 expression would alter the unregulated proliferation of ARF−/− MEFs, ideally we would analyse MEFs that lacked both alleles of Mdm2 and ARF, but were wild-type for p53. However, deletion of Mdm2 results in lethality unless p53 is also absent (Jones et al., 1995; Montes de Oca Luna et al., 1995); therefore, generation of Mdm2/ARF-double null mice was not possible (data not shown). Instead, we measured the growth of early passage ARF−/− MEFs that lacked only one allele of Mdm2. Mdm2+/−ARF−/− MEFs (embryos 3–5 (EM3–5)) derived from three separate embryos had slower rates of growth as compared to the growth rates of MEFs lacking ARF alone (EM1 and EM2), which were derived from littermates of the Mdm2+/−ARF−/− embryos (Figure 1a). As Figure 1a illustrates, one of the Mdm2+/−ARF−/− MEFs (EM5) grew slightly faster than the rest of the Mdm2+/−ARF−/− MEFs. However, Mdm2+/+ARF−/− MEFs consistently grew faster than any of the Mdm2+/−ARF−/− MEFs. Similar results were obtained with other MEF isolates (data not shown).

Figure 1
figure1

ARF-null MEFs heterozygous for Mdm2 have a reduced rate of proliferation. (a) MEFs (EM1 (diamond), EM2 (square), EM3 (triangle), EM4 (cross), EM5 (circle)) were plated in triplicate on day 0. Viable cells were counted every 24 h for 5 days. (b) BrdU incorporation in the indicated MEFs was determined by flow cytometry following 5 (left) or 8 (right) hours incubation with BrdU. Each symbol or bar represents the mean of triplicate samples, and error bars are one standard deviation.

Differences in growth rates are usually due to differences in rates of proliferation and/or apoptosis. To distinguish these two possibilities, proliferation and apoptosis were measured in each of the MEFs. There was increased bromodeoxyuridine (BrdU) incorporation (a measurement of DNA synthesis) in the Mdm2+/+ARF−/− MEFs following a 5 or an 8 h incubation with BrdU, as compared to BrdU incorporation in Mdm2+/−ARF−/− MEFs over the same intervals (Figure 1b). In contrast, the low rates of spontaneous apoptotic cell death were similar among all of the MEFs regardless of genotype (data not shown). Thus, loss of one allele of Mdm2 slows the rate of proliferation of ARF−/− MEFs, but does not appear to alter the rate of spontaneous apoptosis of MEFs in culture. Since we have previously reported that lymphocytes from Mdm2+/− mice had increased p53 activity (Eischen et al., 2004), we postulated that Mdm2+/−ARF−/− MEFs grew more slowly due to this enhanced p53 activity. First, we evaluated the steady state levels of Mdm2 protein in the Mdm2+/−ARF−/− MEFs by Western blot. The protein expression of Mdm2 in Mdm2+/−ARF−/− MEFs was similar to that in ARF-null MEFs with both alleles of Mdm2 (Figure 2). This result is in contrast to Mdm2 expression in cells in vivo that clearly showed lower levels of Mdm2 protein in Mdm2 heterozygous mice (Eischen et al., 2004). We postulated that the elevated Mdm2 levels in the Mdm2+/−ARF−/− MEFs reflected an increase in p53 activity that resulted in the feedback upregulation of Mdm2 expression by p53 in cells in culture, as has been previously reported for MEFs (Mendrysa and Perry, 2000). Therefore, we evaluated p53 levels and activity in both genotypes of MEFs. The low basal levels of p53 protein were similar between the Mdm2+/+ARF−/− (EM1 and EM2) MEFs and the Mdm2+/−ARF−/− (EM4 and EM5) MEFs (Figure 2, lanes 1, 2, 7, and 8). However, the protein levels of the cyclin-dependent kinase inhibitor p21Waf1/Cip1, a transcriptional target of p53, were elevated in the Mdm2+/−ARF−/− MEFs (Figure 2, lanes 2 and 8 as compared to lanes 1 and 7), an indication there was increased basal p53 transcriptional activity in the MEFs lacking one allele of Mdm2. Since p21 functions to block cell cycle progression, elevated levels of p21 should hinder the proliferation of Mdm2+/−ARF−/− MEFs, and this may explain the reduced rates of growth of the Mdm2+/−ARF−/− MEFs.

Figure 2
figure2

Increased p53 activity in Mdm2+/−ARF−/− MEFs. Mdm2+/+ARF−/− (EM1 and EM2) and Mdm2+/−ARF−/− (EM4 and EM5) MEFs were unirradiated (0) or subjected to 10 Grays of gamma irradiation. Cells were harvested 1 and 3 h following irradiation. Whole-cell protein lysates of each were Western blotted with antibodies specific for p53, phosphorylated ser18 of p53, p21, Mdm2, and β-actin.

