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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Oncogene. Author manuscript; available in PMC 2010 July 13.
Published in final edited form as:
PMCID: PMC2903442

p53-dependent senescence delays Eμ-myc-induced B-cell lymphomagenesis


The effect of p53-dependent cell-cycle arrest and senescence on Eμ-myc-induced B-cell lymphoma development remains controversial. To address this question, we crossed Eμ-myc mice with the p53515C mutant mouse, encoding the mutant p53R172P protein that retains the ability to activate the cell-cycle inhibitor and senescence activator p21. Importantly, this mutant lacks the ability to activate p53-dependent apoptotic genes. Hence, Eμ-myc mice that harbor two p53515C alleles are completely defective for p53-dependent apoptosis. Both Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice survive significantly longer than Eμ-myc::p53+/− mice, indicating the importance of the p53-dependent non-apoptotic pathways in B-cell lymphomagenesis. In addition, the p53515C allele is deleted in several Eμ-myc::p53515C/+ lymphomas, further emphasizing the functionality of p53R172P in tumor inhibition. Lymphomas from both Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice retain the ability to upregulate p21, resulting in cellular senescence. Senescence-associated β-galactosidase (SA β-gal) activity was observed in lymphomas from Eμ-myc::p53+/+, Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice but not in lymphomas isolated from Eμ-myc::p53+/− mice. Thus, in the absence of p53-dependent apoptosis, the ability of p53R172P to induce senescence leads to a significant delay in B-cell lymphoma development.

Keywords: p53, apoptosis, senescence, lymphoma, mouse model


The p53 gene encodes a transcription factor activated by oncogenic and proliferative signals and DNA damage that initiates one of four physiological pathways: apoptosis, cell-cycle arrest, DNA repair and senescence (Ko and Prives, 1996; Vogelstein et al., 2000). Because p53 regulates each of the aforementioned pathways, it sits at the nexus of a complex network of signals that regulate proliferation and thus cancer development. However, in vivo, the specific effect that each of these individual pathways have on tumor suppression is unknown.

Analysis of human tumors has led to the identification of a rare point mutation in p53 that results in an arginine-to-proline substitution at amino acid 175 (Ludwig et al., 1996; Rowan et al., 1996). The p53R175P protein retains the ability to induce cell-cycle arrest by transcriptional activation of p21, but is defective in initiating p53-dependent apoptotic programs (Ludwig et al., 1996; Rowan et al., 1996). This rare naturally occurring p53 allele encodes a separation-of-function mutant p53 protein that can be exploited experimentally to understand the effect of different p53 functions on tumor suppression in vivo. A knock-in mouse model, carrying a G-to-C substitution at position 515 (p53515C), mimics precisely the mutation found in humans by altering arginine to proline at the corresponding amino acid 172 in the mouse (Liu et al., 2004). Homozygous mutant p53515C/515C embryos and thymocytes lack p53-dependent apoptosis after ionizing radiation (Liu et al., 2004). Moreover, expression arrays of irradiated mouse embryonic fibroblasts (MEFs) indicate that p53515C/515C MEFs do not induce p53-dependent apoptotic targets (Barboza et al., 2006). p53515C/515C mice show a significant delay in tumor formation when compared with p53−/− mice, through p53R172P-dependent transactivation of the cell-cycle inhibitor p21 (Liu et al., 2004; Barboza et al., 2006). These studies indicate that both cell-cycle arrest and apoptotic programs contribute to suppression of spontaneous tumor development (Ludwig et al., 1996; Liu et al., 2004; Barboza et al., 2006).

