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

Abstract

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.

FIG. 1

Bax does not influence cytokine deprivation-induced apoptosis but does mediate c-Myc-induced apoptosis. (A) Bax^+/+ (squares) and Bax^−/− (triangles) pre-B cells were cultured in medium with (open symbols) or without (solid symbols) IL-7, and their viability was determined at intervals by trypan blue dye exclusion. The data are representative of two independent experiments. (B) 4-HT was added to the indicated primary pre-B-cell cultures to activate Myc-ER, and their viability was determined at intervals thereafter by trypan blue dye exclusion. Apoptosis was confirmed by analysis of subdiploid DNA content after staining with propidium iodide. Steady-state levels of apoptosis in the wild-type primary pre-B cells are indicated at the zero hour time point. (Inset) The protein levels of Myc-ER in Bax ^−/− (−/−) and Bax ^+/+ (+/+) pre-B cells infected with a retrovirus encoding Myc-ER-GFP and pre-B cells infected with the GFP vector control (V) retrovirus were determined by immunoblotting with an antibody for Myc. The data are the mean of three independent experiments, and error bars represent one standard deviation.

FIG. 2

Myc does not upregulate Bax protein expression in B cells. (A) 4-HT was added to wild-type pre-B-cell cultures infected with the Myc-ER encoding retrovirus to activate Myc-ER. At the indicated intervals cells were collected and protein lysates were made. Equal quantities of protein were assessed by immunoblotting with antibodies specific for Bax, p53, or PARP. The 85- kDa caspase cleavage fragment of PARP is denoted by an asterisk. (B) Equal quantities of protein from FACS sorted IgM⁺/CD19⁺ splenic B cells from one wild-type (WT) and two precancerous Eμ-myc transgenics (Tg) were assessed by immunoblotting with an antibody specific for Bax. (C) Total RNA was isolated from Myc-ER-infected wild-type pre-B cells activated with 4-HT for the indicated intervals. Pre-B cells pretreated with cycloheximide are indicated by a plus sign. The expression of bax transcripts was assessed by Northern blot analyses utilizing bax cDNA. (D) 4-HT was added to wild-type, ARF^−/−, p53^−/−, and ARF^−/−p53^−/− pre-B-cell cultures infected with the Myc-ER-encoding retrovirus to activate Myc-ER. At the indicated intervals, cells were collected and protein lysates were prepared. Equal quantities of protein were evident by immunoblotting with antibodies specific for Bax, and the levels of Myc-ER protein expressed in the four genotypes were equivalent (5).

FIG. 3

Myc-induced lymphomagenesis is accelerated by Bax loss. The genotypes of the mice are indicated next to the Kaplan-Meier survival curves, and the numbers of mice in each group are denoted by the n values. Vertical lines indicate ages of surviving mice: 0 Bax^−/−, 3 Bax^+/−, and 10 Bax^+/+ mice. The average life spans of Bax^+/+, Bax^+/−, and Bax^−/− Eμ-myc transgenics were 21.7, 16.0, and 12.6 weeks, respectively. Pre-B- and/or B-cell lymphoma was documented in all of the animals.

FIG. 4

Western blot analysis of lymphomas arising in Bax^+/− and Bax^−/− Eμ-myc transgenic mice. Levels of Bax (A), p53 (B, top), p19^ARF (B, middle), and Mdm2 (B, bottom) protein in whole-cell extracts of tumors from Bax^+/− and Bax^−/− Eμ-myc transgenic mice were assessed by immunoblotting with antibodies specific for each protein. Protein extracts from a tumor arising in a Bax^+/+ Eμ-myc transgenic mouse that contains the p92, p90, and p85 Mdm2 isoforms were run to show the location of these isoforms in panel B and as a blotting control for Bax expression in panel A. The asterisk in panel B marks the position of a nonspecific background band detected with the Mdm2 antibody.

FIG. 5

Southern blot analysis of Bax^+/− and Bax^−/− Eμ-myc lymphomas. AflII and BamHI restriction fragments containing ARF exon 1β and p53 exons 2 to 10, respectively, from genomic DNA isolated from lymphomas arising in Bax^+/− (A) and Bax^−/− (B) Eμ-myc transgenic mice. Genomic DNA from the spleen of a wild-type littermate was used as a control in both panels A and B. Lack of a band denotes biallelic deletion of that gene.

FIG. 6

Schematic describing the outcome of molecular events that occur in lymphomas in Bax^+/+, Bax^+/−, and Bax^−/− Eμ-myc transgenic mice. On the left, the majority of lymphomas that arise in Bax^+/+ and Bax^+/− Eμ-myc transgenic mice have alterations in ARF (deletion, X) or p53 (mutation or deletion, X) and/or Mdm2 (overexpression, ↑) (Fig. 4 and 5 and Table 1; see also reference 5). On right, a similar frequency of alterations in ARF and Mdm2 still occurs in lymphomas from Bax-null Eμ-myc transgenic mice; however, no p53 mutations or deletions are observed in these lymphomas (Fig. 4 and 5, Table 1). Therefore, Myc targets ARF and Mdm2 but not p53 in the absence of Bax during Myc-induced lymphomagenesis.