To more fully evaluate p53 regulation in Mdm2+/−ARF−/− MEFs, ARF-null MEFs with one or both alleles of Mdm2 were subjected to 10 Grays of gamma irradiation. Gamma irradiation leads to stabilization, phosphorylation, and transcriptional activation of p53, which induces the expression of multiple genes, the most notable of which is p21 (el-Deiry et al., 1993; Fiscella et al., 1993; Brugarolas et al., 1995; Deng et al., 1995; Mayr et al., 1995; Canman et al., 1998; Dumaz and Meek, 1999; Vousden, 2002). As expected, 1 h following irradiation, p53 protein levels were significantly increased in both genotypes of MEFs, but the amount of p53 protein was slightly higher in the Mdm2+/+ARF−/− MEFs (Figure 2, lanes 3 and 9 compared to lanes 4 and 10). In contrast, the amount of p53 that is phosphorylated on serine 18, an indicator of activated p53 (Fiscella et al., 1993; Mayr et al., 1995; Canman et al., 1998; Dumaz and Meek, 1999), appeared to be slightly higher in the Mdm2+/−ARF−/− MEFs 1 h postirradiation in comparison to p53 phospho-S18 levels in the Mdm2+/+ARF−/− MEFs. Consequently, p21 protein levels were higher in the Mdm2+/−ARF−/− MEFs (Figure 2, lanes 4 and 10) than in the Mdm2+/+ARF−/− MEFs (lanes 3 and 9). The relative level of p21 1 h after irradiation, as determined by calculating the ratio of p21 protein expression to β-actin protein expression, was 1–2 fold higher in the Mdm2+/−ARF−/− MEFs over the amount of p21 in the Mdm2+/+ARF−/− MEFs. These results indicate there is increased p53 transcriptional activity in the Mdm2+/−ARF−/− MEFs 1 h following irradiation. At 3 h after irradiation, the differences in p53 expression and activity between the Mdm2+/−ARF−/− and the Mdm2+/+ARF−/− MEFs were more evident. Protein levels of p53 in Mdm2+/−ARF−/− MEFs (Figure 2, lanes 6 and 12) were higher when compared to p53 protein expression in Mdm2+/+ARF−/− MEFs (lanes 5 and 11). Importantly, p53 phospho-S18 was also markedly elevated in the Mdm2+/−ARF−/− MEFs 3 h postirradiation, whereas little p53 phospho-S18 was detected in the Mdm2+/+ARF−/− MEFs at this time. Therefore, p53 was active for a longer period of time in the Mdm2+/−ARF−/− MEFs, which resulted in higher levels of p21 protein in the Mdm2+/−ARF−/− MEFs when compared to p21 levels in Mdm2+/+ARF−/− MEFs 3 h postirradiation (Figure 2, lanes 6 and 12 compared to lanes 5 and 11). The relative amount of p21 in the Mdm2+/−ARF−/− MEFs was 1.5- to 3-fold higher than p21 expression in the Mdm2+/+ARF−/− MEFs. The increased p53 stabilization and activity in the Mdm2+/−ARF−/− MEFs was reflected by decreased levels of Mdm2 protein in these MEFs 3 h following irradiation. Similar results were observed in other MEFs of both genotypes (data not shown). Therefore, loss of one allele of Mdm2 in ARF-null MEFs resulted in an increase in the basal levels of p53 activity and in the p53-dependent transcriptional response to gamma irradiation. These results are consistent with the observation that splenocytes from ARF-null mice with reduced levels of Mdm2 protein have higher p21 levels due to increased p53 transcriptional activity (O'Leary et al., 2004). In addition, as observed in other studies, p53 activity did not directly correlate with the total amount of p53 protein present, but instead with levels of Mdm2 (Mendrysa et al., 2003; Eischen et al., 2004) and the amount of activated (phosphorylated) p53 (Fiscella et al., 1993; Mayr et al., 1995; Dumaz and Meek, 1999).

Decreased Mdm2 levels block transformation of ARF−/− cells

To determine whether the reduction in proliferation and the increase in p53 activity of the Mdm2+/−ARF−/− MEFs would alter oncogenic transformation of MEFs lacking ARF, we performed colony assays following RasV12 infection of early passage MEFs. Rates of growth of the different genotypes of RasV12-infected MEFs were measured first to determine whether RasV12 expression would alter the proliferation of Mdm2+/−ARF−/− MEFs. Similar to the uninfected Mdm2+/−ARF−/− MEFs, Mdm2+/−ARF−/− MEFs infected with RasV12 grew more slowly than the RasV12-infected Mdm2+/+ARF−/− MEFs and had decreased BrdU incorporation (Figure 3a and data not shown). Western blots for Ras were also performed, and the different MEFs showed similar levels of RasV12 expression (Figure 3b). In vitro transformation assays showed a reduction in the number of colonies that arose in dishes containing RasV12-infected Mdm2+/−ARF−/− MEFs as compared to dishes with RasV12-infected Mdm2+/+ARF−/− MEFs (Figure 3c and d). RasV12-infected NIH3T3 cells were used as a positive control for these experiments and showed robust colony formation with half the number of cells as used for the MEFs plated. To quantify colony growth, plates were subjected to digital imaging analysis following crystal violet staining. There were more colonies on the plates that contained RasV12 Mdm2+/+ARF−/− MEFs resulting in a greater intensity per surface area analysed as compared to plates with RasV12 Mdm2+/−ARF−/− MEFs (Figure 3d). Therefore, loss of one allele of Mdm2 resulted in a suppression of Ras-induced proliferation and transformation in vitro.