As indicated above, p53R172P transactivates the cell-cycle inhibitor, p21 (Ludwig et al., 1996; Liu et al., 2004; Barboza et al., 2006). Upon activation, p21 disrupts cell-cycle progression by binding to and inhibiting both cyclin-dependent kinase–cyclin complexes (Gu et al., 1993; Harper et al., 1993; Serrano et al., 1993; Xiong et al., 1993; el-Deiry et al., 1993) and proliferating cell nuclear antigen (Waga et al., 1994). In addition, p21 is a potent activator of cell senescence (Noda et al., 1994; Kulju and Lehman, 1995; Tahara et al., 1995; Brown et al., 1997). Owing to the antiproliferative effects of p53 and p21, tumors should inactivate either of these genes to overcome the growth constraints imposed by them. In human tumors, p53 mutations are common but mutations in p21 are rare (Shiohara et al., 1994), suggesting that the selective pressure is predominantly directed toward ablating the pleiotropic effects of p53 rather than uniquely the p21-dependent cell-cycle arrest/ senescence activity. Not surprisingly, most mutations and/or deletions of the p53 allele encountered in human cancer result in loss of all p53 functions.

The Eμ-myc B-cell lymphoma model, in which c-myc is expressed under the control of the immunoglobulin heavy chain enhancer (Adams et al., 1985), has been used extensively to study alterations in the p53 pathway. Eμ-myc transgenic mice overexpress c-myc in B cells and succumb to B-cell lymphomas with a mean survival of 4–6 months. The overexpression of c-myc results in increased levels of the p19Arf tumor suppressor, which in turn, inhibits the function of murine double minute 2, an E3 ubiquitin ligase that degrades p53, thus leading to the stabilization of p53 (Sherr et al., 2005). This sequence of events places selective pressure on the cancer cell to mutate or inactivate the p53 pathway.

The importance of p53-dependent apoptosis in Eμ-myc B-cell lymphomas is well established (Eischen et al., 1999, 2001; Schmitt et al., 2002a). However, the effect of p53-dependent cell-cycle arrest or senescence remains controversial. The progression of lymphomas elicited by c-myc overexpression has been generally attributed to impedance of apoptosis (Schmitt et al., 2002a). On the other hand, other studies have shown that the increased lymphomagenesis observed in Eμ-myc:: p53+/− mice is the result of increased proliferation and not decreased apoptosis (Hsu et al., 1995). The role of proliferation in Eμ-myc lymphomas was further examined in Eμ-myc crosses with Rb heterozygous mice. Retinoblastoma protein binds E2F and inhibits cell-cycle proliferation (Bandara and La Thangue, 1991; Beenken et al., 1991). Eμ-myc::Rb+/− mice showed increased incidence of lymphomagenesis when compared with Eμ-myc mice, further supporting the importance of proliferation in Eμ-myc-induced B-cell lymphomas (Schmitt et al., 1999). In addition, p53-dependent senescence was shown to delay tumor progression in a reconstituted model upon cyclophosphamide treatment or telomere shortening in B-cell leukemia/lymphoma 2-expressing Eμ-myc lymphoma cells (Schmitt et al., 2002b; Feldser and Greider, 2007). It is likely that all three of these p53 activities, that is, apoptosis, senescence and cell-cycle arrest, affect lymphomagenesis to varying degrees.

To directly address the interplay between Eμ-mycand p53-dependent apoptosis and senescence/cell-cycle arrest functions in vivo, we crossed transgenic mice expressing Eμ-myc with mice carrying the p53515C allele. This has allowed us to definitively determine whether the p53515C allele behaves as a p53-null allele (provides no delay in lymphomas) or whether this hypomorphic allele does in fact affect B-cell lymphomagenesis (delays lymphomagenesis compared with the null allele). In this study we show that both Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice survive longer than Eμ-myc:: p53+/ because of establishment of a senescence program. The majority of lymphomas from Eμ-myc:: p53515C/515C and Eμ-myc::p53515C/+ stabilize p53R172P and retain the ability to transactivate p21. Lymphomas isolated from Eμ-myc::p53+/+ and Eμ-myc::p53515C/515C, as well as Eμ-myc::p53515C/+ mice have significantly higher levels of senescence-associated β-galactosidase (SA β-gal) activity than lymphomas from Eμ-myc:: p53+/− mice. These data show that the senescence function of p53 also contributes significantly in delaying B-cell lymphomagenesis in vivo.


