Losses of both products of the Cdkn2a/Arf locus contribute to asbestos-induced mesothelioma development and cooperate to accelerate tumorigenesis

The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1β, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/-) and Arf(+/-) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/-) and Arf(+/-) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/-) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/-) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.     

Paracrine interactions in bone-secreted products of osteoblasts permit osteoclasts to respond to parathyroid hormone

During bone remodeling, activation of resorption is followed by a cycle of formation and this ordered sequence of events has long suggested that local interactions between osteoclasts and osteoblasts are an important regulatory mechanism in bone metabolism. To study this phenomenon, we have prepared bone cells containing primarily osteoclasts by brief digestion of mice calvariae in collagenase, overnight attachment to polystyrene tissue culture flasks in serumless medium supplemented with OB (osteoblast) cell conditioned medium and subsequent growth in low serum. These OC (osteoclast) cells were found to be highly enriched in acid phosphatase activity and expressed cAMP responses to PTH (parathyroid hormone) and prostaglandin E2 but exhibited no PTH-stimulated hyaluronate synthesis in contrast to prostaglandin E2. PTH effects on hyaluronate, however, could be restored upon coculture of OC cells with OB cells (noncontact) or with OB cell conditioned medium, thereby suggesting that OB cells regulate OC cell PTH responsiveness and/or differentiation by soluble cell products secreted into the medium.     

p19Arf suppresses growth, progression, and metastasis of Hras-driven carcinomas through p53-dependent and -independent pathways

Ectopic expression of oncogenes such as Ras induces expression of p19(Arf), which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19(Arf), and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19(Arf)-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19(Arf)-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 (Arf)- and p53-deficient mice. Thus, p19(Arf) inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19(Arf) also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19( Arf)-null mice, and p53 mutations were more frequently seen in wild-type than in p19( Arf)-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19(Arf).     

Tumor suppression by p53 in the absence of Atm

Oncogenes can induce p53 through a signaling pathway involving p19/Arf. It was recently proposed that oncogenes can also induce DNA damage, and this can induce p53 through the Atm DNA damage pathway. To assess the relative roles of Atm, Arf, and p53 in the suppression of Ras-driven tumors, we examined susceptibility to skin carcinogenesis in 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (TPA)-treated Atm- and p53-deficient mice and compared these results to previous studies on Arf-deficient mice. Mice with epidermal-specific deletion of p53 showed increased papilloma number and progression to malignant invasive carcinomas compared with wild-type littermates. In contrast, Atm-deficient mice showed no increase in papilloma number, growth, or malignant progression. gamma-H2AX and p53 levels were increased in both Atm(+/+) and Atm(-/-) papillomas, whereas Arf(-/-) papillomas showed much lower p53 expression. Thus, although there is evidence of DNA damage, signaling through Arf seems to regulate p53 in these Ras-driven tumors. In spontaneous and radiation-induced lymphoma models, tumor latency was accelerated in Atm(-/-)p53(-/-) compound mutant mice compared with the single mutant Atm(-/-) or p53(-/-) mice, indicating cooperation between loss of Atm and loss of p53. Although p53-mediated apoptosis was impaired in irradiated Atm(-/-) lymphocytes, p53 loss was still selected for during lymphomagenesis in Atm(-/-) mice. In conclusion, in these models of oncogene- or DNA damage-induced tumors, p53 retains tumor suppressor activity in the absence of Atm.     

Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.     

Analysis of p53 inactivation in a human T-cell leukemia virus type 1 Tax transgenic mouse model

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). The HTLV-1 Tax protein has been strongly linked to oncogenesis and is considered to be the transforming protein of this virus. A Tax transgenic mouse model was utilized to study the contribution of p53 inactivation to Tax-mediated tumorigenesis. These mice develop primary, peripheral tumors consisting of large granular lymphocytic (LGL) cells, which also infiltrate the lymph nodes, bone marrow, spleen, liver, and lungs. Primary Tax-induced tumors and tumor-derived cell lines exhibited functional inactivation of the p53 apoptotic pathway; such tumors and tumor cell lines were resistant to an apoptosis-inducing stimulus. In contrast, p53 mutations in tumors were found to be associated with secondary organ infiltration. Three of four identified mutations inhibited transactivation and apoptosis induction activities in vitro. Furthermore, experiments which involved mating Tax transgenic mice with p53-deficient mice demonstrated minimal acceleration in initial tumor formation, but significantly accelerated disease progression and death in mice heterozygous for p53. These studies suggest that functional inactivation of p53 by HTLV-1 Tax, whether by mutation or another mechanism, is not critical for initial tumor formation, but contributes to late-stage tumor progression.     