Figure 3
figure3

Loss of one allele of Mdm2 confers resistance to Ras-induced transformation of ARF-null MEFs. (a) Equal numbers of RasV12 or vector control infected MEFs (EM1,2,3,4,5) were plated in triplicate. Viable cells were counted every 24 h for 5 days. Each symbol represents the mean of triplicate samples, and error bars are one standard deviation. (b) Whole-cell protein lysates of each RasV12-infected MEF and NIH3T3 cells were Western blotted for Ras and β-actin protein. (c) The indicated RasV12 or vector control infected MEFs or NIH3T3 cells were plated, allowed to grow for 4 days, fixed and colonies that grew were stained with crystal violet. Digital images of the dishes are shown. (d) Foci on plates were quantified by digital imaging analysis and graphed based on intensity per surface area. Bars represent the mean of triplicate samples, and error bars are one standard deviation. (e) Kaplan–Meier survival curves of athymic nude mice injected with RasV12 (right flank) and control (left flank) expressing MEFs (all solid lines) or NIH3T3 cells (dashed line): EM1 (five mice), EM2 (eight mice), EM3 (five mice), EM4 (five mice), EM5 (five mice), and NIH3T3 (four mice) cells. Mice were killed once tumors reached 1.5 cm in diameter or 40 days had passed. No tumors were observed in the mice killed on day 40.

Since in vitro transformation assays on plastic may not accurately reflect what occurs in vivo during transformation, we subcutaneously injected RasV12-expressing MEFs of both genotypes into athymic nude mice and monitored them for tumor development. For each MEF line, RasV12-infected MEFs were injected into one flank and vector control infected MEFs were injected into the other flank of the same mouse. RasV12-infected NIH3T3 cells were again used as controls for transformation and showed rapid tumor development in nude mice (Figure 3e). Survival curves illustrate the difference in tumor latency between the RasV12-infected Mdm2+/−ARF−/− and the Mdm2+/+ARF−/− MEFs (Figure 3e). Specifically, within 20 days, all 13 mice containing RasV12-expressing Mdm2+/+ARF−/− MEFs developed tumors and had to be killed, whereas none of the 15 mice injected with RasV12-expressing Mdm2+/−ARF−/− MEFs had developed a tumor by day 20 (Figure 3e). RasV12-expressing Mdm2+/−ARF−/− MEFs did eventually form tumors in a fifth of the mice (3 out of 15) by day 40 (one mouse with EM3 and two mice with EM4). Interestingly, the one MEF (EM5) that appeared to have increased proliferation and transforming abilities in vitro, as compared to the other Mdm2+/−ARF−/− MEFs, was the least able to form tumors in vivo. These data highlight the importance of performing in vivo experiments and demonstrate that decreased Mdm2 expression blocks Ras-induced transformation of ARF-null cells in vivo.

Mdm2 heterozygosity inhibits the acquisition of chromosomal aberrations in ARF-null MEFs

Maintenance of chromosomal stability is critical to prevent the acquisition and accumulation of DNA aberrations that lead to transformation. Mdm2+/+ARF−/− MEFs were more easily transformed than Mdm2+/−ARF−/− MEFs, suggesting the possibility that there is a difference in chromosomal stability between the two genotypes of MEFs. To investigate this, metaphase spreads were prepared and analysed from each of the exponentially growing ARF-null MEFs that contained one or both alleles of Mdm2 and that had been passaged a similar number of times (10–14 passages). For each MEF line (EM1-5), over 300 metaphases were evaluated for chromosomal aberrations after being blinded. Notably, the percentage of metaphases from Mdm2+/+ARF−/− MEFs with chromosomal aberrations was more than double the percentage of Mdm2+/−ARF−/− MEFs with chromosomal abnormalities (Figure 4, Table 1). Specifically, an increased number of chromosome and chromatid breaks and gaps and chromosomal fusions were detected in the Mdm2+/+ARF−/− MEFs (Figure 4a, b and d). The percentage of metaphases with at least one break or gap was 2–6 times greater in Mdm2+/+ARF−/− MEFs than in Mdm2+/−ARF−/− MEFs (Table 1). Moreover, there was an increased frequency of metaphases with multiple breaks or gaps in Mdm2+/+ARF−/− MEFs, as compared to Mdm2+/−ARF−/− MEFs (Table 1, Figure 4d–g). Chromatid exchanges were rare for both genotypes, but the percentage was slightly increased in Mdm2+/+ARF−/− MEFs (Table 1, Figure 4c). Therefore, decreased Mdm2 expression inhibits the development of chromosomal aberrations that arise in ARF−/− MEFs in vitro in culture.