The apoptosis-defective p53515C allele delays tumorigenesis

We have previously generated mice containing the p53515C allele that encodes a hypomorphic p53R172P protein that is able to transactivate p21, but not p53-dependent apoptotic genes (Liu et al., 2004; Barboza et al., 2006). p21 is a cell-cycle inhibitor and effector of senescence (Gu et al., 1993; Harper et al., 1993; Serrano et al., 1993; Xiong et al., 1993; el-Deiry et al., 1993; Noda et al., 1994). To determine whether the cellular senescence function of p53 influences the development of Eμ-myc-induced B-cell lymphomas, we crossed Eμ-myc transgenic mice with p53515C/+ mice to generate Eμ-myc::p53515C/+ mice. Eμ-myc:: p53515C/+ male and female mice were infertile because of the fact that they were already unhealthy at breeding age. Therefore, we generated Eμ-myc::p53515C/515C mice through embryo rederivation using 3-week-old Eμ-myc:: p53515C/+ females, in which follicular development was hormonally induced to control the timing of ovulation. Females were then mated with either p53515C/+ or p53515C/515C male mice. Oviducts were harvested from donor females the next morning and embryos at the one-cell stage were collected and implanted in pseudopregnant recipients. The resulting Eμ-myc::p53515C/515C and Eμ-myc:: p53515C/+ mice, similar to the Eμ-myc::p53+/+ and Eμ-myc::p53+/− mice, developed B-cell lymphomas throughout all lymphatic tissues and bone marrow, as determined by immunostaining using the B-cell marker, B-220 (data not shown). Importantly, Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice survived significantly longer than Eμ-myc::p53+/− mice (Figure 1). The median survival of Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice was 62 and 69 days, respectively, and both survived significantly longer than Eμ-myc::p53+/− (35 days, P<0.0001 and P<0.0001, respectively). There was no significant difference between the survival of the Eμ-myc::p53515C/+ and Eμ-myc::p53515C/515C mice (P=0.073). Although the p53515C allele rendered a significant delay in lymphomagenesis, this hypomorphic allele is not as efficacious a tumor suppressor as the wild-type p53 allele, given that Eμ-myc::p53+/+ mice survive significantly longer than Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice, with a median survival of 138 days (P<0.0001; Figure 1).

Figure 1
Eμ-myc mice harboring the p53515C allele have delayed lymphoma development when compared with Eμ-myc::p53+/− mice. Kaplan–Meier curves indicating the survival of Eμ-myc::p53515C/515C, Eμ-myc::p53515C/+, ...

Selection pressure on the p53515C allele in Eμ-myc-driven lymphomagenesis

To examine the molecular events taking place at the p53 locus during lymphomagenesis, we analyzed lymphomas from the Eμ-myc mice with different p53 alleles. Lymphomas that developed in Eμ-myc::p53+/+ mice retained a wild-type p53 allele >70% of the time and rarely underwent biallelic deletions of p53 loci (Eischen et al., 1999). All the lymphomas (100%) arising in Eμ-myc::p53+/+ (n=8) and Eμ-myc::p53515C/515C (n=6) mice that we examined retained a p53 allele. In contrast, all lymphomas from the Eμ-myc::p53+/− mice (n=3) lost the wild-type p53 allele as previously published (Figure 2a; Eischen et al., 1999; Schmitt et al., 1999). As with the lymphomas from the Eμ-myc::p53+/+ and Eμ-myc::p53515C/515C backgrounds, lymphomas isolated from the Eμ-myc::p53515C/+ mice retained at least one p53 allele (Figure 2a). We sequenced the PCR products to distinguish between p53 allele(s) retained in the Eμ-myc::p53515C/+ lymphomas. The p53 alleles are easily distinguishable by the fact that the wild-type p53 allele has a G-nucleotide at position 515, whereas the p53515C allele has a C-nucleotide at this position (Liu et al., 2004). In all, 39% (7/18) of Eμ-myc::p53515C/+ lymphomas lost the wild-type p53 allele, but 44% (8/18) remained heterozygous for both wild-type p53 and p53515C alleles. Moreover, 17% (3/18) of Eμ-myc:: p53515C/+ lymphomas lost the p53515C allele (Figure 2b), indicating the presence of selective pressure to disrupt the function of p53R172P. Sequence analysis of the full-length p53 transcripts revealed no additional mutations in either the wild-type p53 or p53515C allele in 13 Eμ-myc::p53515C/+ lymphomas examined, whereas two of eight (25%) control Eμ-myc::p53+/+ lymphomas had mutated p53 (data not shown), in agreement with previous reports (Eischen et al., 1999). Importantly, there was no statistical difference in the survival of Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice that developed lymphomas that lost the wild-type p53 allele when compared with those that lost the p53515C allele, or those that retained both alleles (Figure 2c), again emphasizing the protective function of the p53515C allele in delaying lymphomagenesis.

