Medical Biology - Research Publications
Permanent URI for this collection
Search Results
1 - 7 of 7
-
ItemUltraviolet radiation triggers apoptosis of fibroblasts and skin keratinocytes mainly via the BH3-only protein Noxa(ROCKEFELLER UNIV PRESS, 2007-02-12)To identify the mechanisms of ultraviolet radiation (UVR)-induced cell death, for which the tumor suppressor p53 is essential, we have analyzed mouse embryonic fibroblasts (MEFs) and keratinocytes in mouse skin that have specific apoptotic pathways blocked genetically. Blocking the death receptor pathway provided no protection to MEFs, whereas UVR-induced apoptosis was potently inhibited by Bcl-2 overexpression, implicating the mitochondrial pathway. Indeed, Bcl-2 overexpression boosted cell survival more than p53 loss, revealing a p53-independent pathway controlled by the Bcl-2 family. Analysis of primary MEFs lacking individual members of its BH3-only subfamily identified major initiating roles for the p53 targets Noxa and Puma. In the transformed derivatives, where Puma, unexpectedly, was not induced by UVR, Noxa had the dominant role and Bim a minor role. Furthermore, loss of Noxa suppressed the formation of apoptotic keratinocytes in the skin of UV-irradiated mice. Collectively, these results demonstrate that UVR activates the Bcl-2-regulated apoptotic pathway predominantly through activation of Noxa and, depending on cellular context, Puma.
-
ItemMitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak(ROCKEFELLER UNIV PRESS, 2007-04-23)The Bcl-2 family regulates apoptosis by controlling mitochondrial integrity. To clarify whether its prosurvival members function by sequestering their Bcl-2 homology 3 (BH3)-only ligands or their multidomain relatives Bak and Bax, we analyzed whether four prosurvival proteins differing in their ability to bind specific BH3 peptides or Bak could protect isolated mitochondria. Most BH3 peptides could induce temperature-dependent cytochrome c release, but permeabilization was prevented by Bcl-x(L), Bcl-w, Mcl-1, or BHRF1. However, their protection correlated with the ability to bind Bak rather than the added BH3 peptide and could be overcome only by BH3 peptides that bind directly to the appropriate prosurvival member. Mitochondria protected by both Bcl-x(L)-like and Mcl-1 proteins were disrupted only by BH3 peptides that engage both. BH3-only reagents freed Bak from Bcl-x(L) and Mcl-1 in mitochondrial and cell lysates. The findings support a model for the control of apoptosis in which certain prosurvival proteins sequester Bak/Bax, and BH3-only proteins must neutralize all protective prosurvival proteins to allow Bak/Bax to induce mitochondrial disruption.
-
ItemBcl-2-regulated apoptosis and cytochrome c release can occur independently of both caspase-2 and caspase-9(ROCKEFELLER UNIV PRESS, 2004-06-21)Apoptosis in response to developmental cues and stress stimuli is mediated by caspases that are regulated by the Bcl-2 protein family. Although caspases 2 and 9 have each been proposed as the apical caspase in that pathway, neither is indispensable for the apoptosis of leukocytes or fibroblasts. To investigate whether these caspases share a redundant role in apoptosis initiation, we generated caspase-2(-/-)9(-/-) mice. Their overt phenotype, embryonic brain malformation and perinatal lethality mirrored that of caspase-9(-/-) mice but were not exacerbated. Analysis of adult mice reconstituted with caspase-2(-/-)9(-/-) hematopoietic cells revealed that the absence of both caspases did not influence hematopoietic development. Furthermore, lymphocytes and fibroblasts lacking both remained sensitive to diverse apoptotic stimuli. Dying caspase-2(-/-)9(-/-) lymphocytes displayed multiple hallmarks of caspase-dependent apoptosis, including the release of cytochrome c from mitochondria, and their demise was antagonized by several caspase inhibitors. These findings suggest that caspases other than caspases 2 and 9 can promote cytochrome c release and initiate Bcl-2-regulated apoptosis.
