Different mechanisms for suppression of apoptosis by cytokines and calcium mobilizing compounds

Overexpression of wild-type p53 in M1 myeloid leukemia cells induces apoptotic cell death that was suppressed by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin (TG). This suppression of apoptosis by A23187 or TG was associated with suppression of caspase activation but not with suppression of wild-type-p53-induced expression of WAF-1, mdm-2, or FAS. In contrast to suppression of apoptosis by the cytokines interleukin 6 (IL-6) and interferon γ, a protease inhibitor, or an antioxidant, suppression of apoptosis by A23187 or TG required extracellular Ca2+ and was specifically abolished by the calcineurin inhibitor cyclosporin A. IL-6 induced immediate early activation of junB and zif/268 (Egr-1) but A23187 and TG did not. A23187 and TG also suppressed induction of apoptosis by doxorubicin or vincristine in M1 cells that did not express p53 by a cyclosporin A-sensitive mechanism. Suppression of apoptosis by A23187 or TG was not associated with autocrine production of IL-6. Apoptosis induced in IL-6-primed M1 cells after IL-6 withdrawal was not suppressed by A23187 or TG but was suppressed by the cytokines IL-6, IL-3, or interferon γ. The results indicate that these Ca2+-mobilizing compounds can suppress some pathways of apoptosis suppressed by cytokines but do so by a different mechanism.

Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation

The yeast translation factor eIF4G associates with both the cap-binding protein eIF4E and the poly(A)-binding protein Pab1p. Here we report that the two yeast eIF4G homologs, Tif4631p and Tif4632p, share a conserved Pab1p-binding site. This site is required for Pab1p and poly(A) tails to stimulate the in vitro translation of uncapped polyadenylylated mRNA, and the region encompassing it is required for the cap and the poly(A) tail to synergistically stimulate translation. This region on Tif4631p becomes essential for cell growth when the eIF4E binding site on Tif4631p is mutated. Pab1p mutations also show synthetic lethal interactions with eIF4E mutations. These data suggest that eIF4G mediates poly(A) tail stimulated translation in vitro, and that Pab1p and the domain encompassing the Pab1p-binding site on eIF4G can compensate for partial loss of eIF4E function in vivo.

Cytokine suppression of protease activation in wild-type p53-dependent and p53-independent apoptosis

M1 myeloid leukemic cells overexpressing wild-type p53 undergo apoptosis. This apoptosis can be suppressed by some cytokines, protease inhibitors, and antioxidants. We now show that induction of apoptosis by overexpressing wild-type p53 is associated with activation of interleukin-1β-converting enzyme (ICE)-like proteases, resulting in cleavage of poly(ADP- ribose) polymerase and the proenzyme of the ICE-like protease Nedd-2. Activation of these proteases and apoptosis were suppressed by the cytokine interleukin 6 or by a combination of the cytokine interferon γ and the antioxidant butylated hydroxyanisole, and activation of poly(ADP-ribose) polymerase and apoptosis were suppressed by some protease inhibitors. In a clone of M1 cells that did not express p53, vincristine or doxorubicin induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by interleukin 6. In another myeloid leukemia (7-M12) doxorubicin also induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by granulocyte–macrophage colony-stimulating factor. The results indicate that (i) overexpression of wild-type p53 by itself or treatment with cytotoxic compounds in wild-type p53-expressing or p53-nonexpressing myeloid leukemic cells is associated with activation of ICE-like proteases; (ii) cytokines exert apoptosis-suppressing functions upstream of protease activation; (iii) the cytotoxic compounds induce additional pathways in apoptosis; and (iv) cytokines can also suppress these other components of the apoptotic machinery.

Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development

Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR). It has been postulated that TR-mediated gene regulation involves chromatin remodeling. In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation. During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation. Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles. We further show that TH treatment leads to histone deacetylase release from TH response gene promoters. Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes. On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation. These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

Differential Regulation of Enolase during Anaerobiosis in Maize1

It was reported previously that enolase enzyme activity and ENO1 transcript levels are induced by anaerobic stress in maize (Zea mays). Here we show that not all isoforms of maize enolase are anaerobically induced. We cloned and sequenced a second enolase cDNA clone (pENO2) from maize. Sequence analysis showed that pENO2 shares 75.6% nucleotide and 89.5% deduced amino acid sequence identity with pENO1 and is encoded by a distinct gene. Expression of ENO2 is constitutive under aerobic conditions, whereas ENO1 levels are induced 10-fold in maize roots after 24 h of anaerobic treatment. Western-blot analysis and N-terminal sequencing of in vivo-labeled maize roots identified two major proteins selectively synthesized upon anaerobic stress as isozymes of enolase. We describe the expression of enolase in maize roots under anaerobic stress.

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells1

Anoxia induces a rapid elevation of the cytosolic Ca2+ concentration ([Ca2+]cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca2+ release is important for gene activation and survival in O2-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca2+ to the anoxic [Ca2+]cyt elevation in maize cells. Imaging of intramitochondrial Ca2+ levels showed that a majority of mitochondria released their Ca2+ in response to anoxia and took up Ca2+ upon reoxygenation. We also investigated whether the mitochondrial Ca2+ release contributed to the increase in [Ca2+]cyt under anoxia. Analysis of the spatial association between anoxic [Ca2+]cyt changes and the distribution of mitochondrial and other intracellular Ca2+ stores revealed that the largest [Ca2+]cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca2+]cyt under aerobic conditions but prevented a [Ca2+]cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca2+]cyt and participate in the signaling of O2 deprivation.

Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents.

Apoptosis induced in myeloid leukemic cells by wild-type p53 was suppressed by different cleavage-site directed protease inhibitors, which inhibit interleukin-1 beta-converting enzyme-like, granzyme B and cathepsins B and L proteases. Apoptosis was also suppressed by the serine and cysteine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) [corrected], but not by other serine or cysteine protease inhibitors including N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), E64, pepstatin A, or chymostatin. Protease inhibitors suppressed induction of apoptosis by gamma-irradiation and cycloheximide but not by doxorubicin, vincristine, or withdrawal of interleukin 3 from interleukin 3-dependent 32D non-malignant myeloid cells. Induction of apoptosis in normal thymocytes by gamma-irradiation or dexamethasone was also suppressed by the cleavage-site directed protease inhibitors, but in contrast to the myeloid leukemic cells apoptosis in thymocytes was suppressed by TLCK but not by TPCK. The results indicate that (i) inhibitors of interleukin-1 beta-converting enzyme-like proteases and some other protease inhibitors suppressed induction of apoptosis by wild-type p53 and certain p53-independent pathways of apoptosis; (ii) the protease inhibitors together with the cytokines interleukin 6 and interferon-gamma or the antioxidant butylated hydroxyanisole gave a cooperative protection against apoptosis; (iii) these protease inhibitors did not suppress induction of apoptosis by some cytotoxic agents or by viability-factor withdrawal from 32D cells, whereas these pathways of apoptosis were suppressed by cytokines; (iv) there are cell type differences in the proteases involved in apoptosis; and (v) there are multiple pathways leading to apoptosis that can be selectively induced and suppressed by different agents.

Cellular oxidative stress and the control of apoptosis by wild-type p53, cytotoxic compounds, and cytokines.

Apoptosis induced by wild-type p53 or cytotoxic compounds in myeloid leukemic cells can be inhibited by the cytokines interleukin 6, interleukin 3, granulocyte-macrophage colony-stimulating factor, and interferon gamma and by antioxidants. The antioxidants and cytokines showed a cooperative protective effect against induction of apoptosis. Cells with a higher intrinsic level of peroxide production showed a higher sensitivity to induction of apoptosis and required a higher cytokine concentration to inhibit apoptosis. Decreasing the intrinsic oxidative stress in cells by antioxidants thus inhibited apoptosis, whereas increasing this intrinsic stress by adding H2O2 enhanced apoptosis. Induction of apoptosis by wild-type p53 was not preceded by increased peroxide production or lipid peroxidation and the protective effect of cytokines was not associated with a decrease in these properties. The results indicate that the intrinsic degree of oxidative stress can regulate cell susceptibility to wild-type p53-dependent and p53-independent induction of apoptosis and the ability of cytokines to protect cells against apoptosis.