Figure 2
The p53515C allele undergoes similar selection pressure as the wild-type p53 allele in Eμ-myc-dependent lymphomas. (a) PCR analysis of the p53 locus using primers designed to amplify exons 5–7. The p53 pseudogene (Ψ) serves as ...

p53 is expressed in most Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ lymphomas

The Eμ-myc mice used in this study overexpress c-myc in B cells that result in wild-type p53 stabilization. As a result, a disruption of p53-dependent pathways occurs at high frequency in Eμ-myc-driven lymphomas (Eischen et al., 1999, 2001; Schmitt et al., 1999). Analysis of control Eμ-myc::p53+/+ lymphomas showed a high percentage of lymphomas with detectable p53 levels as previously reported (Figure 3b; Eischen et al., 1999). Conversely, Eμ-myc::p53+/− lymphomas, which have lost the remaining wild-type p53 allele, express no p53 as expected (Figures 3a and b; Eischen et al., 1999). Analysis of Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ lymphomas revealed detectable levels of p53 in most tumors (Figures 3c and d).

Figure 3
p53 stability in Eμ-myc::p53515C/515C and Eμ-myc::p53515C lymphomas. (a) Immunohistochemistry of Eμ-myc::p53515C/515C, Eμ-myc::p53515C/+ and Eμ-myc::p53+/− lymphomas using a p53 antibody. (b) Lysates were ...

We also wished to determine the stability of the p53R172P protein. These experiments were performed in vivo as standard culture conditions stabilize p53 whether it is mutant or wild type (Terzian et al., 2008). Ionizing radiation was used to stimulate p53R172P stability. We therefore irradiated three wild-type and three p53515C/515C mice with 5Gy ionizing radiation and killed individual mice at 0, 2, 4 and 8 h time points (Figure 4). At this dose, wild-type p53 was clearly stable at 4 h after irradiation, as was p53R172P. However, wild-type p53 levels had decreased by 8 h after irradiation, whereas p53R172P remained stabilized. These data indicate that the p53R172P levels are normally regulated, similar to wild-type p53, but in response to DNA damage (and likely oncogenic activation, for example, c-Myc expression) remained stable for a longer time.

Figure 4
p53R172P is stabilized in response to DNA damage. Wild type or p53515C/515C mice, 6 weeks old, were untreated or treated with 5Gy ionizing radiation. Spleens were harvested at 2, 4 or 8 h after treatment. Spleens from untreated (−) mice serve ...

p21 is activated in most Eμ-myc::p53515C/+ lymphomas

As the majority (83%) of Eμ-myc::p53515C/+ and 100% of Eμ-myc::p53515C/515C lymphomas retained a p53515C allele and expressed detectable levels of p53, we next determined the levels of p21 in these lymphomas, as p53R172P is able to transactivate p21 but cannot transactivate p53-dependent apoptotic targets (Ludwig et al., 1996; Liu et al., 2004; Barboza et al., 2006). To this end, we analyzed p21 protein levels in Eμ-myc lymphomas using western blot analysis. Eμ-myc::p53+/+ lymphomas expressed variable but detectable amounts of p21 (Figure 5a). However, the levels of p21 were undetectable in Eμ-myc::p53+/− lymphomas as they had lost the wild-type p53 allele. High but variable amounts of p21 were also observed in most Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ lymphomas when compared with Eμ-myc::p53+/− lymphomas (Figures 5b and c). Importantly, lymphomas from Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice, some of which retained only the p53515C allele, also had high levels of p21 (Figure 5b). Taken together, these data show that the Eμ-myc:: p53515C/515C and Eμ-myc::p53515C/+ lymphomas retain the ability to activate p21 in vivo and suggest that the delay in the onset of Eμ-myc-dependent lymphoma observed in these mice may be dependent on p53R172P.