Decreased Mdm2 Expression Inhibits Tumor Development and Extends Survival Independent of Arf and Dependent on p53

Inactivation of the Arf-Mdm2-p53 tumor suppressor pathway is a necessary event for tumorigenesis. Arf controls Mdm2, which in turn regulates p53, but Arf and Mdm2 also have p53-independent functions that affect tumor development. Moreover, inhibition of oncogene-induced tumorigenesis relies on Arf and p53, but the requirements of Arf and p53 in tumor development initiated in the absence of overt oncogene overexpression and the role of Mdm2 in this process remain unclear. In a series of genetic experiments in mice with defined deficiencies in Arf, Mdm2 and/or p53, we show Mdm2 haploinsufficiency significantly delayed tumorigenesis in mice deficient in Arf and p53. Mdm2 heterozygosity significantly inhibited tumor development in the absence of Arf, and in contrast to Myc oncogene-driven cancer, this delay in tumorigenesis could not be rescued with the presence of one allele of Arf. Notably, Mdm2 haploinsufficieny blocked the accelerated tumor development in Arf deficient mice caused by p53 heterozygosity. However, tumorigenesis was not inhibited in Mdm2 heterozygous mice lacking both alleles of p53 regardless of Arf status. Surprisingly, loss of Arf accelerated tumor development in p53-null mice. Tumor spectrum was largely dictated by Arf and p53 status with Mdm2 haploinsufficiency only modestly altering the tumor type in some of the genotypes and not the number of primary tumors that arose. Therefore, the significant effects of Mdm2 haploinsufficiency on tumor latency were independent of Arf and required at least one allele of p53, and an Mdm2 deficiency had minor effects on the types of tumors that developed. These data also demonstrate that decreased levels of Mdm2 are protective in the presence of multiple genetic events in Arf and p53 genes that normally accelerate tumorigenesis.     

Critical role of filamin-binding LIM protein 1 (FBLP-1)/migfilin in regulation of bone remodeling

Bone remodeling is a complex process that must be precisely controlled to maintain a healthy life. We show here that filamin-binding LIM protein 1 (FBLP-1, also known as migfilin), a kindlin- and filamin-binding focal adhesion protein, is essential for proper control of bone remodeling. Genetic inactivation of FBLIM1 (the gene encoding FBLP-1) in mice resulted in a severe osteopenic phenotype. Primary FBLP-1 null bone marrow stromal cells (BMSCs) exhibited significantly reduced extracellular matrix adhesion and migration compared with wild type BMSCs. Loss of FBLP-1 significantly impaired the growth and survival of BMSCs in vitro and decreased the number of osteoblast (OB) progenitors in bone marrow and OB differentiation in vivo. Furthermore, the loss of FBLP-1 caused a dramatic increase of osteoclast (OCL) differentiation in vivo. The level of receptor activator of nuclear factor κB ligand (RANKL), a key regulator of OCL differentiation, was markedly increased in FBLP-1 null BMSCs. The capacity of FBLP-1 null bone marrow monocytes (BMMs) to differentiate into multinucleated OCLs in response to exogenously supplied RANKL, however, was not different from that of WT BMMs. Finally, we show that a loss of FBLP-1 promotes activating phosphorylation of ERK1/2. Inhibition of ERK1/2 activation substantially suppressed the increase of RANKL induced by the loss of FBLP-1. Our results identify FBLP-1 as a key regulator of bone homeostasis and suggest that FBLP-1 functions in this process through modulating both the intrinsic properties of OB/BMSCs (i.e., BMSC-extracellular matrix adhesion and migration, cell growth, survival, and differentiation) and the communication between OB/BMSCs and BMMs (i.e., RANKL expression) that controls osteoclastogenesis.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

