Among the more prominent regulators of Programmed cell death and apoptosis are Bcl-2 and its homologs. Gene transfer studies have shown that elevated levels of Bcl-2 protein can block or delay apoptotic cell death induced by a wide variety of stimuli and insults, suggesting that Bcl-2 controls a distal step in what may represent a evolutionarily conserved, final common pathway for cell death (reviewed in (1) ). The biochemical details of how the 25-26-kDa Bcl-2 protein controls cell life and death remain enigmatic, due at least in part to its lack of amino acid sequence homology with other proteins whose mechanism of action is known.

The Bcl-2 protein physically interacts with several other proteins, including several homologous proteins that constitute the Bcl-2 protein family(2, 3, 4) . Some of the members of this protein family are blockers of cell death akin to Bcl-2, whereas others promote apoptosis and antagonize the function of Bcl-2 (reviewed in (1) ). The Bax protein shares 20% amino acid identity with Bcl-2 and accelerates apoptosis when overexpressed by gene transfer methods(3) . Bax forms heterodimers (or oligomers) with Bcl-2, and mutagenesis studies have demonstrated that the ability of Bcl-2 to heterodimerize with Bax is critical for the function of Bcl-2 as a suppressor of cell death(5) . Immunohistochemical studies have demonstrated that the BAX gene is widely expressed in vivo, but is subject to tissue-specific and differentiation stage-dependent regulation, which sometimes but not always correlates with the apoptotic tendencies of cells in vivo(6) . Expression of BAX is induced in some types of tumor cells by radiation and chemotherapeutic drugs, in a p53-dependent manner, suggesting an important role for the Bax protein in apoptosis induced by genotoxic stress(7, 8, 9) . Furthermore, reductions in Bax protein levels have been observed in 35% of adenocarcinomas of the breast and correlate with poor responses to chemotherapy in some subgroups of patients(10) . In addition, rapid and striking elevations in p21 protein levels have been described in ischemia-damaged neurons, implying a role for Bax in at least some types of neuronal cell death that occur in the setting of stroke (11) .

Taken together, these observations suggest that BAX plays an important role in the control of cell death in several clinically relevant contexts and suggest a need for a greater understanding of the mechanisms that regulate the expression of this gene. At present, however, relatively little is known about the mechanisms that control the levels of the Bax protein. Clearly, transcriptional mechanisms can play an important role, given that the BAX gene promoter contains several p53-binding sites and can be strongly trans-activated by p53, at least in some types of cells(9) . Here, we demonstrate that the levels of Bax protein can also be controlled by post-translational mechanisms, with the Bcl-2 protein reducing the rate of p21 protein degradation in some but not all types of cells.

MATERIALS AND METHODS

Cells and Metabolic Labeling

()

Immunoprecipitations and Immunoblot Assays

Immunoblotting was performed as described in detail previously(6, 17) . Briefly, cells were lysed in 1% Triton X-100 solution as above, normalized for total protein content, and subjected to SDS-PAGE/immunoblot assay using either 0.1% (v/v) or 0.05% (v/v) rabbit anti-mouse Bax or anti-human Bax antiserum, respectively. Antibodies were detected using either biotinylated goat anti-rabbit Ig and an avidin-biotin-complex (ABC) reagent (Vector Laboratories, Inc.) containing horseradish peroxidase, followed by Vector SG-substrate or 3,3`-diaminobenzidine for colorimetric detection, or using horseradish peroxidase-conjugated goat anti-rabbit Ig and an ECL kit from Amersham, Inc. for autoradiographic detection.