Figure 5
Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ lymphomas retain the ability to activate p21. (a) p21 is expressed in splenic lymphomas of Eμ-myc::p53+/− and Eμ-myc::p53+/+ mice. Western blots of lysates isolated from ...

p53R172P-dependent activation of cellular senescence in Eμ-myc-driven lymphomas

As p53R172P is unable to activate p53-dependent apoptosis, yet Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice showed delayed lymphomagenesis, we next examined the net contribution of apoptosis for this delay. To this end, we examined lymphomas obtained from Eμ-myc::p53+/−, Eμ-myc::p53+/+, Eμ-myc::p53515C/ 515C and Eμ-myc::p53515C/+ mice. No statistically significant differences were observed in the levels of apoptosis in any of the lymphomas, regardless of p53 status (P=0.1; Figures 6a and b), suggesting that apoptosis observed in each of these lymphomas is independent of p53. The observations that Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice survive longer (Figure 1) and their lymphomas have higher p21 levels than Eμ-myc:: p53+/− lymphomas (Figures 5b and c) suggest that the increased survival of the Eμ-myc::p53515C/515C and Eμ-myc:: p53515C/+ mice when compared with the Eμ-myc::p53+/− mice is a consequence of p53R172P-dependent activity.

Figure 6
Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ lymphomas have similar levels of apoptosis when compared with Eμ-myc::p53+/− and Eμ-myc::p53+/+ mice. (a) Apoptosis was determined using hematoxylin and eosin (H & ...

To explore the mechanism of p53R172P-dependent increase in survival, lymphomas from Eμ-myc:: p53515C/515C and Eμ-myc::p53515C/+ mice were tested for SA β-gal activity (Figure 7a). Lymphomas from Eμ-myc:: p53+/+, Eμ-myc::p53515C/+ and Eμ-myc::p53515C/515C mice had 6.83±1.90, 4.35±1.13 and 5.10±1.06% SA β-gal-positive cells, respectively (Figure 7b). Importantly, two of four lymphomas from Eμ-myc::p53515C/+ mice that were examined retained only the p53515C allele, and showed increased SA-β-gal activity (4.76%) when compared with Eμ-myc::p53+/− lymphomas. Lymphomas from Eμ-myc:: p53+/− mice that had lost the wild-type p53 allele had few (0.74±0.62%) SA β-gal-positive cells, indicating the p53-dependent nature of this phenotype (Figure 7b). Altogether, these findings suggest that the delay in the onset of lymphoma in the Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice is at least partly due to the ability of the p53515C allele to trigger a cellular senescence program.

Figure 7
Senescence delays tumorigenesis in Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice. (a) Senescence-associated β-galactosidase activity was assayed at pH 6.0 in Eμ-myc::p53+/+, Eμ-myc::p53515C/515C, Eμ-myc::p53 ...

Last, we examined whether p53R172P affects lymphoma development before high-grade lymphoma formation. To this end, we killed mice at 4 to 5 weeks, before palpable tumor formation, and analyzed their spleens for apoptosis and senescence. Apoptosis was detected in all spleens, regardless of genotype (Supplementary Figure 1a). Spleens from the Eμ-myc::p53+/+ and Eμ-myc::p53515C/+ mice had slightly lower levels of apoptosis when compared with spleens from one Eμ-myc::p53515C/515C mouse and three Eμ-myc::p53+/− mice. With regard to senescence, spleens from the Eμ-myc::p53+/+ and Eμ-myc::p53515C/+ were indistinguishable. Eμ-myc::p53515C/515C spleens had an intermediate SA β-gal activity, whereas Eμ-myc::p53+/− mice had little activity (Supplementary Figure 1b). As expected, these data suggest that the hypomorphic p53R12P protein is not as robust as wild-type p53 in initiating senescence.


