Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry - PubMed (original) (raw)

Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry

Yelena M Janumyan et al. EMBO J. 2003.

Abstract

Bcl-x(L) and Bcl-2 inhibit both apoptosis and proliferation. In investigating the relationship between these two functions of Bcl-x(L) and Bcl-2, an analysis of 24 Bcl-x(L) and Bcl-2 mutant alleles, including substitutions at residue Y28 previously reported to selectively abolish the cell cycle activity, showed that cell cycle delay and anti-apoptosis co-segregated in all cases. In determining whether Bcl-2 and Bcl-x(L) act in G(0) or G(1), forward scatter and pyronin Y fluorescence measurements indicated that Bcl-2 and Bcl-x(L) cells arrested more effectively in G(0) than controls, and were delayed in G(0)-G(1) transition. The cell cycle effects of Bcl-2 and Bcl-x(L) were reversed by Bad, a molecule that counters the survival function of Bcl-2 and Bcl-x(L). When control and Bcl-x(L) cells of equivalent size and pyronin Y fluorescence were compared, the kinetics of cell cycle entry were similar, demonstrating that the ability of Bcl-x(L) and Bcl-2 cells to enhance G(0) arrest contributes significantly to cell cycle delay. Our data suggest that cell cycle effects and increased survival both result from intrinsic functions of Bcl-2 and Bcl-x(L).

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Figures

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Fig. 1. Y28F-Bcl-2 inhibits Myc-induced cell cycle entry and Myc-induced apoptosis. Rat1MycER cells expressing pBabe/wtBcl-2, pBabe/Y28F-Bcl-2 or pBabe vector were cultured in αMEM media containing 0.05% FBS for 3 days, then 1 µM 4-OHT was added. (A) Cells were harvested before serum starvation (10%), after 3 days of serum starvation just prior to addition of 4-OHT (0 h) and at the indicated times after MycER induction, and cell cycle profiles were obtained by PI/FACS analysis. (B) Graph of the percentages of cells in S phase obtained from Modfit analysis of data points in (A). (C) Percentages of apoptotic cells as determined by PI exclusion assays are compared between asynchronously growing cells and cells that have been serum-starved and treated with 4-OHT. Data represent averages of two experiments. Similar results were obtained with Annexin V staining and measuring sub-G1 DNA content.

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Fig. 2. Y28F-Bcl-2 and Y28A-Bcl-2 delay serum-induced cell cycle entry. Rat1MycER cells were serum starved as in Figure 1, but were induced to re-enter the cell cycle by adding 10% FBS. (A) PI/FACS cell cycle profiles before serum starvation (10%), after serum starvation (0 h) and at the indicated times after serum addition are shown. (B) Graph of percentages of S phase cells by Modfit analysis of data from (A). (C) NIH 3T3 cells expressing wtBcl-2 or Y28A-Bcl-2 were serum-starved and released with 10% serum. The percentages of cells incorporating BrdU at the indicated times, normalized against control cells at 36 h, are shown. One of several representative experiments is shown.

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Fig. 3. Cell cycle delay co-segregated with anti-apoptosis in Bcl-xL and Bcl-2 mutants. Rat1MycER cells harboring retrovirally introduced Bcl-xL or Bcl-2 wild-type or mutant cDNAs were serum-starved and induced to enter the cell cycle by 4-OHT. (A) Cells were assayed by BrdU incorporation and/or PI/FACS analysis or after (0 h) serum starvation and at the indicated times after 4-OHT addition. BrdU/PI profiles for cells expressing pBabe vector alone, wtBcl-xL, and representative mutations that had no effect (G187A), significant effect (G138E,R139L,I149N) or partial effect (ΔC21) on the cell cycle delay activity of Bcl-xL are shown. (B) Percentages of BrdU-positive cells from (A) are plotted against duration of MycER induction (top). The percentages of cells with sub-G1 DNA content after MycER induction, normalized to 0 h, are plotted as apoptotic cells (bottom). (C) Results of all the mutants analyzed similarly are shown. ‘+’ denotes that the ability to inhibit MycER-induced apoptosis or MycER-induced cell cycle entry is similar to wt Bcl-xL or wt Bcl-2; ‘–’ denotes that the mutant construct is unable to rescue MycER-induced cell death or delay cell cycle entry. MH refers to mutations generated in Marie Hardwick’s laboratory (Cheng et al., 1996). (D) Western blotting was performed to compare expression of mutant proteins, using 100 µg of total protein and antibodies H5 (Santa Cruz) for Bcl-xL and 6C8 (PharMingen) for Bcl-2.

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Fig. 4. Bcl-2 targeted to the ER or the mitochondria outer membrane retains cell cycle delay activity. Rat1MycER cells expressing wtBcl-2, Bcl-2 fused with the ActA mitochondria targeting sequence (Bcl-2-acta), Bcl-2 fused with the ER-targeting sequence from cytochrome _b_5 (Bcl-2-cyb5) or pBH vector alone were cultured in 0.05% FBS for >48 h, then stimulated with (A) addition of 10% serum or (B) treatment with 1 µM 4-OHT. DNA content was measured by PI staining and FACS analysis. The percentages of S phase cells were obtained from Modfit program. Representative experiments are shown. Percent apoptosis was derived from PI exclusion assays. Standard deviations were obtained from three experiments. Similar results were obtained scoring Annexin V positivity and cells with sub-G1 DNA.