Figure 4
figure4

ARF-null MEFs with reduced levels of Mdm2 have increased chromosomal stability. Metaphase spreads were prepared from exponentially growing MEFs (EM1, 2, 3, 4, 5). Blinded samples were screened for chromosomal aberrations. (a) The percentage of metaphases of each MEF that contained breaks, gaps, fusions, and/or exchanges is illustrated. The number of metaphases evaluated for each MEF is indicated at the bottom of each bar. (bg) Photographs of representative examples of metaphases of each MEF that contained chromosomal aberrations are displayed. (b) Two centromere–centromere chromosomal fusions in EM1. (c) Radial-type chromosomes with multiple exchanges between nonhomologous chromosomes in EM2. (d) Two chromatid breaks and one chromosomal break in EM2. Arrow indicates one of the chromatid breaks. (e, f) Chromosomal breaks in EM3 (e) and EM4 (f). (g) Chromatid break in EM5. A magnified view of the specific aberrant chromosome(s) in each metaphase is (are) shown in the top left of each photograph.

Table 1 Genetic aberrations in Mdm2+/+ARF−/− and Mdm2+/−ARF−/− MEFs

Loss of one allele of Mdm2 inhibits tumor development and alters tumor numbers in ARF-null mice

To determine whether the decreased transforming ability and the reduced frequency of chromosomal alterations of Mdm2+/−ARF−/− MEFs reflects a reduction in the overall susceptibility of spontaneous tumor development in vivo, we generated ARF-null mice that contained either one or both alleles of Mdm2. Both genotypes of mice were monitored for tumor development. Consistent with our observations above, loss of one allele of Mdm2 significantly inhibited tumorigenesis and increased tumor latency in ARF-null mice (Figure 5). Mice that lacked ARF and were wild-type for Mdm2 had the same average survival (296 days or 9.7 months) that was previously reported for ARF-null mice (Kamijo et al., 1997). In contrast, the average survival of Mdm2+/−ARF−/− littermates was 492 days (or 16.1 months), 1.7 times longer than Mdm2+/+ARF−/− mice. In fact, only 25% (6/24) of the Mdm2+/+ARF−/− mice survived longer than a year, whereas 78% (18/23) of the Mdm2+/−ARF−/− mice lived beyond a year old (Figure 5). These striking results indicate that reduced levels of Mdm2 inhibit the tumor development initiated by the absence of ARF.

Figure 5
figure5

Mdm2+/−ARF−/− mice have a prolonged survival. Kaplan–Meier survival curves of ARF-null mice with one or both alleles of Mdm2. The genotypes of the mice are indicated next to each curve, and the numbers of mice in each group are denoted by the n values. The average life spans of Mdm2+/+ARF−/− and Mdm2+/−ARF−/− mice were 296 and 492 days, respectively. The three surviving mice are indicated by vertical lines. The significance in the difference in survival between the two genotypes of mice was determined by log-rank test (P<0.001).

Although tumor latency is delayed in Mdm2+/−ARF−/− mice, the types of tumors that developed in these mice were not grossly altered. Mice from each genotype were randomly chosen and tissue sections from each were blinded and subjected to an extensive pathological/histological analysis. There was a similar frequency of lymphomas, sarcomas, and carcinomas that emerged in both genotypes of mice (Table 2). Lymphoma and sarcoma were the most frequent tumor types observed, which is consistent with previous publications (Kamijo et al., 1997, 1999; Eischen et al., 2002; Moore et al., 2003). The lymphomas identified in both genotypes were B-cell, T-cell, or of indeterminate lineage. The B-cell lymphomas stained positive for B220 (B-cell marker) (Figure 6g and h) and showed low or no staining for CD3 (T-cell marker), Ter119 (erythrocyte marker), and Mac2 (monocyte/macrophage marker). The T-cell lymphomas stained positive for CD3 (Figure 6c and d), and were low or negative for B220, Ter119 and Mac2. Lymphomas of indeterminate lineage showed an equal proportion of B- and T-cell markers (sometimes termed pleomorphic lymphomas) or neither marker. The latter is most likely due to a problem with fixation of the tissues, but could reflect a tumor of lymphocyte precursor origin.

Table 2 Tumor spectrum in Mdm2+/+ARF−/− and Mdm2+/−ARF−/− mice
Figure 6
figure6

A third of the Mdm2+/−ARF−/− mice developed two primary malignancies. The histology of tumors that arose in two Mdm2+/−ARF−/− mice is shown. (ad) A mouse that had both a sarcoma and a T-cell lymphoma. (a and b) Low and high power H&E stained sections of a subcutaneous sarcoma with smooth muscle differentiation that expressed SMA (c) H&E stained section of a T-cell lymphoma that stained positively for CD3 (d). (eh) A mouse that had both an adenocarcinoma and a B-cell lymphoma. (e and f) Low and high power H&E stained sections of an adenocarcinoma in the lung. (g) H&E stained section of a B-cell lymphoma from a lymph node that stained positively for B220 (h). The magnification of each picture is indicated.