RNA Blot Assays

RESULTS

We noticed that stable transfectants of the Jurkat human T-cell leukemia cell line which overexpress Bcl-2 contained markedly higher steady-state levels of endogenous p21 protein compared to control transfectants (``Neo''). Fig. 1shows a representative example of immunoblot results obtained for Jurkat-Bcl-2 and Jurkat-Neo cells. For this experiment, detergent lysates were prepared from Jurkat-Bcl-2 and Jurkat-Neo cells, normalized for total protein content (50 µg/lane), and subjected to SDS-PAGE/immunoblot assay using an anti-human Bax antiserum(10) . As shown, a strong 21-kDa band was seen in the BCL-2 transfectants, whereas only a very faint 21-kDa band was observed in the Neo control transfectants (50-fold less). In contrast to Jurkat cells which contained very low levels of p21 protein prior to transfection with BCL-2 expression plasmids, the basal steady-state levels of p21 protein were much higher in another T-cell leukemia line, CEM, and transfection with BCL-2 did not produce a further elevation in Bax protein levels (Fig. 1). The blot shown in Fig. 1was then reincubated with an anti-Bcl-2 antibody, confirming the presence of 10-fold elevations in the relative levels of p26-Bcl-2 protein in the BCL-2 transfectants of Jurkat and CEM relative to the Neo control transfectants.

Figure 1: Immunoblot analysis of Bcl-2 and Neo transfectants of Jurkat and CEM leukemia lines reveals differential regulation of p21 levels. Detergent lysates were prepared from Bcl-2 (B) and Neo (N) transfectants of Jurkat and CEM cells, normalized for total protein content (50 µg/lane), and subjected to SDS-PAGE/immunoblot assay using anti-human Bax antiserum followed by biotinylated secondary antibody, ABC-horseradish peroxidase reagent, and 3,3`-diaminobenzidine for color detection (brown), which revealed a 21-kDa band corresponding to the human Bax protein (closed arrowhead). The same blot was then incubated with anti-human Bcl-2 antiserum(24) , and colorimetric detection was achieved similarly, except that Vector SG substrate was employed (black), revealing the presence of the expected 26-kDa band corresponding to human Bcl-2 (open arrowhead). (The original brown (Bax) and black (Bcl-2) colors are not retained in the black and white photograph shown.) Staining of the top of the blot (>50 kDa) with Ponceau S confirmed loading of equivalent amounts of protein samples for the pairs of BCL-2 and Neo transfectants (not shown). In addition, a faint spurious band of 40-45 kDa can be seen at equal intensity in Jurkat-Bcl-2 and Jurkat-Neo cells, thus providing further confirmation that equivalent amounts of protein samples were loaded.

This analysis of steady-state p21 protein levels was then extended to additional pairs of BCL-2- and Neo-transfected cell lines, using immunoblot assays and antibodies specific for either the human or mouse Bax proteins. As shown in Fig. 2, BCL-2 transfection resulted in increases in the steady-state levels of p21 only in the Jurkat T-cell and 697 pre-B-cell leukemia cell lines. In contrast, the relative steady-state levels of p21 in BCL-2-transfected PC12, NIH-3T3, 32D.3, and CEM cells were not detectably different from their Neo control transfectant counterparts. Incubation of these blots with control antibodies such as anti-F--ATPase or anti-actin confirmed the loading of equivalent amounts of total protein for the pairs of samples (not shown). These results therefore suggest that the influence of Bcl-2 on p21 protein levels is cell type-specific.

Figure 2: Immunoblot analysis of p21 levels in several pairs of Bcl-2- and Neo-transfected cell lines. Detergent lysates were prepared from pairs of Bcl-2 and Neo transfectants of human, rat, and mouse cell lines, normalized for total protein content, and subjected to SDS-PAGE/immunoblot analysis using either anti-mouse Bax (mouse and rat samples) or anti-human Bax antiserum. Antibody detection was accomplished either by a colorimetric method using Vector SG substrate (NIH-3T3, 32D.3, PC12, LN, Jurkat) or by an ECL method (697, CEM). LN refers to detergent lysates prepared from lymph nodes derived from either Bcl-2 transgenic (TG) or control nontransgenic littermate (C).

