[CANCER RESEARCH 62, 2184 –2191, April 1, 2002] Loss of Bax Alters Tumor Spectrum and Tumor Numbers in ARF-deficient Mice 1 Christine M. Eischen, 2 Jerold E. Rehg, Stanley J. Korsmeyer, and John L. Cleveland 3 Departments of Biochemistry [C. M. E., J. L. C.] and Pathology [J. E. R.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, and Department of Cancer Immunology and AIDS and Howard Hughes Medical Institute, Dana Farber Cancer Institute [S. J. K.], Boston, Massachusetts 02115 ABSTRACT p19 ARF is a key regulator of the p53-mediated apoptotic and tumor suppressor pathway. The proapoptotic Bax gene is a transcription target of p53, yet genetic studies in some animal models have suggested that Bax and p53 loss may cooperate in tumorigenesis. ARF-deficient mice are tumor prone, and to determine whether Bax loss could cooperate in the development of these tumors, we generated mice null for both ARF and Bax. The tumor latency of Bax / ARF / , Bax / ARF / , and Bax / ARF / mice was similar with a mean survival of 48.9, 48.1, and 47.6 weeks, respectively. In Bax / ARF / mice, the predominant tumor type was B- and T-cell lymphoma followed by sarcomas and a lack of carcinomas. However, the frequency of lymphoma development dramat- ically decreased, whereas that of sarcomas and carcinomas increased, in a gene dosage-dependent manner in Bax / ARF / and Bax / ARF / mice. Furthermore, uncommon tumors of ARF -/- mice (osteosarcoma and hemangiosarcoma) were observed in Bax/ARF-double null mice, and tumor types not described previously in ARF-null mice (mixed germ cell tumor, Triton tumor, and histiocytic sarcoma) also developed in Bax / ARF / animals. Importantly, multiple primary malignant tumors of different lineage arose in 25% of the Bax / ARF / mice, whereas only one tumor type per animal was observed in Bax / ARF-null littermates. Finally, the wild-type Bax allele was retained in tumors arising in Bax / ARF / mice. Thus, Bax appears to function as a tumor modifier rather than as a classic tumor suppressor, and the combined loss of Bax and the ARF allows for the emergence of multiple malignant tumor types, an alteration of the tumor spectrum, and tumors not observed previously in ARF-null mice. INTRODUCTION Cells that have sustained excessive genetic damage or are exposed to inappropriate proliferative signals undergo apoptosis that is de- pendent on p53 and/or ARF (1– 6). One function of ARF is to regulate p53 activity by binding to and sequestering the p53 regulator Mdm2 into the nucleolus (7, 8). The ability of cells that have acquired genetic changes to evade apoptosis leads to malignant transformation, e.g., many oncoproteins, such as Myc, E2F-1, and E1A, induce apoptosis by up-regulating ARF and p53 (4, 9, 10). Consequently, the majority of lymphomas that arise in mice genetically engineered to overexpress Myc in the B-cell compartment have alterations in the ARF-p53 pathway, including biallelic deletion of ARF or mutation or deletion of p53 (5, 6). Therefore, p53- and ARF-dependent apoptosis is a critical step in controlling tumorigenesis. After p53 inactivation, the deletion of the INK4A/ARF locus is the second most common alteration in human tumors (11, 12). The tumor suppressor activities of ARF and p53 were established in mice lacking ARF or p53, which spontaneously develop tumors with a 100% penetrance (13, 14). ARF-deficient mice on a C57Bl/6  129/SvJ mixed background preferentially develop undifferentiated sarcomas (43%), followed by T-cell lymphomas (29%), carcinomas (17%), and tumors of the nervous system (11%), with an average survival of 38 weeks (14, 15). By contrast, p53-null mice preferentially develop T-cell lymphomas (70%), followed by fibrosarcomas (30%), and have a mean survival of 19 weeks (13, 16). However, unlike ARF-null mice, p53-deficient mice rarely develop carcinomas or nervous sys- tem tumors (13, 16). Therefore, inactivation of ARF or p53 is a critical step in tumor development, but their loss seems to have context- specific effects on tumorigenesis. The Bcl-2 family of proteins includes both antiapoptotic and pro- apoptotic members that are essential for regulating cell survival (re- viewed in Ref. 17), and these have also been linked to tumor devel- opment (reviewed in Ref. 18). The antiapoptotic proteins Bcl-2 and Bcl-X L are overexpressed in a wide variety of human and murine malignancies (18 –20), whereas inactivating mutations in the pro- apoptotic bax and bak genes have been reported in colon cancers and other cancer types (21–26). Despite these findings, the overexpression of antiapoptotic Bcl-2 family members or inactivation of pro- apoptotic members alone only rarely results in cancer (27–30), e.g., unlike p53- and ARF-deficient mice, Bax-, Bak-, and Bax/Bak-double null mice do not develop spontaneous tumors (29, 30). Additionally, mice engineered to overexpress Bcl-2 have a very low incidence of malignancy (27, 28). When combined with oncogenes or loss of tumor suppressors, alterations in Bcl-2 family members cooperate to enhance transfor- mation and accelerate tumorigenesis, e.g., E-bcl-2/E-myc double transgenic mice develop lymphoma much more rapidly than mice expressing either transgene alone (31). Furthermore, Bax-null MEFs 4 are more susceptible to transformation than wild-type MEFs (32). Finally, loss of Bax accelerates brain and breast tumor development in SV40 large T antigen transgenic mice (33, 34), suggesting that Bax loss can collaborate with inactivation of p53 and/or Rb in tumorigen- esis. Cooperation of Bax loss with the Rb pathway appears more likely, as Bax is a transcription target of p53 (35), and Bax loss does not alter the tumor spectrum or tumor latency in p53-null mice (36). In contrast, here we establish that Bax loss cooperates with ARF loss in tumorigenesis. Mice lacking both ARF and Bax have a marked alteration in their spectrum of tumors compared with those that arise in ARF-null mice, without altering the mean tumor latency. Further- more, Bax/ARF-double null mice develop uncommon tumors and multiple primary malignancies, which are not observed in ARF-null mice. These results support the concept that there are cooperative effects of tumor suppressor loss and alterations in the Bcl-2 family of apoptotic regulators on tumor development. MATERIALS AND METHODS Mice. Both ARF- and Bax-null mice were generated using RW4 embryonic stem cells (14, 29). ARF-null mice (C57BL/6  129SvJ) were kindly provided by Dr. Charles J. Sherr. Bax heterozygous mice (C57BL/6  129SvJ) were crossed to ARF-null mice, and the F1s were then interbred to generate F2 Bax +/+ ARF -/- , Bax +/- ARF -/- , and Bax -/- ARF -/- mice. All F2 littermates Received 10/16/01; accepted 2/13/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH Grants DK44158 and CA76379 (to J. L. C.) Cancer Center Core Grant CA-21765, NIH Postdoctoral Grant CA81695 (to C. M. E.), and by the American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children’s Re- search Hospital. 2 Present address: The Eppley Institute for Cancer Research, 987696 University of Nebraska Medical Center, Omaha, NE 68198-7696. 3 To who request for reprints should be addressed, at Department of Biochemistry, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-2398; Fax: (901) 525-8025; E-mail: john.cleveland@stjude.org. 4 The abbreviations used are: MEF, mouse embryo fibroblast; Rb, retinoblastoma ; MSA, muscle-specific actin; SMA, smooth muscle actin. 2184 on May 6, 2016. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from