ALBERT

Description: Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.

Description: Numerous p53 target genes have been implicated in DNA damage–induced apoptosis signaling, but proapoptotic Bcl-2 (B-cell leukemia 2) family members of the BH3 (Bcl-2 homolog region [BH] 3)–only subgroup appear to play the critical initiating role. In various types of cultured cells, 3 BH3-only proteins, namely Puma (p53 up-regulated modulator of apoptosis), Noxa, and Bim (Bcl-2 interacting mediator of cell death), have been shown to initiate p53-dependent as well as p53-independent apoptosis in response to DNA damage and treatment with anticancer drugs or glucocorticoids. In particular, the absence of Puma or Bim renders thymocytes and mature lymphocytes refractory to varying degrees to death induced in vitro by growth factor withdrawal, DNA damage, or glucocorticoids. To assess the in vivo relevance of these findings, we subjected mice lacking Puma, Noxa, or Bim to whole-body γ-radiation or the glucocorticoid dexamethasone and compared lymphocyte survival with that in wild-type and BCL2–transgenic mice. Absence of Puma or Bcl-2 overexpression efficiently protected diverse types of lymphocytes from the effects of γ-radiation in vivo, and loss of Bim provided lower but significant protection in most lymphocytes, whereas Noxa deficiency had no impact. Furthermore, both Puma and Bim were found to contribute significantly to glucocorticoid-induced killing. Our results thus establish that Puma and Bim are key initiators of γ-radiation– and glucocorticoid-induced apoptosis in lymphoid cells in vivo.

Description: Imatinib mesylate (imatinib) exerts the anti-Philadelphia-positive (Ph1+) leukemia activity both by the inhibition of cell proliferation and by the induction of apoptosis. Recent studies demonstrate that the induction of cell death is essential for eradication of Ph1+ leukemic clones in imatinib treatment; however, the molecular mechanisms have not yet been clearly described. By examining the effect of imatinib on parental K562 and subclones overexpressing either Bcl-2, Bcl-XL or a dominant interfering mutant of FADD/MORT1, which blocks death receptor apoptosis signalling, we found that imatinib triggers apoptosis exclusively via the Bcl-2 family-regulated intrinsic apoptotic pathway. We investigated the involvement of BH3-only proteins as apoptotic initiators in imatinib-induced cell death, because the cell life-or-death decision is arbitrated by the balance between pro-apoptotic BH3 only-proteins and anti-apoptotic Bcl-2 proteins. We found that imatinib treatment upregulated Bim in Ph1+ leukemic cell lines and bcr-c-abl transformed murine fetal liver cells (FLCs)-derived cell lines both by transcriptional and post-translational mechanisms. Imatinib also activated Bad through dephosphorylation and upregulated Bmf transcriptionally. To examine the role of Bim in imatinib-induced apoptosis, we examined the cell killing activity of imatinib in subclones of K562 and BV173 Ph1+ cells expressing abnormally reduced levels of Bim using stable RNA interference system. This revealed that the cell killing activity of imatinib largely dependent on Bim expression levels in these cell lines, although significant apoptosis was still evident. To further define the role of Bim, Bad and Bmf in imatinib-induced cell death, we examined the effect of imatinib on retrovirally bcr-c-abl transformed cell lines derived from FLCs from wild type C57BL/6, Bim-/-, Bad-/-, Bim-/-Bad-/- double KO and Bcl-2 transgenic fetuses. The bim-/-bcr-c-abl+ FLCs were shown to be more resistant to imatinib-induced cell death than wt.bcr-c-abl+ FLCs, however, bim-/-bcr-c-abl+ FLCs were eventually induced into cell death, indicating that Bim is not the only initiator of apoptosis. The bad-/-bcr-c-abl+ FLCs were also partially resistant to imatinib-induced cell death. Intriguingly, like in vav.bcl-2.bcr-c-abl+ FLCs, the cell death induction by imatinib (~5.0μM) was largely abrogated in bim-/-bad-/-bcr-c-abl+ FLCs, indicating that Bim collaborates with Bad for the apoptotic induction by imatinib. Importantly, we found that Bim was inducible by ex vivo imatinib treatment in primary Ph1+ leukemic cells only from clinically good responders but not from patients refractory to imatinib treatment. Collectively, these results demonstrate that Bim is the critical but not the only initiator required for imatinib-induced apoptosis of Bcr/Abl-positive hematopoietic cells; Bad and Bmf may be the ancillary BH3-only proteins in this process. Our results provide evidence for the therapeutic significance of regulation of BH3-only proteins, particularly Bim, for the eradication of Ph1+ leukemic cells.

Description: Although myc and bcl-2 synergize in tumor development, particularly lymphomagenesis, it is not known whether endogenous bcl-2 is required for myc-induced tumorigenesis. To investigate the role of endogenous Bcl-2 in myc-induced lymphomagenesis, we bypassed the early death of Bcl-2–deficient mice by reconstituting lethally irradiated wild-type (wt) mice with a hematopoietic system from fetal liver–derived stem cells of Eμ-myc/bcl-2−/− or control Eμ-myc transgenic embryos. In premalignant (healthy) recipients, loss of Bcl-2 caused a moderate decrease in pre-B and immature B cells, and a dramatic reduction of mature B lymphocytes expressing the Eμ-myc transgene. Furthermore, cultured preneoplastic Eμ-myc/bcl-2−/− mature B cells displayed accelerated apoptosis compared with Eμ-myc B cells. However, despite the striking reduction in B-cell numbers in vivo, ablation of endogenous Bcl-2 did not prevent or even delay development of Eμ-myc lymphoma. Moribund mice presented with similar degrees of splenomegaly, blood leukocyte numbers, and tumor dissemination at death. These findings demonstrate that the initiation, development, continued growth, and severity of Eμ-myc lymphoma do not depend upon endogenous Bcl-2, nor upon the total number of B lymphoid cells driven by the Eμ-myc transgene. These results have implications for the treatment of hematopoietic tumors, particularly those that are not caused by Bcl-2 overexpression.