Despite conflicting reports, the prevailing paradigm contends that, although p53 regulates a plethora of processes that modulate cellular proliferation and survival, apoptosis is the most important (if not the only) function used by p53 for suppression of lymphomagenesis (Schmitt et al., 2002a). To determine the specific contribution of p53-dependent cell-cycle arrest/ senescence functions on lymphomagenesis in vivo, we took advantage of a knock-in mouse model previously generated in our laboratory that expresses the p53R172P mutant protein. This mutant mouse model recapitulates the rare human p53 mutation that is unable to activate apoptotic genes but retains the ability to activate other p53-dependent activities such as cell-cycle arrest (Ludwig et al., 1996; Rowan et al., 1996). Using this mouse, we previously showed that p53-dependent apoptosis was ablated in both the central nervous system and lymphatic tissues (Liu et al., 2004). Furthermore, microarray expression analysis from p53515C/515C mouse embryonic fibroblasts revealed the absence of p53-dependent apoptotic targets after treatment with ionizing radiation (Barboza et al., 2006). In our study, no significant changes in apoptosis were observed in lymphomas, regardless of genotype. Altogether, these data indicate that p53R172P is functionally null for p53-dependent apoptotic activities. By taking advantage of this p53 mutant, we have now shown in vivo that p53-dependent activities, other than apoptosis, significantly extend the survival of Eμ-myc-expressing mice. Both Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice showed significantly longer life spans than Eμ-myc::p53+/− mice. This observation, coupled with the fact that some B-cell lymphomas from Eμ-myc::p53515C/+ mice lose the p53515C allele, indicates the functionality of p53R172P in inhibition of lymphomagenesis. It must be noted, however, that the Eμ-myc::p53515C/+ and Eμ-myc::p53515C/515C mice do ultimately succumb to lymphomas earlier than Eμ-myc::p53+/+ mice, reflecting the fact that the p53515C allele is only partially functional. Interestingly, there was no statistical difference in the survival of Eμ-myc:: p53515C/515C and Eμ-myc::p53515C/+ mice, regardless of the p53 status in the lymphomas. This may be due to several possibilities. First, in response to Eμ-myc expression, p53R172P may be dominant over wild-type p53. This possibility is supported by the observation that once stabilized, the p53R172P mutant remains stable for a longer time. Thus, it is plausible that having either one (Eμ-myc::p53515C/+ lymphomas that have lost the wild-type p53 allele) or two (Eμ-myc::p53515C/515C) p53515C alleles may provide equivalent delays in lymphoma development, rendering their survival indistinguishable. Second, loss of one p53 allele may be a late event that dampens the effect on development of these tumors. This idea is supported by our observation that four healthy spleens from 4-week-old Eμ-myc::p53515C/+ mice still contained both p53 alleles, and expressed p53 and p21 (data not shown).

In Eμ-myc tumors, B cells overexpress c-myc, which in turn, activates p53, and likely the p53R172P protein, given what we know about mutant p53 stabilization (Figure 4; Terzian et al., 2008)). Eμ-myc cells are thus under immense pressure to bypass the antiproliferative effects of p53, as shown by the fact that Eμ-myc::p53+/− cells circumvent p53 activity by almost universally deleting the wild-type allele (Eischen et al., 1999; Schmitt et al., 1999). However, cells in the Eμ-myc:: p53515C/515C mouse activate p53-dependent senescence without activating p53-dependent apoptosis. Therefore, it is conceivable that the selective pressure against the p53515C allele in Eμ-myc::p53515C/515C lymphoma cells is much more attenuated when compared with the negative selection pressure aimed at ablating a fully functional p53 gene. Nevertheless, the overall effect is a delay in the onset of lymphoma, suggesting that B-cell lymphomas in the Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice ultimately escape this senescence barrier. The Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+in vivo data suggest several non-exclusive possibilities: (1) cells that have never entered senescence have a selective growth advantage and therefore can outgrow cells with an intact senescence response, (2) senescence may not be a dead-end event but rather a stopping point that can be bypassed if secondary mutations occur in the senescent cells, and (3) senescence may only slow the rate of lymphomagenesis, but it is unable to completely arrest tumor formation, which ultimately causes the mice to succumb to lymphomas.