To explore the in vivo relevance of the observation that Bcl-2 can produce elevations in the steady-state levels of p21 in some cell lines, we compared the relative levels of Bax protein in lymph nodes derived from Bcl-2/IgH transgenic and nontransgenic littermate control mice(18) . Fig. 2shows a representative experiment demonstrating markedly elevated levels of p21 protein in Bcl-2/Ig transgenic lymph nodes compared to the nontransgenic littermate control. Similar results were obtained in 3 of 3 transgenic/nontransgenic pairs of age-matched animals (not shown).

Northern blot analysis was performed to explore the effects of BCL-2 transfection on the steady-state levels of BAX mRNA in Jurkat and 697 cells, where gene transfer-mediated elevations in Bcl-2 resulted in substantial increases in p21. As shown in Fig. 3(left panel), the relative levels of the major 1.0-kbp as well as the minor 1.5-kbp BAX mRNAs were not higher in the BCL-2 transfectants compared with the Neo control transfectants. Reprobing the same RNA blot using a [P]glyceraldehyde phosphate dehydrogenase cDNA confirmed loading of equivalent amounts of total RNA for all samples (Fig. 3, right panel). Bcl-2, therefore, does not cause increases in p21 protein levels by stimulating elevations in BAX mRNAs.

Figure 3: RNA blot analysis of Bcl-2 and Neo transfectants of Jurkat and 697 leukemia cells. Total cellular RNA (20 µg/lane) was subjected to Northern blot analysis using a P-labeled human BAX cDNA probe (left panel). The arrowhead indicates the major 1.0-kbp BAX mRNA species. A minor 1.5-kbp BAX mRNA is also seen, consistent with previous reports (3) . The same blot was stripped and rehybridized with a human [P]glyceraldehyde phosphate dehydrogenase (GAPDH) probe (right panel) to verify loading of equivalent amounts of RNA in all lanes.

In the absence of alterations in the relative levels of BAX mRNA, it seemed likely that Bcl-2 causes elevations in p21 protein levels by either increasing the rate of BAX mRNA translation or by decreasing the rate of p21 degradation. To explore this possibility, 697-Bcl-2 and 697-Neo cells were metabolically labeled with L-[S]methionine for 2 h, and p21 protein was immunoprecipitated immediately (0 h) or at various times after chasing with unlabeled L-methionine. As shown in Fig. 4A, the relative rates of [S]methionine incorporation into p21 were not reduced in 697-Neo cells relative to 697-Bcl-2 cells, based on analysis of the Bax protein at 0 h before beginning the chase with unlabeled L-methionine. Thus, the rate of synthesis of p21 protein was not appreciably different for 697-Neo and 697-Bcl-2 cells. In contrast to 0 h, at 6 h after chasing out L-[S]methionine, S-Bax protein levels were substantially reduced in 697-Neo cells but not in 697-Bcl-2 cells (Fig. 4A). By 12 h, S-Bax had almost disappeared from 697-Neo cells and by 24 h no S-Bax was detected, whereas S-Bax protein levels were nearly constant for the first 24 h after chasing out L-[S]methionine in the 697-Bcl-2 cells (t 4 h for Neo versus >24 h for Bcl-2). In parallel with the disappearance of the 21-kDa Bax band in 697-Neo cells upon chasing with unlabeled methionine, a new species of 17 to 18 kDa appeared, suggesting a possible precursor-product relation. Immunoprecipitations performed using normal rabbit serum(N) instead of anti-Bax antiserum or using anti-Bax antiserum that had been preadsorbed with Bax peptide (P) confirmed the specificity of these results, in that no 21-kDa band corresponding to Bax was detected. An additional 26-kDa band was also co-immunoprecipitated with p21 in 697-Bcl-2 but not 697-Neo lysates (Fig. 4A). This 26-kDa band represents Bcl-2 in physical association with p21, based on experiments where the Bax immunoprecipitates were analyzed by immunoblotting using Bcl-2-specific antibodies (data not presented). (Note that a slightly faster migrating protein at the 25-kDa band is also seen in 697-Neo cells which appears to be nonspecific, since it was also present when normal rabbit serum was employed instead of anti-Bax antiserum. This nonspecific protein is also seen to a lesser extent in 697-Bcl-2 cells just below the 26-kDa band corresponding to Bcl-2.) Similar results were obtained for Jurkat-Neo and Jurkat-Bcl-2 cells, except that the rate of degradation of Bax in Jurkat-Neo cells was faster than in 697 cells, precluding an accurate estimate of the half-life of Bax protein under the experimental conditions employed here (not shown).

