A comparison of Akt-and Bcl-x L -dependent cell survival was undertaken using interleukin-3-dependent FL5.12 cells. Expression of constitutively active Akt allows cells to survive for prolonged periods following growth factor withdrawal. This survival correlates with the expression level of activated Akt and is comparable in magnitude to the protection provided by the antiapoptotic gene Bcl-x L . Although both genes prevent cell death, Akt-protected cells can be distinguished from Bclx L -protected cells on the basis of increased glucose transporter expression, glycolytic activity, mitochondrial potential, and cell size. In addition, Akt-expressing cells require high levels of extracellular nutrients to support cell survival. In contrast, Bcl-x L -expressing cells deprived of interleukin-3 survive in a more vegetative state, in which the cells are smaller, have lower mitochondrial potential, reduced glycolytic activity, and are less dependent on extracellular nutrients. Thus, Akt and Bcl-x L suppress mitochondrion-initiated apoptosis by distinct mechanisms. Akt-mediated survival is dependent on promoting glycolysis and maintaining a physiologic mitochondrial potential. In contrast, Bcl-x L maintains mitochondrial integrity in the face of a reduced mitochondrial membrane potential, which develops as a result of the low glycolytic rate in growth factordeprived cells.There is increasing evidence that tissue homeostasis in multicellular organisms is controlled by the availability of growth factors (1). Within a given tissue, high levels of relevant growth factors promote increased cellular mass, metabolism, and proliferation. In contrast, when the availability of growth factor becomes limiting, cellular atrophy and an increased rate of apoptosis are observed. Many growth factors affect cellular responses through receptor-mediated recruitment and activation of the phosphoinositide 3-kinase (PI3K) 1 and the serine/ threonine kinase Akt (2, 3). Once activated, Akt can phosphorylate substrates involved in controlling a variety of cellular processes, including cellular metabolism and survival (4). The PI3K/Akt pathway is an important regulator of cellular homeostasis in vivo, as activating mutations of this pathway are correlated with multiple types of cancer. Akt itself was first identified as a viral oncogene, and deficiency in PTEN, an inositol phosphatase that opposes the activity of PI3K, is frequently found in numerous types of advanced cancers (5-7).Akt activation by growth factor receptors prevents apoptosis by blocking the release of cytochrome c (8). A number of molecular targets for the inhibition of apoptosis by Akt have been proposed. Akt phosphorylates and inactivates pro-apoptotic proteins, including Bad and Forkhead family transcription factors (9, 10). In addition, Akt has been reported to stimulate the expression of anti-apoptotic Bcl-2 proteins, such as Bcl-x L and Mcl-1, through the activation of NF-B (11, 12). However, it is not clear if these targets entirely account for the effects of Akt on cell s...

To identify genes that contribute to apoptotic resistance, IL-3 dependent hematopoietic cells were transfected with a cDNA expression library and subjected to growth factor withdrawal. Transfected cells were enriched for survivors over two successive rounds of IL-3 withdrawal and reconstitution, resulting in the identification of a full-length elongation factor 1 alpha (EF-1a) cDNA. Ectopic EF-1a expression conferred protection from growth factor withdrawal and agents that induce endoplasmic reticulum stress, but not from nuclear damage or death receptor signaling. Overexpression of EF-1a did not lead to growth factor independent cell proliferation or global alterations in protein levels or rates of synthesis. These findings suggest that overexpression of EF-1a results in selective resistance to apoptosis induced by growth factor withdrawal and ER stress.

To analyse individual factors that may contribute to leukemic transformation in vivo, we have developed a murine model of leukemogenesis based on the early hematopoietic precursor cell FL5.12. FL5.12 cells are interleukin-3 (IL-3) dependent for growth, proliferation, and survival. Relative resistance to cell death following IL-3 withdrawal can be conferred by either overexpression of the Bcl-x L apoptotic inhibitor, or constitutive activation of the serine/threonine kinase Akt. The ability of Bcl-x L or a constitutively active myristylated Akt to promote leukemic transformation of FL5.12 cells was compared in athymic nu + /nu + mice. Bcl-x L alone could not promote leukemic transformation, but mice injected with FL5.12 cells overexpressing Bcl-x L and a dominant-negative p53 construct developed leukocytosis and blastic infiltration of lymph nodes, spleen, and liver with features of a highgrade lymphoid malignancy. In contrast to the cells injected into these animals, cell lines derived from the mice were able to proliferate in the absence of IL-3, and were found to have constitutively activated Akt. This constitutive activation was associated with a variety of alterations of the signaling pathway regulating Akt activity, including alterations of PTEN mRNA and protein expression. In addition, some of these leukemic clones demonstrated concurrent constitutive upregulation of ERK activity. A constitutively active Akt construct introduced into FL5.12 cells promoted similar clonal expansion in vivo, with emergence of clonal IL-3-independent proliferation. Bclx L and Akt appeared to function cooperatively in this model, enhancing rapid clonal outgrowth in vivo relative to Akt alone. These results implicate activated Akt and growth-factor independence in leukemogenic transformation, and demonstrate the potential for in vivo analysis of genetic determinants of leukemogenesis.