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Diminished Cell Proliferation Associated with the Death-protective Activity of Bcl-2*

The oncogene product Bcl-2 effectively spares cells from programmed cell death (apoptosis). The molecular mechanism underlying this death-protective activity has, however, remained enigmatic. Here we show that induction of Bcl-2 expression is consistently associated with a retardation of mammalian cell proliferation due to a prolongation of the G 1 phase of the cell cycle. Whereas cells lacking Bcl-2 expression die from any point of the cell cycle in response to apoptotic agents, Bcl-2-overexpressing cells accumulate in the G 0 /G 1 phase and are protected from cell death. Co-expression of Bax, a negative regulator of Bcl-2, reverts both the cell death protective and proliferation retarding activities of Bcl-2. Moreover, a Bcl-2 mutant defective in death protection does not affect cell division. These findings indicate that Bcl-2 contributes to cell survival by dimin-ishing the rate of cell proliferation.

The oncogene product Bcl-2 effectively spares cells from programmed cell death (apoptosis). The molecular mechanism underlying this death-protective activity has, however, remained enigmatic. Here we show that induction of Bcl-2 expression is consistently associated with a retardation of mammalian cell proliferation due to a prolongation of the G 1 phase of the cell cycle. Whereas cells lacking Bcl-2 expression die from any point of the cell cycle in response to apoptotic agents, Bcl-2-overexpressing cells accumulate in the G 0 /G 1 phase and are protected from cell death. Co-expression of Bax, a negative regulator of Bcl-2, reverts both the cell death protective and proliferation retarding activities of Bcl-2. Moreover, a Bcl-2 mutant defective in death protection does not affect cell division. These findings indicate that Bcl-2 contributes to cell survival by diminishing the rate of cell proliferation.
Programmed cell death (apoptosis) exhibits a series of morphological features in common with mitosis. These include cell rounding, surface blebbing, nuclear lamina disassembly, and chromatin condensation (1)(2)(3). In addition, a variety of gene products involved in the transition from quiescence to proliferation (immediate early genes) are activated during apoptosis (4,5). Fundamental differences have, however, been noted between mitosis and apoptosis. For a proper proceeding of mitosis, cell cycle components such as cyclins, protein kinases, and phosphatases have to be activated and inactivated in limited amounts at specific time points (6), and cells can pass to the next phase of the cell cycle only when the previous one has been successfully completed (checkpoint controls) (7). By contrast, during apoptosis, cell cycle components are frequently activated prematurely, leading to an abortive cell cycle that fails to enter S phase (8,9) or to perturb G 2 or M phases (10). Moreover, DNA damage is mostly left unrepaired due to an improper functioning of the repair machinery and/or the activation of specific endonucleases that cleave DNA into large (50 -300-kilobase) or small (200-base pair) fragments (11,12). These observations indicate that cycling cells often encounter a crossroads of apoptosis and mitosis where the decision for the subsequent fate is made. If anti-apoptotic survival factors act at this point, cells are rescued from apoptosis (13). This can be due to a direct inhibition of positive regulators of apoptosis such as proteases and endonucleases, as has been shown for the survival factors cowpox virus serpin cytokine response modifier A (crmA) (14) or the baculovirus p35 (15). Alternatively, survival factors may actively interfere with cell proliferation in order to diminish the chance of a cell to encounter the crossroads of mitosis and apoptosis.
The oncogene product Bcl-2 effectively saves cells from apoptosis (16). The molecular basis of its death-sparing activity has been proposed to be an antioxidant function (17). However, the findings that Bcl-2 still protects cells from apoptosis under anaerobic conditions (18,19) suggest yet other molecular functions of Bcl-2.
Here we test a possible anti-proliferative function of Bcl-2 and find that overexpression of Bcl-2 indeed markedly retards cell division of several mammalian cell lines under nonstressed and apoptotically stressed conditions. This effect is specific since co-expression of the Bcl-2 antagonist Bax mitigates both the anti-proliferative and anti-apoptotic activities of Bcl-2, and a survival-deficient Bcl-2 mutant has no effect on cell division.