Figure 4: [S]Methionine pulse-chase analysis of p21 degradation rates in Bcl-2 and Neo transfectants of 697 and CEM leukemia line. In A, Bcl-2 and Neo transfectants of 697 cells were metabolically labeled with L-[S]methionine and either lysed immediately (0 h) or cultured for 6-24 h in medium containing unlabeled methionine prior to preparing detergent lysates and performing immunoprecipitations using anti-human Bax antiserum. In some cases, either preimmune serum (N) was used for immunoprecipitations or the anti-Bax antibody was preadsorbed with 10 µg of Bax peptide (P) as controls for verifying specificity of the antibodies. In the Bax immune complexes prepared from 697-Bcl-2 cells, an additional 26-kDa band was seen which probably represents Bcl-2 protein. In B, Bcl-2 and Neo transfectants of 697 and CEM cells were labeled with L-[S]methionine, and immunoprecipitates were prepared using anti-human Bax antibody immediately (0 h) or after 4 h of culture in medium containing unlabeled methionine. Gels were analyzed by autoradiography (top), and p21 bands were quantified using a -scanner, with the results expressed relative to the 0-h value for each stable transfectant. Data are representative of two experiments.

In addition, the effects of Bcl-2 transfection on p21 protein stability were compared in 697 cells with CEM cells. Since Bcl-2 did not cause elevations in p21 in CEM cells, we would not expect the half-life of p21 to be prolonged in these leukemic cells, unlike in 697 cells where Bcl-2 did increase the relative levels of p21. Again, a [S]methionine pulse-chase analysis was performed, comparing the relative amounts of S-Bax at 0 h before chasing L-[S]methionine and at 4 h after chase, which was chosen for these comparisons because it represents the approximate t of Bax in 697-Neo cells. As shown in Fig. 4B, the relative levels of S-Bax remained essentially unchanged in CEM-Neo and CEM-Bcl-2 cells at 4 h after chasing with unlabeled L-methionine, suggesting that the t of Bax is comparatively long in these cells irregardless of their levels of Bcl-2 protein. In contrast, relative levels of S-Bax were reduced by approximately one-half at 4 h after chasing with unlabeled [S]methionine in 697-Neo cells but not in 697-Bcl-2 cells.

DISCUSSION

The data presented here provide the first evidence that levels of a Bcl-2 family protein, in this case p21, can be regulated through post-translational mechanisms. Gene transfer-mediated elevations in Bcl-2 protein resulted in increases in p21 by decreasing the rate of Bax protein degradation in some types of cell lines. A reasonable speculation therefore is that the interaction of Bcl-2 with p21, leading to formation of heterodimers or complexes with other stoichiometry, somehow stabilizes the Bax protein in some types of cells, thus leading to increases in the steady-state levels p21 protein. We are currently attempting to express a variety of Bcl-2 mutants in Jurkat T-cells that either do or do not retain the ability to bind to Bax as an approach to testing this hypothesis directly. Consistent with this notion, a 26-kDa protein representing Bcl-2 co-immunoprecipitated with Bax in 697-Bcl-2 cells where p21 had a long half-life (t > 24 h) but not in 697-Neo cells where p21 turnover was considerably more rapid (t 4 h).