Sarcomas frequently arise in both old and young ARF-null mice (Kamijo et al., 1997, 1999), and we observed similar results in ARF−/− mice regardless of their Mdm2 status. The sarcomas that emerged in Mdm2+/+ARF−/− mice were primarily (5 of 6) of neural origin due to expression of S-100 and/or neurofilaments (NF). No smooth-muscle derived sarcomas were identified in Mdm2+/+ARF−/− mice. One of the sarcomas in the Mdm2+/−ARF−/− mice was of neural origin, but the majority (3 of 5) were of smooth-muscle origin. The smooth-muscle sarcomas had a typical spindle cell appearance and often grouped in intersecting bundles (Figure 6a and b). Sarcomas of smooth-muscle origin stained positive for smooth muscle actin (SMA) and/or muscle-specific actin (MSA). The cellular origin of two sarcomas arising in Mdm2+/+ARF−/− and Mdm2+/−ARF−/− mice (one each) were of indeterminate cell origin; a mixed expression pattern or a lack of a predominate marker was observed in these two sarcomas.

The carcinomas that developed in both genotypes of mice were all adenocarcinomas, most likely of lung cell origin (Figure 6e and f). They were composed of columnar cells ranging from disorganized infiltrations to well-differentiated glandular structure. All carcinomas stained positive with AE1/3, a pan-keratin antibody. Two Mdm2+/−ARF−/− mice developed benign hemangiomas, whereas none of the Mdm2+/+ARF−/− mice in this study did. However, it has been previously reported that ARF-null mice do develop hemangiomas (Kamijo et al., 1999; Eischen et al., 2002), so this is likely not significant or specific for Mdm2+/−ARF−/− mice.

An unexpected finding was that a third of the Mdm2+/−ARF−/− mice developed two primary cancers. Five of the 15 Mdm2+/−ARF−/− mice analysed had a sarcoma or a carcinoma and also a lymphoma (Table 2). Tissue sections of one mouse with a sarcoma and a T-cell lymphoma and one mouse with a carcinoma and a B-cell lymphoma are shown (Figure 6). The majority of the tumor load in four of the five Mdm2+/−ARF−/− mice with two primary malignancies was from the solid organ cancers rather than the lymphomas. All but one of the mice that had two different malignancies were over a year old (135, 373, 397, 450, 526 days old), suggesting age may have played a role. Although ARF-null mice have not been previously reported to develop more than one primary cancer (Kamijo et al., 1999), only a small fraction live longer than a year old. The Mdm2+/+ARF−/− mice in this study that lived past a year also only developed one primary cancer. Another factor that was considered was gender. However, there were similar numbers of male and female mice analysed between the two genotypes (6 male/10 female Mdm2+/+ARF−/− versus 4 male/11 female Mdm2+/−ARF−/−). Moreover, three female and two male Mdm2+/−ARF−/− mice developed two primary malignancies, so gender differences are not a likely cause. Therefore, loss of one allele of Mdm2 may have led to the development of two primary cancers, but due to the small numbers of ARF-null mice surviving past a year, the possibility that age may be a contributing factor to the development of two primary malignancies observed in Mdm2+/−ARF−/− mice cannot be ruled out and is instead, likely.

Discussion

ARF is a potent tumor suppressor whose inactivation is a common occurrence in malignancies that arise in both humans and mice (Lowe and Sherr, 2003). Loss of ARF causes immortalization and unregulated cell growth, which can lead to the accumulation of genetic alterations that ultimately result in cellular transformation. Mechanisms to suppress transforming events brought about by ARF inactivation have not been previously investigated. Therefore, the data presented here provide important new insight into tumor development initiated by ARF loss by showing that decreased levels of Mdm2 can profoundly inhibit events leading to the transformation of cells lacking ARF, and consequently suppress tumorigenesis in ARF-null mice. Decreased Mdm2 expression resulted in a decrease in proliferation, an increase in p53 activity, a suppression of chromosomal alterations that lead to transformation, and a reduced ability of ARF−/− cells to be transformed in vitro and in vivo. Moreover, our results provide essential genetic evidence that in the presence or absence of oncogene overexpression, Mdm2 functions to regulate tumor development independent of ARF. These results open the door for new treatment regiments that target Mdm2 in tumors that lack ARF expression.