The control of hematopoiesis and leukemia: from basic biology to the clinic.

Hematopoiesis gives rise to blood cells of different lineages throughout normal life. Abnormalities in this developmental program lead to blood cell diseases including leukemia. The establishment of a cell culture system for the clonal development of hematopoietic cells made it possible to discover proteins that regulate cell viability, multiplication and differentiation of different hematopoietic cell lineages, and the molecular basis of normal and abnormal blood cell development. These regulators include cytokines now called colony-stimulating factors (CSFs) and interleukins (ILs). There is a network of cytokine interactions, which has positive regulators such as CSFs and ILs and negative regulators such as transforming growth factor beta and tumor necrosis factor (TNF). This multigene cytokine network provides flexibility depending on which part of the network is activated and allows amplification of response to a particular stimulus. Malignancy can be suppressed in certain types of leukemic cells by inducing differentiation with cytokines that regulate normal hematopoiesis or with other compounds that use alternative differentiation pathways. This created the basis for the clinical use of differentiation therapy. The suppression of malignancy by inducing differentiation can bypass genetic abnormalities that give rise to malignancy. Different CSFs and ILs suppress programmed cell death (apoptosis) and induce cell multiplication and differentiation, and these processes of development are separately regulated. The same cytokines suppress apoptosis in normal and leukemic cells, including apoptosis induced by irradiation and cytotoxic cancer chemotherapeutic compounds. An excess of cytokines can increase leukemic cell resistance to cytotoxic therapy. The tumor suppressor gene wild-type p53 induces apoptosis that can also be suppressed by cytokines. The oncogene mutant p53 suppresses apoptosis. Hematopoietic cytokines such as granulocyte CSF are now used clinically to correct defects in hematopoiesis, including repair of chemotherapy-associated suppression of normal hematopoiesis in cancer patients, stimulation of normal granulocyte development in patients with infantile congenital agranulocytosis, and increase of hematopoietic precursors for blood cell transplantation. Treatments that decrease the level of apoptosis-suppressing cytokines and downregulate expression of mutant p53 and other apoptosis suppressing genes in cancer cells could improve cytotoxic cancer therapy. The basic studies on hematopoiesis and leukemia have thus provided new approaches to therapy.

A mutant p53 antagonizes the deregulated c-myc-mediated enhancement of apoptosis and decrease in leukemogenicity.

Myeloid leukemic M1 cells that do not express p53 and transfected M1 clones that constitutively express the [Val135]p53 mutant or deregulated c-myc or coexpressing both genes grew autonomously in culture with a similar growth rate and cloning efficiency. Expression of deregulated c-myc in M1 leukemic cells enhanced susceptibility to induction of apoptotic cell death and resulted in a reduced leukemogenicity when injected into isologous mice. Expression of the [Val135]p53 mutant did not change cell susceptibility to induction of apoptosis or leukemogenicity, but expression of this mutant p53 suppressed the effects of deregulated c-myc on these properties. The results indicate that the [Val135]p53 mutant can show a gain of function for susceptibility to apoptosis and leukemogenicity in leukemic cells with deregulated c-myc and, thus, enhance tumor development.

Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells.

Signals transduced by the met tyrosine kinase, which is the receptor for scatter factor/hepatocyte growth factor, are of major importance for the regulation of epithelial cell motility, morphogenesis, and proliferation. We report here that different sets of tyrosine residues in the cytoplasmic domain of the met receptor affect signal transduction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues 13-16 (Y1311, Y1347, Y1354, and Y1363) reduced or abolished ligand-induced cell motility and branching morphogenesis. In contrast, mutation of the juxtamembrane tyrosine residue 2 (Y1001) produced constitutively mobile, fibroblastoid cells. Furthermore, the gain-of-function mutation of tyrosine residue 2 suppressed the loss-of-function mutations of tyrosine residue 15 or 16. The opposite roles of the juxtamembrane and C-terminal tyrosine residues may explain the suggested dual function of the met receptor in both epithelial-mesenchymal interactions and tumor progression.