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Fig. 5. Bcl-2 expression retains cells in G0 during serum-induced cell cycle entry. NIH 3T3 cells stably infected with pBabe (NIH 3T3/pBabe) or pBabeBcl-2 (NIH 3T3/Bcl-2) were arrested by culturing in media containing 0.5% serum for 72 h and released by adding 10% serum. Cells were collected at indicated times and stained with both Hoechst and pyronin Y for FACS analysis. (A) Dot-plot of Hoechst against pyronin Y fluorescence for cells before serum starvation (10%), after serum starvation (0 h) and at consecutive time points after serum re-addition. Populations of G0, G1 and G2 cells are indicated. The box gates on cells with 2N DNA content. (B) Histograms of pyronin Y fluorescence of Bcl-2 and controls cells gated for 2N DNA content from (A). (C) Comparison of mean pyronin Y fluorescence of Bcl-2 and control cells with 2N DNA content from (A).

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Fig. 6. Bcl-2 and Bcl-xL prolong G0 during induction of cell cycle entry after release from contact inhibition. Retrovirally infected NIH 3T3 cells stably expressing Bcl-2, Bcl-xL, or pBabe vector were arrested by contact inhibition and released by replating at low density. (A) Cells were stained with Hoechst and pyronin Y for FACS analysis in subconfluent cultures (ctrl), after contact inhibition (C.I.), and at the indicated times after replating in low density. (B) Histograms of pyronin Y fluorescence of cells gated for 2N DNA content from (A).

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Fig. 7. Bad reverses the delay of cell size and RNA increases, and the delay of S phase progression by Bcl-xL. NIH 3T3 cells expressing Bcl-xL, Bad and Bcl-xL, BadL151A and Bcl-xL, Bad alone, BadL151A alone, or vector alone were arrested by serum starvation and released by serum addition. Cells were stained for Hoechst and pyronin Y as in Figures 5 and 6. [(A), top] FSC of cells gated for 2N DNA content at selected time points are compared. Mean FSC channel are compared between cell lines in tabular form. [(A), bottom] Mean pyronin Y fluorescence of cells gated for 2N DNA at selected time points are compared. (B) Cell cycle profiles of cells arrested by serum starvation and released by serum addition, and the percentage of S phase cells at each time point are shown. (C) Western blot Bcl-xL and Bad expression in the indicated cell lines, using 250 000 cells per lane, the antibodies 2A1 for Bcl-xL and 10929 for BAD.

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Fig. 7. Bad reverses the delay of cell size and RNA increases, and the delay of S phase progression by Bcl-xL. NIH 3T3 cells expressing Bcl-xL, Bad and Bcl-xL, BadL151A and Bcl-xL, Bad alone, BadL151A alone, or vector alone were arrested by serum starvation and released by serum addition. Cells were stained for Hoechst and pyronin Y as in Figures 5 and 6. [(A), top] FSC of cells gated for 2N DNA content at selected time points are compared. Mean FSC channel are compared between cell lines in tabular form. [(A), bottom] Mean pyronin Y fluorescence of cells gated for 2N DNA at selected time points are compared. (B) Cell cycle profiles of cells arrested by serum starvation and released by serum addition, and the percentage of S phase cells at each time point are shown. (C) Western blot Bcl-xL and Bad expression in the indicated cell lines, using 250 000 cells per lane, the antibodies 2A1 for Bcl-xL and 10929 for BAD.

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Fig. 8. Enhanced G0 arrest contributes significantly to cell cycle delay. (A) FSC profiles of contact inhibited NIH 3T3 cells expressing Bcl-xL or vector were compared (a) and cells were sorted by FSC into ‘small’ and ‘large’ populations. Markers indicate the FSC windows used for sorting. The result of sorting was checked by re-analyzing FSC and pyronin Y staining of the sorted cells. FSC of sorted small cells (b) and large cells (c), and pyronin Y fluorescence of sorted small cells (e) were overlayed to confirm that the sorted pBabe and Bcl-xL cells were comparable. Pyronin Y fluorescence of unsorted pBabe and Bcl-xL cells is also shown for comparison (d). (B) Sorted small and large cells were replated at low density, collected at the times indicated, and analyzed by PI/FACS. Small pBabe and small Bcl-xL cells were compared by overlaying DNA profiles (top panel). DNA profiles of small and large pBabe cells were compared with each other (lower panel). The profiles shown are from one representative experiment among several.

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References

    1. Asoh S., Ohtsu,T. and Ohta,S. (2000) The super anti-apoptotic factor Bcl-xFNK constructed by disturbing intramolecular polar interactions in rat Bcl-xL. J. Biol. Chem., 275, 37240–37245. - PubMed
    1. Borner C., Martinou,I., Mattmann,C., Irmler,M., Schaerer,E., Martinou,J.C. and Tschopp,J. (1994) The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis. J. Cell Biol., 126, 1059–1068. - PMC - PubMed
    1. Brady H.J., Gil-Gomez,G., Kirberg,J. and Berns,A.J. (1996) Bax alpha perturbs T cell development and affects cell cycle entry of T cells. EMBO J., 15, 6991–7001. - PMC - PubMed
    1. Chattopadhyay A., Chiang,C.W. and Yang,E. (2001) BAD/BCL-XL heterodimerization leads to bypass of G0/G1 arrest. Oncogene, 20, 4507–4518. - PubMed
    1. Cheng E.H., Levine,B., Boise,L.H., Thompson,C.B. and Hardwick,J.M. (1996) Bax-independent inhibition of apoptosis by Bcl-XL. Nature, 379, 554–556. - PubMed

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