Tumorigenesis in ARF−/− mice has been reported to be in part independent of p53 (Weber et al., 2000; Moore et al., 2003). Similarly, Mdm2 can function in a p53-dependent and a p53-independent manner (reviewed in Ganguli and Wasylyk, 2003). Therefore, the Mdm2 haploinsufficiency effects on tumor development in ARF-null mice may also be mediated by both p53-dependent and p53-independent pathways. Evidence to support the former includes reports by us and others that show decreased Mdm2 expression results in a reduced ability of cells in mice to regulate p53 activity induced by oncogene overexpression or gamma irradiation, known activators of p53 (Alt et al., 2003; Mendrysa et al., 2003; Eischen et al., 2004; O'Leary et al., 2004). Specifically, loss of one allele of Mdm2 led to an increase in p53-dependent apoptosis of B-cells that overexpressed the c-Myc oncogene (Eμ-myc transgenic mice), resulting in the inhibition of Myc-induced B-cell lymphoma development (Alt et al., 2003). Mdm2+/− mice and mice expressing low levels of Mdm2 (Mdm2 hypomorphic mice) had increased sensitivity to whole body gamma irradiation, the effects of which were p53-dependent (Mendrysa et al., 2003; O'Leary et al., 2004). Splenocytes from Mdm2+/− and Mdm2 hypomorphic mice showed higher levels of p53 activity induced by gamma irradiation as compared to controls (Mendrysa et al., 2003; Eischen et al., 2004; O'Leary et al., 2004). Moreover, loss of ARF did not rescue the radiation sensitivity or the developmental defects of Mdm2 hypomorphic mice (Mendrysa et al., 2003). Consistent with these reports, we also observed greater p53 activity in the Mdm2+/−ARF−/− MEFs in comparison to Mdm2+/+ARF−/− MEFs following gamma irradiation. These results more firmly establish that both alleles of Mdm2 are required to regulate p53 under stressful conditions, such as gamma irradiation and possibly during tumor development in ARF-null mice, and that ARF is not required for this function of Mdm2.

During the development of a cancer, cells experience both internal and external stresses, such as disregulated growth signals and nutrient and oxygen limitations. Consequently, these stresses should activate p53 to suppress tumor development. Thus, it is interesting to speculate that in vivo, in cells expressing reduced levels of Mdm2 that are under stress, the inability to regulate p53 activity should result in reduced rates of cell growth due to increased p53-mediated cell cycle checkpoint activation and/or increased rates of apoptosis. We observed an increase in the basal levels of p53 activity in Mdm2+/−ARF−/− MEFs induced by the stress of tissue culture, which lead to higher p21 protein expression in Mdm2+/−ARF−/− MEFs when compared to p21 levels in Mdm2+/+ARF−/− MEFs. This observation provides an explanation for the decreased rates of proliferation detected for the Mdm2+/−ARF−/− MEFs. In vivo, the outcome of increased p53 activity is the prevention of transformation itself or possibly a delay in tumor cell expansion (Garcia-Cao et al., 2002; Tyner et al., 2002), resulting in a prolonged tumor latency, as we observed in the Mdm2+/−ARF−/− mice. Moreover, the development of two tumors of different cellular origin in a third of the Mdm2+/−ARF−/− mice can also be explained through this mechanism. Specifically, the delay in tumor cell development and/or expansion/proliferation should allow for the emergence of more than one independent tumor, since neither malignancy would emerge quickly. Therefore, the reduced ability of cells in Mdm2 heterozygous mice to regulate p53 may explain both the delayed tumorigenesis and the development of two different tumors in the Mdm2+/−ARF−/− mice.

The contribution of p53-dependent apoptosis to tumorigenesis in ARF-null mice was partially addressed when mice lacking Bax, a downstream mediator of p53-dependent apoptosis, were crossed to ARF-null mice. Deletion of Bax did not alter tumor latency but did change the tumor spectrum in ARF−/− mice (Eischen et al., 2002), indicating that apoptosis or at least Bax-dependent apoptosis does not appear to be a major determinant of spontaneous tumor development in ARF−/− mice. Similarly, we observed that decreased levels of Mdm2 in ARF-null MEFs resulted in reduced proliferation and not an increase in apoptosis, which suggests that regulation of cell cycle rather than apoptosis by p53 may be the critical factor in tumor development in Mdm2+/−ARF−/− mice. However, mice lacking the entire p16INK4a/ARF locus and p21 have been generated and showed no change in tumor latency, although these mice did have an altered tumor spectrum (Martin-Caballero et al., 2004). Therefore, the contribution p53-dependent cell cycle regulation has on tumorigenesis in ARF-null mice remains unclear.

Although the preponderance of the evidence points to a p53-dependent effect caused by decreased Mdm2 levels, a p53-independent component of Mdm2 functioning may also be playing a role in the inhibition of tumor development in Mdm2+/−ARF−/− mice. Consistent with this idea, Mdm2+/−p53−/− mice had an altered tumor spectrum and a slightly longer survival as compared to Mdm2+/+p53−/− mice (McDonnell et al., 1999). We observed that loss of one allele of Mdm2 in ARF-null mice profoundly extended survival, but did not appear to grossly alter the tumor spectrum. In another study, deletion of both alleles of Mdm2 altered the tumor spectrum of ARF−/−p53−/− mice but not the tumor latency (Weber et al., 2000). Moreover, half of the Mdm2−/−ARF−/−p53−/− mice and only a third of the ARF−/−p53−/− mice developed two primary tumors of different cellular origin, and only Mdm2−/−ARF−/−p53−/− mice developed three primary tumors. We observed two primary cancers in a third of the Mdm2+/−ARF−/− mice as compared to ARF-null only mice, which only developed one malignancy. Taken together these studies suggest that a deficiency in Mdm2 has both ARF and p53-independent consequences during transformation and that Mdm2 has functions independent of p53 during tumor development. It is likely that the interaction of Mdm2 with one or more of the other proteins to which it has been shown to bind (reviewed in Iwakuma and Lozano, 2003), such as the DNA repair protein Nbs1 we recently identified (Alt et al., 2005), is likely responsible for the p53-independent functions of Mdm2 that contribute to tumor development. Therefore, since a haploinsufficiency in Mdm2 may have both p53-dependent and p53-independent effects on tumorigenesis, further investigation is needed to define these pathways.

