BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.

The protooncogene Bcl-2 functions as a suppressor of apoptosis in growth factor-dependent cells, but a postreceptor signaling mechanism is not known. We recently reported that interleukin 3 (IL-3) and erythropoietin, or the protein kinase C activator bryostatin-1 (Bryo), not only suppresses apoptosis but also stimulates the phosphorylation of Bcl-2 (May, W. S., Tyler, P. G., Ito, T., Armstrong, D. K., Qatsha, K. A., and Davidson, N. E. (1994) J. Biol. Chem. 269, 26865-26870). To test whether phosphorylation is required for Bcl-2 function, conservative serine 3 alanine mutations were produced at the seven putative protein kinase C phosphorylation sites in Bcl-2. Results indicate that the S70A Bcl-2 mutant fails to be phosphorylated after IL-3 or Bryo stimulation and is unable to support prolonged cell survival either upon IL-3 deprivation or etoposide treatment when compared with wild-type Bcl-2. In contrast, a Ser 3 Glu mutant, S70E, which may mimic a potential phosphate charge, more potently suppressed the etoposide-induced apoptosis than wild type in the absence of IL-3. Since the loss of function S70A mutant can heterodimerize with its partner protein and death effector Bax, these findings demonstrate that Bcl-2:Bax heterodimerization is not sufficient and Bcl-2 phosphorylation is required for full Bcl-2 death suppressor signaling activity.

Adults maintain a reservoir of hematopoietic stem cells that can enter the circulation to reach organs in need of regeneration. We developed a novel model of retinal neovascularization in adult mice to examine the role of hematopoietic stem cells in revascularizing ischemic retinas. Adult mice were durably engrafted with hematopoietic stem cells isolated from transgenic mice expressing green fluorescent protein. We performed serial long-term transplants, to ensure activity arose from self-renewing stem cells, and single hematopoietic stem-cell transplants to show clonality. After durable hematopoietic engraftment was established, retinal ischemia was induced to promote neovascularization. Our results indicate that self-renewing adult hematopoietic stem cells have functional hemangioblast activity, that is, they can clonally differentiate into all hematopoietic cell lineages as well as endothelial cells that revascularize adult retina. We also show that recruitment of endothelial precursors to sites of ischemic injury has a significant role in neovascularization.

Phosphorylation of Bcl2 at serine 70 may result from activation of a classic protein kinase C (PKC) isoform and is required for functional suppression of apoptosis by Bcl2 in murine growth factor-dependent cell lines (Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673). Human pre-B REH cells express high levels of Bcl2 yet remain sensitive to the chemotherapeutic agents etoposide, cytosine arabinoside, and Adriamycin. In contrast, myeloid leukemiaderived HL60 cells express less than half the level of Bcl-2 but are >10-fold more resistant to apoptosis induced by these drugs. The mechanism responsible for this apparent dichotomy appears to involve a deficiency of mitochondrial PKC␣ since 1) HL60 but not REH cells contain highly phosphorylated Bcl2; 2) PKC␣ is the only classical isoform co-localized with Bcl2 in HL60 but not REH mitochondrial membranes; 3) the natural product and potent PKC activator bryostatin-1 induces mitochondrial localization of PKC␣ in association with Bcl2 phosphorylation and increased REH cell resistance to drug-induced apoptosis; 4) PKC␣ can directly phosphorylate wild-type but not phosphorylation-negative and loss of function S70A Bcl2 in vitro; 5) stable, forced expression of exogenous PKC␣ induces mitochondrial localization of PKC␣, increased Bcl2 phosphorylation and a >10-fold increase in resistance to drug-induced cell death; and (6) PKC␣-transduced cells remain highly sensitive to staurosporine, a potent PKC inhibitor. Furthermore, treatment of the PKC␣ transformants with bryostatin-1 leads to even higher levels of mitochondrial PKC␣, Bcl2 phosphorylation, and REH cell survival following chemotherapy. While these findings strongly support a role for PKC␣ as a functional Bcl2 kinase that can enhance cell resistance to antileukemic chemotherapy, they do not exclude the possibility that another Bcl2 kinase(s) may also exist. Collectively, these findings identify a functional role for PKC␣ in Bcl2 phosphorylation and in resistance to chemotherapy and suggest a novel target for antileukemic strategies.