It remains to be determined what the explanation is for the cell type specificity observed, where Bcl-2-mediated up-regulation of p21 protein levels in some cell lines but not others. Interestingly, even closely related cells such as the human T-cell acute lymphocytic leukemia lines Jurkat and CEM exhibited markedly different responses to Bcl-2 in terms of effects on p21 protein levels. Despite the fact that Jurkat and CEM are both T-cell acute lymphocytic leukemias, however, they possess immunophenotypes consistent with different stages of thymic development, i.e. CD4CD8 (mature thymocytes; Jurkat) and CD4CD8 (immature thymocytes; CEM). Presumably, whatever factors that contribute to rapid degradation of Bax in cell lines such as 697 and Jurkat are not operative in CEM and other cell lines where Bcl-2 did not induce increases in Bax protein levels. These factors could include differences in the levels or activity of (i) proteases, (ii) kinases or phosphatases that theoretically could control phosphorylation of p21, or (iii) non-Bcl-2 family proteins that might bind to p21. Of particular note with regard to mechanisms of protein degradation is the observation that several recently described Bcl-2 binding proteins contain either PEST sequences (Nip-1, Nip-2, Nip-3) or ubiquitin-like domains (BAG-1) which conceivably might target protein complexes that contain these Bcl-2 binding proteins for proteolytic degradation(19, 20) . Moreover, the Bcl-2 homologous protein Mcl-1 contains PEST sequences and has been shown to bind to p21(4, 21) . Alternatively, it could be that in addition to Bcl-2, other antiapoptotic members of the Bcl-2 protein family such as Bcl-X or Mcl-1, which can also heterodimerize with Bax(2, 4) , are constitutively present at higher levels in cell lines such as CEM and these proteins perhaps may similarly stabilize p21 in a manner similar to Bcl-2. In this case, the supposition would be that these other Bcl-2-like proteins are already present at optimal levels for suppressing p21 degradation and that gene transfer-mediated increases in Bcl-2 protein therefore provide no further benefit. Immunoblot experiments not shown here indicate that all of the cell lines tested here express Bcl-X, Mcl-1, or both, although no correlations with Bax levels were apparent. Nevertheless, the more rapid degradation of Bax may be to some extent analogous to the rapid degradation of unpaired subunits of multipolypeptide complexes(22, 23) .

Gene transfer studies in mammalian cells have shown that elevations in Bcl-2 protein increase resistance of cells to various apoptotic stimuli, whereas increases in Bax can accelerate cell death in the setting of growth factor deprivation. Presumably, therefore, the ratio of Bcl-2 to Bax controls the relative sensitivity or resistance of cells to apoptosis. One prediction of the finding that Bcl-2 can induce elevations in p21, therefore, is that it may represent a feedback mechanism for maintaining Bcl-2/Bax ratios within a physiologically appropriate range. Since experimental manipulations that increase Bcl-2 relative to Bax have been shown to prolong cell survival and block apoptosis induced by many insults, the positive effects of Bcl-2 on p21 half-life may help to limit cell longevity and thus reduce the risk of tumorigenesis. Alternatively, it has not been excluded that Bcl-2/Bax heterodimers represent a molecular entity that promotes cell survival, not unlike the - and -chains of some cell surface receptors such as integrins and T-cell antigen receptors. In this case, Bcl-2-mediated up-regulation of p21 would constitute a positive feedback loop. Thus, until the functional properties of Bax/Bax homodimers, Bax/Bcl-2 heterodimers, and Bcl-2/Bcl-2 homodimers are better understood in terms of their biochemical influence on cell death pathways, it is difficult to predict the physiological consequences of the Bcl-2-mediated stabilization of the p21 protein described here. Nevertheless, these findings indicate that it may be important to determine the effects of Bcl-2 on Bax protein levels before interpreting the biological significance of experiments where gene transfer-mediated elevations in Bcl-2 are employed to assess the relative Bcl-2 dependence or independence of various apoptotic stimuli and pathways.

FOOTNOTES

*

This work was supported in part by NCI, National Institutes of Health Grant CA-60381. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Scholar of the Leukemia Society of America. To whom correspondence should be addressed: La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-455-6480 (Ext. 430); Fax: 619-450-4693.

()

The abbreviations used are: FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); kbp, kilobase pair(s).

ACKNOWLEDGEMENTS

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