-
ItemFas ligand, Bcl-2, granulocyte colony-stimulating factor, and p38 mitogen-activated protein kinase: Regulators of distinct cell death and survival pathways in granulocytes(ROCKEFELLER UNIV PRESS, 2000-09-04)The short life span of granulocytes, which limits many inflammatory responses, is thought to be influenced by the Bcl-2 protein family, death receptors such as CD95 (Fas/APO-1), stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), and proinflammatory cytokines like granulocyte colony-stimulating factor (G-CSF). To clarify the roles of these various regulators in granulocyte survival, we have investigated the spontaneous apoptosis of granulocytes in culture and that induced by Fas ligand or chemotherapeutic drugs, using cells from normal, CD95-deficient lpr, or vav-bcl-2 transgenic mice. CD95-induced apoptosis, which required receptor aggregation by recombinant Fas ligand or the membrane-bound ligand, was unaffected by G-CSF treatment or Bcl-2 overexpression. Conversely, spontaneous and drug-induced apoptosis occurred normally in lpr granulocytes but were suppressed by G-CSF treatment or Bcl-2 overexpression. Although activation of p38 MAPK has been implicated in granulocyte death, their apoptosis actually was markedly accelerated by specific inhibitors of this kinase. These results suggest that G-CSF promotes granulocyte survival largely through the Bcl-2-controlled pathway, whereas CD95 regulates a distinct pathway to apoptosis that is not required for either their spontaneous or drug-induced death. Moreover, p38 MAPK signaling contributes to granulocyte survival rather than their apoptosis.
-
ItemPro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-xL(ROCKEFELLER UNIV PRESS, 2000-05-01)How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-x(L) could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.
-
ItemTHE E-MU-MYC TRANSGENIC MOUSE - A MODEL FOR HIGH-INCIDENCE SPONTANEOUS LYMPHOMA AND LEUKEMIA OF EARLY B-CELLS(ROCKEFELLER UNIV PRESS, 1988-02-01)Mice transgenic for a c-myc gene driven by the IgH enhancer (E mu-myc) were shown to almost invariably develop lymphomas, 90% succumbing in the first 5 mo of life. The tumors typically presented as rapidly progressive lymphadenopathy with thymic involvement and were highly malignant by transplantation assay. Morphologically, they were lymphoblastic lymphomas, usually accompanied by lymphoid leukemia and granulocytosis, and were distinct from the tumors that arose much later in 37% of nontransgenic mice of the same (C57BL/6 x SJL)F2 genetic background. Cell-surface markers on 31 E mu-myc tumors identified 52% as pre-B lymphomas, 29% as mixed pre-B and B lymphomas, and 19% as B lymphomas. The tumors appeared to arise at random from a population of pre-B cells expanded by constitutive expression of the myc transgene. A majority of the animals initiated malignancy at the rate of 17% per week. The rate at which the cycling, benign pre-B cells spontaneously convert to malignancy was estimated to about 10(-10) per cell per generation. A transient leukocytosis identified in young E mu-myc mice was developed into a rapid assay for inheritance of the transgene.
-
ItemA SEARCH FOR MESSENGER-RNA MOLECULES BEARING IMMUNOGLOBULIN VH NUCLEOTIDE-SEQUENCES IN T-CELLS(ROCKEFELLER UNIV PRESS, 1982)Expression of VH-coded mRNA molecules in T cells, antigen-specific T cell lines, or T cell hybridomas was not detected using four different VH DNA probes under conditions that permitted cross-hybridization between distantly related VH genes. In contrast, VH gene expression was readily detected in two B cell lymphomas and in splenic B cells. Less than one molecule per cell of RNA, exactly complementary to the DNA probes used, would have been detected in these T cell populations. The results thus seriously question the proposition that T cells use the B cell VH repertoire to code for antigen receptors.