The U937 cells co-expressing Bax and Bcl-2 (U937-Bax/wtBcl-2mix) were generated as follows: 5 ϫ 10 5 U937-wtBcl-2mix cells were transfected with the expression plasmid pCB6neo (21) containing the human Bax cDNA. A mixed population of G418-resistant transfectants was established and probed for the expression of Bax by immunoblotting using a polyclonal anti-human Bax antiserum provided by J. C. Reed (22). U937-Bax/wtBcl-2 cells were re-examined for Bcl-2 expression by immunoblotting and revealed similar basal and zinc-induced Bcl-2 levels as U937-wtBcl-2mix cells. Anti-Bcl-2 and anti-Bax immunoblots were performed with extracts from cells treated with or without zinc for 24 h.
Analysis of Cell Proliferation and Viability-Cells were seeded at a * This work was supported by Swiss National Science Foundation Grants 31-34600.92 and 31-36152.92 and Swiss Cancer League Grant 421. 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. density of 0.5-1 ϫ 10 5 cells per 35-mm plate (diameter) in 3 ml of Dulbecco's modified Eagle's medium plus 10% fetal calf serum (without hygromycin). They were counted on triplicate plates with media changes every 2nd day. Cell viability was determined by trypan blue exclusion. Extraction of total protein and immunoblotting were performed as described previously (20).
Generation of Mutant Bcl-2-The mutant Bcl-2 (G142E) was created by site-directed mutagenesis using the Transformer TM kit of Clontech according to the protocol provided by the manufacturer. The mutagenic primer was GGGGTGAACTGGGAGAGGATTGTGGCC which changed glycine to glutamic acid at position 142 of murine Bcl-2. This point mutation lies within the conserved BH1 region of the Bcl-2 family members and has previously been shown to mitigate the survival activity of Bcl-2 (24). The mutant Bcl-2 cDNA was subcloned into pMEP and transfected into U937 cells creating U937-mutBcl-2mix cells as described above.
Cell Cycle Analysis-For flow cytometry, subconfluent cells were untreated or treated with 100 nM staurosporine for 24 h. Subsequently, cells were harvested, permeabilized, stained with 20 M propidium iodide for 15 min, and analyzed by flow cytometry as described (25). Cell viability was determined by trypan blue exclusion. For the induction of DNA synthesis by thymidine incorporation, serum-starved subconfluent R6pMV12#5 and R6-Bcl-2#9 cells were either stimulated or not with 10% fetal calf serum, and, 12-24 h later, [ 3 H]thymidine incorporation into the DNA of these cells was determined exactly as described (26).

RESULTS AND DISCUSSION
We initially observed that slowly proliferating subclones of the rat 6 embryo fibroblast (R6) cell line were more resistant to cell death (apoptosis) than rapidly proliferating subclones of the same cell line (data not shown). To substantiate this inverse correlation between cell proliferation and cell survival, we introduced into five different mammalian cell lines the mouse counterpart of the survival factor Bcl-2. Cloned cell lines and mixed cell populations which stably overexpressed Bcl-2 ( Fig. 1, insets) all grew slower than their vector control counterparts as measured by cell counting (Fig. 1). A direct corre-lation between the degree of proliferation and the level of Bcl-2 overexpression was difficult to assess because the endogenous expression of factors which impede Bcl-2 function were unknown. The most dramatic step down of cell proliferation following Bcl-2 overexpression was seen in transformed cell lines (L929 fibroblastoid, JILY lymphoblastoid, or U937 monocytic lymphoma) (Fig. 1B). In these cells, only low to moderate expression levels of Bcl-2 could be achieved following Bcl-2 cDNA transfer (Fig. 1B) suggesting that the clonal outgrowth of transformed cells is impeded by Bcl-2 overexpression.
