Bax promotes cell death by permeabilizing mitochondrial outer membranes by an unresolved mechanism. However, in cells lacking the gene c-myc, membrane permeabilization by Bax is blocked by changes in the mitochondria that prevent Bax oligomerization. Drug-treated c-myc null cells and cells expressing Myc were used to map the topology of Bax in membranes prior to and after mitochondrial permeabilization. Chemical labeling of single cysteine mutants of Bax using a membrane bilayer impermeant cysteine-specific modifying agent revealed that Bax inserted both the 'pore domain' (helices alpha5-alpha6), and the tail-anchor (helix alpha9) into membranes prior to oligomerization and membrane permeabilization. Additional topology changes for Bax were not required in Myc-expressing cells to promote oligomerization and cytochrome c release. Our results suggest that unlike most pore-forming proteins, Bax membrane permeabilization results from oligomerization of transmembrane monomers rather than concerted insertion of the pore domains of a preformed oligomer.

No abstract

In metazoan organisms, terminal differentiation is generally tightly linked to cell cycle exit, whereas the undifferentiated state of pluripotent stem cells is associated with unlimited self-renewal. Here, we report that combined deficiency for the transcription factors MafB and c-Maf enables extended expansion of mature monocytes and macrophages in culture without loss of differentiated phenotype and function. Upon transplantation, the expanded cells are nontumorigenic and contribute to functional macrophage populations in vivo. Small hairpin RNA inactivation shows that continuous proliferation of MafB/c-Maf deficient macrophages requires concomitant up-regulation of two pluripotent stem cell-inducing factors, KLF4 and c-Myc. Our results indicate that MafB/c-MafB deficiency renders self-renewal compatible with terminal differentiation. It thus appears possible to amplify functional differentiated cells without malignant transformation or stem cell intermediates.

Differentiated macrophages can self-renew in tissues and expand long-term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. Single cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.

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¯uorescence 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 Ȳ uorescence 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 signi®cantly 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 .

The ability of the c-Myc oncoprotein to potentiate apoptosis has been well documented; however, the mechanism of action remains ill defined. We have previously identified spatially distinct apoptotic pathways within the same cell that are differentially inhibited by Bcl-2 targeted to either the mitochondria (Bcl-acta) or the endoplasmic reticulum (Bcl-cb5). We show here that in Rat1 cells expressing an exogenous c-myc allele, distinct apoptotic pathways can be inhibited by Bcl-2 or Bcl-acta yet be distinguished by their sensitivity to Bcl-cb5 as either susceptible (serum withdrawal, taxol, and ceramide) or refractory (etoposide and doxorubicin). Myc expression and apoptosis were universally associated with Bcl-acta and not Bcl-cb5, suggesting that Myc acts downstream at a point common to these distinct apoptotic signaling cascades. Analysis of Rat1 c-myc null cells shows these same death stimuli induce apoptosis with characteristic features of nuclear condensation, membrane blebbing, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation; however, this Myc-independent apoptosis is not inhibited by Bcl-2. Among the known proto-oncogenes, the cellular myc gene (c-myc) is one of those most frequently implicated in carcinogenesis. Deregulated expression of the structurally unaltered Myc protein is sufficient to drive continuous cell proliferation and programmed cell death in response to growth-promoting and growth-inhibitory signals, respectively. As a regulator of gene transcription, Myc is thought to drive such disparate activities by controlling distinct subsets of target genes. The ability of Myc to trigger apoptosis is key to the control of tumor development. Apoptosis is thought to function as an intrinsic safety mechanism to limit the life of a cell that acquires deregulated c-myc expression, thus preventing further transformation. However, loss of this function through any additional mutation that prevents Myc from triggering apoptosis will promote survival and strongly cooperate with Myc to allow the continued proliferation, mutation, and carcinogenic evolution of the affected clone (4,5,12,18,19,25,27,37,47,49).The apoptotic process can be divided into three interdependent phases: induction, decision, and execution. For simplicity, inducers have been categorized as those that trigger apoptosis through death receptor activation (e.g., CD95 and tumor necrosis factor receptor) and those that stimulate apoptosis by a non-death receptor mechanisms (e.g., chemotherapeutic drugs, metabolic inhibitors, and withdrawal of survival factors). The decision phase is largely regulated by the Bcl-2 family of apoptotic regulators, including both pro-and antiapoptotic members. These molecules integrate a wide variety of signaling cascades and, through protein-protein interaction and subcellular localization, determine whether the balance of signals dictates that cell death will proceed or terminate. A hallmark of the execution phase is the activation of caspases and their substrates that essentially dissolve the norma...

No abstract

No abstract