The role of p21 in tumor suppression has been the subject of intense debate. Transfection of oncogenic Ras induces senescence in MEFs carrying wild-type p53, which results in low transformation potential (Serrano et al., 1997; Pantoja and Serrano, 1999). This transformation potential is dependent on p53 and largely independent of p21. However, in vivo models using oncogenic Ras-infected keratinocytes showed that transformation potential was greatly increased in the absence of p21 (Missero et al., 1996). In addition, using a similar model, a less robust but statistically significant p21-dependent effect on transformation potential was also observed (Weinberg et al., 1997). In a murine leukemia virus-infected model (mainly T-cell lymphoma), loss of p21 does not affect a tumor phenotype (Martins and Berns, 2002). In addition, alterations of p21 are also rare in human cancers (Shiohara et al., 1994). A critical point in the aforementioned studies is that p53 remains completely functional to initiate other p53-dependent pathways, including apoptotic pathways. MEFs derived from p53515C/515C mice form fewer foci than MEFs derived from p53 / mice when infected with oncogenic Ras (Barboza et al., 2006), suggesting that p53R172P also reduces transformation potential and induces senescence. Furthermore, deletion of p21 in p53515C/515C mice results in increased tumor incidence (mostly T-cell lymphomas and sarcomas) when compared with p53515C/515C mice (Barboza et al., 2006). In our model, Eμ-myc::p53515C/515C lymphomas posses only the p53515C allele, and hence show the ability to activate a p53R172P-dependent pathway. This results in increased survival and increased senescence when compared with the Eμ-myc::p53+/− lymphomas that lost their lone wild-type p53 allele. Collectively, these data confirm the important role of p53-dependent senescence in delaying Eμ-myc B-cell lymphomas. Although, the p53-dependent apoptotic pathway is clearly important in Eμ-myc B-cell lymphomas, the effect of senescence in delaying B-cell lymphoma development, and other tumors for that matter, must not be overlooked.

Materials and methods

Generation of mice

Generation of Eμ-myc, p53515C/+ and p53+/− mice has been previously described (Adams et al., 1985; Jacks et al., 1994a; Liu et al., 2004). Eμ-myc::p53515C/515C mice were generated by injecting 3-week-old Eμ-myc::p53515C/+ female mice with pregnant mare serum gonadatropin followed by injection of human chorionic gonadatropin 46 h later. These female mice were then mated with either p53515C/+ or p53515C/515C males. Female mice were killed 18 h after introduction with the male mice and their oviducts were removed. Fertilized eggs at the one-cell stage were harvested and transplanted into pseudopregnant females. The background of Eμ-myc::p53+/+, Eμ-myc::p53515C/515C, Eμ-myc::p53515C/+ or Eμ-myc::p53+/− mice was 75% C57BL/6 and 25% 129Sv. All animal studies were performed according to the guidelines of the institutional animal care and use committee of MD Anderson, protocol number 079906634.

Histology and immunohistochemistry

Spleens, lymph nodes, thymuses and other tissues with gross tumor involvement were harvested from tumor-bearing mice and fixed in 10% phosphate-buffered formalin (Sigma, St Louis, MO, USA) followed by paraffin embedding. Serial sections were either stained with hematoxylin and eosin or subjected to immunohistochemistry. For immunohistochemistry, sections were deparaffinized and antigens were retrieved using citric acid and steam. It was performed using rabbit α-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), rat α-B220 (R&D Systems, Minneapolis, MN, USA) and rabbit α-p53 (CM5) (Vector Laboratories, Burlingame, CA, USA) and visualized using ABC and DAB kits from Vector Laboratories. Slides were counterstained with nuclear fast red.