To test whether diminished cell proliferation is a direct consequence of Bcl-2 expression, we created U937 cells in which Bcl-2 expression was inducible. For that purpose we used the expression vector pMEP which contains a zinc-inducible metallothionein promoter. When U937 cells transfected with the vector alone were treated with 100 M ZnSO 4 , the rate of proliferation was the same as with untreated cells (compare Figs. 1B and 2A, control ϩ zinc). U937 cells carrying the pMEP vector with the wild-type murine Bcl-2 cDNA were slightly compromised in cell division due to a basal expression level of Bcl-2 ( Fig. 2A, wtBcl-2). This impediment on cell proliferation was strongly accentuated when the latter cells were exposed to 100 M ZnSO 4 to further induce Bcl-2 expression ( Fig. 2A,  wtBcl-2 ϩ zinc). Importantly, induced expression of the inactive Bcl-2 mutant G142E (24) did not cause retardation of U937 cell proliferation ( Fig. 2A, mutBcl-2 ϩ zinc).
Bax is a Bcl-2 homolog that mitigates the survival activity of Bcl-2, presumably through heterodimerization with Bcl-2 (27). We therefore generated cell lines which constitutively overexpressed Bax to investigate whether this would revert the inhibitory effect of Bcl-2 on cell proliferation. Parental, vector control, and Bcl-2-overexpressing U937 cells all displayed low endogenous expression levels of Bax (Fig. 2B, inset). Transfec-

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tion of human Bax into U937-Bcl-2mix cells yielded a mixed population of hygromycin-and G418-resistant cells (U937-Bax/ wtBcl-2mix) which constitutively overexpressed Bax (Fig. 2B,  inset) and could still be induced by zinc to express high levels of Bcl-2 (data not shown). In the presence of 100 M ZnSO 4 , the U937-Bax/wtBcl-2mix cells grew at a rate similar to vector control cells, indicating that Bax had reverted the inhibitory effect of Bcl-2 on cell proliferation (Fig. 2B, Bax/wtBcl-2 ϩ  zinc). This is most likely due to a neutralization of the Bcl-2 activity by heterodimerization with Bax since overexpression of Bax alone did not accelerate U937 cell proliferation (data not shown). If Bax neutralized the negative impact of Bcl-2 on cell proliferation, it should also allow the cloning of transformed cells with high Bcl-2 expression levels. Indeed, Bcl-2 cDNA transfection into Bax-overexpressing L929 cells now yielded hygromycin-resistant clones which expressed Bcl-2 at 4 -10 times higher levels than in the absence of Bax overexpression (data not shown).
Next we wanted to know whether the impact of Bcl-2 on the retardation of cell proliferation is linked to its survival activity. For that purpose, we concomitantly monitored the survival capacity and the cell cycle state of cells which had been treated with the apoptotic agent staurosporine. Similar results were obtained when cells were exposed to other apoptotic substances such as brefeldin A, okadaic acid or tumor necrosis factor ␣. As evidenced by thymidine incorporation unstressed, Bcl-2-overexpressing R6 cells (R6-Bcl-2#9) exhibited a longer G 1 phase of the cell cycle than vector control cells (R6-pMV12#5) (Fig. 3C). This was confirmed by flow cytometry of the DNA content of asynchronous cell populations (Fig. 3A). Whereas vector control cells randomly distributed throughout the cell cycle with 58% in G 1 phase, 24% in S phase, and 18% in G 2 /M phase, Bcl-2overexpressing cells displayed a higher accumulation in the G 1 phase (70%) with lower mitotic figures (18%). This G 1 prolongation effect was not seen with either the overexpression of the inactive Bcl-2 mutant G142E (data not shown) or the co-overexpression of Bcl-2 and Bax (R6-Bax/Bcl-2#1) (60% G 1 , 22% S,