DNA damage induces MDMX nuclear translocation by p53-dependent and -independent mechanisms

The MDM2 homolog MDMX is an important regulator of p53 activity during embryonic development. MDMX inactivation in mice results in embryonic lethality in a p53-dependent fashion. The expression level of MDMX is not induced by DNA damage, and its role in stress response is unclear. We show here that ectopically expressed MDMX is mainly localized in the cytoplasm. DNA damage promotes nuclear translocation of MDMX in cells with or without p53. Coexpression of MDM2 or p53 is sufficient to induce MDMX nuclear translocation, suggesting that activation of p53 and induction of MDM2 expression can contribute to this process. Stable transfection of MDMX into U2OS cells does not alter p53 level but results in reduced p53 DNA-binding activity and reduced MDM2 expression. The ability of ARF (alternate reading frame of INK4a) to activate p53 is also significantly inhibited by expression of MDMX. These results suggest that MDMX function may be regulated by DNA damage. Furthermore, MDMX may complement MDM2 in regulating p53 during embryonic development due to its ability to inhibit p53 in the presence of ARF.     

Obligate Roles for p16Ink4a and p19Arf-p53 in the Suppression of Murine Pancreatic Neoplasia

Epithelial tumors of the pancreas exhibit a wide spectrum of histologies with varying propensities for metastasis and tissue invasion. The histogenic relationship among these tumor types is not well established; moreover, the specific role of genetic lesions in the progression of these malignancies is largely undefined. Transgenic mice with ectopic expression of transforming growth factor alpha (TGF-alpha) in the pancreatic acinar cells develop tubular metaplasia, a potential premalignant lesion of the pancreatic ductal epithelium. To evaluate the cooperative interactions between TGF-alpha and signature mutations in pancreatic tumor genesis and progression, TGFalpha transgenic mice were crossed onto Ink4a/Arf and/or p53 mutant backgrounds. These compound mutant mice developed a novel pancreatic neoplasm, serous cystadenoma (SCA), presenting as large epithelial tumors bearing conspicuous gross and histological resemblances to their human counterpart. TGFalpha animals heterozygous for both the Ink4a/Arf and the p53 mutation showed a dramatically increased incidence of SCA, indicating synergistic interaction of these alleles. Inactivation of p16(Ink4a) by loss of heterozygosity, intragenic mutation, or promoter hypermethylation was a common feature in these SCAs, and correspondingly, none of the tumors expressed wild-type p16(Ink4a). All tumors sustained loss of p53 or Arf, generally in a mutually exclusive fashion. The tumor incidence data and molecular profiles establish a pathogenic role for the dual inactivation of p16(Ink4a) and p19(Arf)-p53 in the development of SCA in mice, demonstrating that p16(Ink4a) is a murine tumor suppressor. This genetically defined model provides insights into the molecular pathogenesis of SCA and serves as a platform for dissection of cell-specific programs of epithelial tumor suppression.     

Similar Tumor Suppressor Gene Alteration Profiles in Asbestos-Induced Murine and Human Mesothelioma

The status of tumor suppressor genes (TSGs) relevant to human malignant mesothelioma (HMM) pathogenesis was examined in cultures of mesothelioma cells from tumoral ascites developed in mice exposed to asbestos (asb) fibers. The status of the respective hortologous human genes was also investigated in 12 HMM cell cultures. Eleven primary cultures from mice hemizygous for N?2 (asb-Nf2KO3/+) and 4 wild type counterparts (asb-Nf2+/+) were analyzed for mutations in Nf2, p16/Cdkn2a, p19/Arf and Trp53 genes and protein expression of p15/Cdkn2b and Cdk4. TSG alterations in both mouse and human mesothelioma cells consisted in frequent inactivation of p16/Cdkn2a, p19/Arf (or P14/ARF) and p15/Cdkn2b, co-inactivation of p16/Cdkn2a and p15/Cdkn2b and low rate of Trp53 mutations in both asb-Nf2KO3/+ and asb-Nf2+/+ mesothelioma cells. In both mouse and human mesothelioma cells, inactivation of the hortologous genes p16/Cdkn2a or P16/CDKN2A was due to deletions at the Ink4/Arf locus encompassing p19/Arf or P14/ARF, respectively. Loss of heterozygosity at the Nf2 locus was detected in 10 of 11 asb-Nf2KO3/+ cultures and Nf2 gene rearrangement in one asb-Nf2+/+ culture. These data show that the profile of TSG alterations in asbestos-induced mesothelioma is similar in mice and humans. Thus, the mouse mesothelioma model could be useful for human risk assessment, taking into account interindividual variations in genetic sensitivity to carcinogens.     