Materials and methods

Mice

Mdm2 heterozygous mice (Mdm2+/− C57Bl/6 × 129/Sv) (Montes de Oca Luna et al., 1995) and ARF-null mice (ARF−/− C57Bl/6 × 129/Sv) (Kamijo et al., 1997) were generously provided by Dr Guillermina Lozano (MD Anderson Cancer Center, Houston, TX, USA) and Drs Martine Roussel and Charles Sherr (St Jude Children's Research Hospital, Memphis, TN, USA), respectively. Originally, the Mdm2+/− mice were crossed to ARF−/− mice to generate F1's. The F1's were then crossed to ARF−/− mice to generate F2 Mdm2+/−ARF−/− and Mdm2+/+ARF−/− mice. The F2 offspring were then interbred for 4 years resulting in all the mice being 58–64% C57Bl/6 as determined by microsatellite analysis (Charles River Laboratories, Troy, NY, USA). All Mdm2+/−ARF−/− mice and their Mdm2+/+ARF−/− littermates were closely monitored, and at signs of disease, mice were killed, necropsied, and tissues were collected and formalin fixed for histological evaluation (see below). A Kaplan–Meier survival analysis was performed for the Mdm2+/−ARF−/− mice and their Mdm2+/+ARF−/− littermates. Only mice with tumors were included in these analyses. The statistical significance of the survival between the different genotypes of ARF−/− mice was determined by log-rank test.

Athymic nude (nu/nu) female mice (5 weeks old) were purchased from the National Cancer Institute. In total, 2 × 106 MEFs (see below) or NIH3T3 cells infected with retroviruses expressing RasV12 or empty vector control (see below) in 50 μl of PBS were injected subcutaneously into the right and left flanks, respectively, of the mice. Mice were carefully monitored for tumor development and killed when tumors reached 1.5 cm in diameter. All remaining mice were killed on day 40, which is twice the number of days the last mouse with Mdm2+/+ARF−/− MEFs was killed. None of the mice killed on day 40 contained tumors. All research with mice complied with federal, state, and institutional guidelines and was preapproved by the institutional IACUC committee.

Primary cell culture, retroviral infection, cell growth and apoptosis analysis

MEFs from E13.5–14.5 embryos from crosses with Mdm2+/−ARF−/− and Mdm2+/+ARF−/− mice were collected and grown in tissue culture as previously described (Zindy et al., 1998). Early passage (<6) MEFs (EM1, 2, 3, 4, and 5 from one pregnant female) were seeded at 3 × 104 cells/well in six-well plates in triplicate. Viable (Trypan blue vital dye negative) MEFs were counted daily for 5 days. The percentage of apoptotic MEFs (cells with less than 2N DNA content) were measured by flow cytometry, following propidium iodide or 7-amino-actinomycin D (7AAD) staining of DNA. RasV12 and empty pBabe retroviruses were produced as previously described (Zindy et al., 1998). MEFs were infected with the pBabe-RasV12 retrovirus or the control empty pBabe retrovirus and selected in puromycin containing media, as previously described (Zindy et al., 1998). Following 3–4 days of expansion, MEFs were seeded at 3 × 104 in six-well plates in triplicate. Viable (trypan blue vital dye negative) MEFs were counted daily for 5 days.

In vitro transformation assay

RasV12 or control infected MEFs (4 × 104) were placed in 35 mm cell culture dishes. NIH3T3 cells were used as controls and plated at half the density (2 × 104 cells). Following 4 days of culture, all cells were fixed with 95% ethanol and stained with crystal violet. Colony growth was quantified by a digital imaging reader (NucleoVision; Nucleotech Corp., SanMateo, CA, USA) that measures light intensity per surface area. The more foci present, the more crystal violet staining there is and the more light that is absorbed resulting in a higher value of intensity. Digital images of the dishes were also captured and presented in Figure 3.

Bromodeoxyuridine (BrdU) incorporation

Early passage MEFs (5 × 105) were grown in triplicate in six-well plates. Cells were pulsed with 1 mM BrdU for 5 or 8 h, fixed, and stained with FITC conjugated anti-BrdU antibody (BD pharmingen BrdU flow kit; San Diego, CA, USA). BrdU incorporation was measured by flow cytometry (FACSCalibur; BD Immunocytometry Systems, San Jose, CA, USA) and analysed by CellQuest software (San Jose, CA, USA).