Genotyping and lymphoma analysis

Tails from Eμ-myc::p53+/+, Eμ-myc::p53515C/515C, Eμ-myc:: p53515C/+ or Eμ-myc::p53+/− mice were genotyped using primers previously described (Jacks et al., 1994b; Eischen et al., 1999; Liu et al., 2004). Loss of heterozygosity at the p53 locus was determined by PCR amplification using primers 5′-TACTCTCCTCCCCTCAATAAGCTATTC-3′ (exon 5) and 5′-GGATGGTGGTATACTCAGAGCC-3′ (exon 7). The PCR reactions were separated using electrophoresis followed by DNA purification of the appropriate PCR products. Sequencing was performed using the exons 5 and 7 primers and analyzed using Chromas software. Full-length p53 was analyzed for secondary mutations by first performing first-strand synthesis according to the manufacturer’s protocol (Amersham, Buckinghamshire, UK) on total RNA isolated from lymphomas using the Qiagen RNeasy kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). The resulting first-strand DNA was amplified by PCR using primers 5′-ATGGAGGAGTCACAGTCGGAT-3′ (exon 2) and 5′-AGTCAGGCC CCACTTTCTTGAC-3′ (exon 11). The PCR reactions were purified as above. Sequencing, using each of the aforementioned primers, was performed as above.

Immunoblotting analysis

Spleens, lymph nodes and thymuses were collected and frozen in liquid nitrogen. Protein extracts were prepared by passing the lymphomas through a series of needles in radioimmunoprecipitation assay buffer supplemented with complete protease and phosphatase inhibitors. Proteins were boiled in 2× sodium dodecyl sulfate sample buffer, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (Amersham). Membranes were incubated with rabbit α-p53 (CM5) (Vector Laboratories), mouse α-p21 (BD Pharmingen, San Diego, CA, USA), rabbit α-γ tubulin (Sigma) and mouse α-β-actin (Sigma) antibodies and antigen–antibody complexes were detected using enhanced chemiluminescence kit (Amersham) or BCIP/NBT Color Development Substrate (Promega, Madison, WI, USA).

Ionizing radiation

Healthy 6-week-old wild-type and p53515C/515C mice were treated with 5Gy ionizing radiation. Spleens were collected from the mice at 2, 4, or 8 h.

Senescence-associated β-galactosidase (SA β-gal) assay

Lymph nodes were harvested and fixed in 4% paraformaldyhde for 2 h followed by overnight incubation in Hanks’ balanced salt solution, without calcium and magnesium, supplemented with 10% sucrose (w/v) at 4 °C. The tissues were then incubated at room temperature for 6 h in 15% (w/v) sucrose/Hanks’ balanced salt solution and then in 20% (w/v) sucrose/Hanks’ balanced salt solution. Tissues were subsequently frozen in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and 10 μm sections were mounted on slides. SA β-gal assays were then performed as described (Dimri et al., 1995). Slides were counterstained with nuclear fast red.

Statistical analysis

Comparisons of mean values between the groups were analyzed using GraphPad Instat software (GraphPad Prism, GraphPad Software Inc., San Diego, CA). Statistical significance of the differences was analyzed using unpaired Student’s t-test for comparisons of two groups or by one-way analysis of variance for comparisons of more than two groups. Survival curves were plotted using the Kaplan–Meier method and compared by the log-rank (Mantel–Cox) test using GraphPad Prism. All P-values were two sided and the level of statistical significance was set at <0.05.

Supplementary Material

Figure Legend


We thank Dr Tomoo Iwakuma, Arlette Audiffred and Kristina Castro for helpful discussions and technical assistance, and Dr Clifton Stephens for pathological analysis. We thank Drs Jim Jackson and Vinod Pant for review of this paper. Veterinary support and DNA sequencing core facilities were supported by NCI Cancer Center Support Grant CA16672. SMP is supported by a Ruth L Kirschstein NRSA fellowship F32CA119616 and is a recipient of the Dowdy P Hawn postdoctoral fellowship. This study was supported by NIH Grant CA82577 (to GL).


Conflict of interest

The authors declare no conflict of interest.

Supplementary Information accompanies the paper on the Oncogene website (


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