GFAP-Cre-mediated transgenic activation of Bmi1 results in pituitary tumors

Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16(INK4A)/Rb rather than on regulation of p19(ARF)/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.     

C/EBPbeta represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19(Arf)

CCAAT/enhancer-binding protein-beta (C/EBPbeta) is a mediator of cell survival and tumorigenesis. When C/EBPbeta(-/-) mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19(Arf) and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19(Arf) is dramatically elevated in C/EBPbeta(-/-) epidermis and that C/EBPbeta represses a p19(Arf) promoter reporter. To determine whether p19(Arf) is responsible for the proapoptotic phenotype in C/EBPbeta(-/-) mice, C/EBPbeta(-/-);p19(Arf-/-) mice were generated. C/EBPbeta(-/-);p19(Arf-/-) mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19(Arf) is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPbeta(-/-) epidermis, we generated K14-ER:Ras;C/EBPbeta(-/-) mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPbeta(-/-) mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPbeta represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPbeta may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents.     

p16(Ink4a) interferes with Abelson virus transformation by enhancing apoptosis

Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of p53 or down-modulation of p19(Arf), a p53 regulatory protein. Consistent with these data, pre-B cells from either p53 or Ink4a/Arf null mice bypass crisis. However, the Ink4a/Arf locus encodes both p19(Arf) and a second tumor suppressor, p16(Ink4a), that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16(Ink4a) plays a role in Ab-MLV transformation, primary transformants derived from Arf(-/-) and p16(Ink4a(-/-)) mice were compared. A fraction of those derived from Arf(-/-) animals underwent crisis, and even though all p16(Ink4a(-/-)) primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of Ink4a/Arf(-/-) cells infected with a virus that expresses both v-Abl and p16(Ink4a) revealed that p16(Ink4a) expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the Ink4a/Arf locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16(Ink4a) can suppress transformation by inducing apoptosis.     

Bax loss impairs Myc-induced apoptosis and circumvents the selection of p53 mutations during Myc-mediated lymphomagenesis

The ARF and p53 tumor suppressors mediate Myc-induced apoptosis and suppress lymphoma development in E mu-myc transgenic mice. Here we report that the proapoptotic Bcl-2 family member Bax also mediates apoptosis triggered by Myc and inhibits Myc-induced lymphomagenesis. Bax-deficient primary pre-B cells are resistant to the apoptotic effects of Myc, and Bax loss accelerates lymphoma development in E mu-myc transgenics in a dose-dependent fashion. Eighty percent of lymphomas arising in wild-type E mu-myc transgenics have alterations in the ARF-Mdm2-p53 tumor suppressor pathway characterized by deletions in ARF, mutations or deletions of p53, and overexpression of Mdm2. The absence of Bax did not alter the frequency of biallelic deletion of ARF in lymphomas arising in E mu-myc transgenic mice or the rate of tumorigenesis in ARF-null mice. Furthermore, Mdm2 was overexpressed at the same frequency in lymphomas irrespective of Bax status, suggesting that Bax resides in a pathway separate from ARF and Mdm2. Strikingly, lymphomas from Bax-null E mu-myc transgenics lacked p53 alterations, whereas 27% of the tumors in Bax(+/-) E mu-myc transgenic mice contained p53 mutations or deletions. Thus, the loss of Bax eliminates the selection of p53 mutations and deletions, but not ARF deletions or Mdm2 overexpression, during Myc-induced tumorigenesis, formally demonstrating that Myc-induced apoptotic signals through ARF/Mdm2 and p53 must bifurcate: p53 signals through Bax, whereas this is not necessarily the case for ARF and Mdm2.