Western blotting and gamma irradiation

RasV12 infected MEFs of both genotypes were lysed as previously described (Zindy et al., 1998). Equal amounts of protein from each MEF were separated on 10% SDS-acrylamide gels, transferred onto nitrocellulose, and Western blotted with anti-Ras (F235, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-actin (Sigma, St Louis, MO, USA). For the irradiation experiments, 1.5 × 106 MEFs were plated and then subjected to 10 Grays of gamma irradiation from a 137cesium source. Cells were harvested at different intervals postirradiation and equal amounts of protein were Western blotted for p53 (Ab7, Oncogene Research Products, Cambridge, MA, USA), p53-ser18P (Cell Signaling Technology, Danvers, MA, USA), p21 (F5, Santa Cruz Biotechnology), Mdm2 (SMP14, Santa Cruz Biotechnology), and β-actin (Sigma). Following Western blotting and detection by enhanced chemiluminescence, quantitation of p21 and β-actin protein expression in the MEFs was determined by digital analysis on the Kodak imaging station 440 (Perkin Elmer Life Sciences, Downers Grove, IL, USA) with Kodak 1D 3.5.5 software (Scientific Imaging Systems, New Haven, CT, USA). Unirradiated MEFs were also analysed as described here.

Chromosome stability analysis

Exponentially growing Mdm2+/−ARF−/− and Mdm2+/+ARF−/− MEFs at passage 10–14 were treated with colcemid (Gibco, Grand Island, NY, USA) for 4 h. Metaphase spreads were then prepared by standard protocols. Following staining with propidium iodide (PI) (Sigma, St Louis, MO, USA) and 4′,6′-diamidino-2-phenyl-indo-dihydrochloride (DAPI) (Sigma), slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). Slides were blinded and a minimum of 300 separate metaphases were analysed by fluorescence microscopy (Nikon, Melville, NY, USA) with MetaVue software (Molecular Devices Corp., Sunnyvale, CA, USA). Each metaphase was evaluated for chromosomal aberrations (breaks, gaps, fusions, and/or exchanges). Photographs were taken of representative abnormal metaphases.

Histological analyses

Tissues from mice were collected, fixed in 10% buffered formalin, and then embedded in paraffin. Tumors were classified on blinded, fixed tissue sections by standard H&E staining and immunohistochemistry techniques. Tumors where 75% or more of the tumor cells expressed the B-lymphocyte CD45R/B220 antigen were classified as a B-cell lymphoma. Tumors where 75% or more of the tumor cells expressed the T-lymphocyte CD3 antigen were classified as a T-cell lymphoma. Lymphomas where neither B- nor T-cell antigens were detectable, likely due to problems in fixation, or that expressed both B- and T-cell markers were classified as lymphomas of indeterminate lineage.

Antigen retrieval was performed with 10 mM citrate at 95–115°C for 10–60 min for CD3, CD45R/B220, Ter119/Ly-76, Mac2, and desmin or with 0.5% pepsin at 37°C for 20 min for lysozyme and MPO. Protease 2 pretreatment at 37°C for 8–16 min for S-100, NF, and cytokeratin was performed on the Ventana ES instrument (Tucson, AZ, USA). Endogenous peroxidase activity was inactivated with H2O2.

The following polyclonal antibodies were purchased from Dako (Carpinteria, CA, USA): lysozyme, CD3, S-100, and myeloperoxidase (MPO). The following monoclonal antibodies were purchased from Dako: smooth muscle actin (SMA, clone 1A4), muscle-specific actin (MSA, clone HHF35), desmin (clone D33), neurofilaments (NF, clone 2F11), and cytokeratin (clone AE1/AE3). CD45R/B220 (RA3-6B2) and Ter119/Ly-76 (Ter-119) monoclonal antibodies were purchased from BD Pharmingen (San Diego, CA, USA). The Mac2 (M3/38) monoclonal antibody was purchased from Accurate Antibodies (Westbury, NY, USA). Biotinylated secondary antibodies were obtained from Vector (Burlingame, CA, USA) or Ventana (Tucson, AZ, USA). Negative controls were prepared by omitting primary antibody and incubating tissue sections with the secondary antibody. Tissue sections were counter-stained with haematoxylin (Dako Corp.), dehydrated and coverslipped, following the streptavidin–biotin–peroxidase complex method for antigen visualization.

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Acknowledgements

We would like to thank Jane Kennedy, Silvia Plaza, and Gregg Cochran for expert technical assistance with the mice, genotyping, and immunohistochemisty, respectively, Dr Jane Meza for Kaplan–Meier analysis, Drs Hua Xiao and Timothy McKeithan for helpful discussion, Dr Guillermina Lozano for the Mdm2+/− breeder mice and Drs Martine Roussel and Charles Sherr for ARF−/− breeder mice. This work was supported by NCI grant CA098139, the Eppley Institute for Research in Cancer, and the Wanda Rizzo Memorial fund. TCG is a Mantle Cell Lymphoma grantee of the Lymphoma Research Foundation. CME is a Leukemia & Lymphoma Society Scholar.

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Correspondence to C M Eischen.

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Wang, P., Greiner, T., Lushnikova, T. et al. Decreased Mdm2 expression inhibits tumor development induced by loss of ARF. Oncogene 25, 3708–3718 (2006). https://doi.org/10.1038/sj.onc.1209411

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Keywords

  • Mdm2
  • ARF
  • p53
  • transformation
  • tumor

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