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18% G 2 /M) (Fig. 3A). Treatment of R6-pMV12#5 with 100 nM staurosporine led to a drastic decrease in cell survival within the first 24 h (Fig. 3D). Flow cytometry after 24 h revealed a high percentage of cells with less than 2n DNA indicating that nuclear/cellular fragmentation into apoptotic bodies had occurred ( Fig. 3B) (25). A significant amount of cells still had DNA contents corresponding to the S and G 2 /M phases of the cell cycle (Fig. 3B). This result is consistent with previous reports that stressed cells do not necessarily pass through G 0 /G 1 before they undergo apoptosis but die in any phase of the cell cycle (13,28). However, R6 cells overexpressing Bcl-2 did not display DNA fragmentation and were spared from staurosporine-induced cell death for the first 24 h (Fig. 3D). These cells exclusively accumulated in the G 0 /G 1 phase during cell death protection (Fig. 3B). Again, Bax co-expression mitigated the effect of a G 0 /G 1 accumulation in Bcl-2-overexpressing cells (Fig. 3B), and cells died in a way similar to vector control cells (Fig. 3D). Thus, cells appear to survive better if the G 0 /G 1 phase is prolonged and Bcl-2 actively contributes to achieve this condition. Indeed, arresting R6-pMV12#5 cells in G 0 /G 1 phase by thymidine block or isoleucine starvation partially rescued the cells from staurosporine-induced apoptosis (data not shown) as had been reported previously for other cell death systems (29,30). Cell death protection by cell cycle blockers has, however, never been as efficient as if Bcl-2 was overexpressed indicating that Bcl-2 controls yet other molecular mechanisms than the cell cycle to save cells from apoptosis. Our findings support the concept that the susceptibility of a cell to apoptosis is influenced by its proliferative state (29 -34). Apoptosis is associated with the activation of growth-related immediate early gene products (c-Fos, c-Myc, c-Jun, etc.) (4,5), and the ectopic expression of positive regulators of cell proliferation such as c-Myc (32,33), ornithine decarboxylase (35), E2F (36), or the adenovirus E1A (37) promotes apoptosis. Moreover, in some cell systems (38,39), including postmitotic neurons (40), cell death appears to be caused by driving the cells from a quiescent G 0 state into the cell cycle, which they are then unable to complete (abortive mitosis). Conversely, arresting cells early in G 1 offers protection from apoptosis (29,30). This is best illustrated by the retinoblastoma gene product (41) and the Abl tyrosine kinase (34,42), both of which keep cells in G 0 /G 1 and act as potent cell survival factors. Here we add the survival factor Bcl-2 to the list of proteins which can prolong the G 0 /G 1 phase. Indeed, it has previously been seen that colon cancer cells overexpressing Bcl-2 are inhibited in their proliferation (43), and that IL-3 removal from Bcl-2-overexpressing BAF3 cells irreversibly arrests the cells in G 1 phase (44). We show here that the retardation in cell proliferation is a direct consequence of Bcl-2 overexpression and inhibitable by its negative regulator Bax. How Bcl-2 performs this action is unknown. Due to its nuclear membrane localization (45), Bcl-2 may interfere with the nuclear transport of critical S phase progression factors as has been shown for Cdc2 and Cdk2 (30). Alternatively, Bcl-2 could impede the Ca 2ϩ signaling required for both apoptosis and proliferation since Bcl-2-overexpressing cells were found to display diminished Ca 2ϩ fluxes across the endoplasmic reticulum membrane (46). By whatever mechanism, by slowing down cell division Bcl-2 may diminish the chance of a cell to encounter a crossroads of mitosis and apoptosis or to allow cells to better deal with the repair of protein, lipid, and DNA which are damaged during apoptotic stresses.
The present results also provide an explanation why hematopoietic tumors such as non-Hodgkin follicular lymphomas and B-CLL which overexpress Bcl-2 are typically of low-grade nature and contain a high portion of cells in a resting or prolonged G 1 state (47). Perhaps Bcl-2 plays a crucial role in promoting the survival of slowly proliferating tumors.