Artin Mahboubi

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

DNA Damaging Agents Induce Expression of Fas Ligand and Subsequent Apoptosis in T Lymphocytes via the Activation of NF-κB and AP-1

Apoptosis induced by DNA damage and other stresses can proceed via expression of Fas ligand (FasL) and ligation of its receptor, Fas (CD95). We report that activation of the two transcription factors NF-kappa B and AP-1 is crucially involved in FasL expression induced by etoposide, teniposide, and UV irradiation. A nondegradable mutant of I kappa B blocked both FasL expression and apoptosis induced by DNA damage but not Fas ligation. These stimuli also induced the stress-activated kinase pathway (SAPK/JNK), which was required for the maximal induction of apoptosis. A 1.2 kb FasL promoter responded to DNA damage, as well as coexpression with p65 Rel or Fos/Jun. Mutations in the relevant NF-kappa B and AP-1 binding sites eliminated these responses. Thus, activation of NF-kappa B and AP-1 contributes to stress-induced apoptosis via the expression of FasL.

The end of the (cell) line: Methods for the study of apoptosis in vitro

Publisher Summary This chapter describes techniques commonly used to study cell death using in vitro cell culture systems. These techniques include a range of simple dye exclusion assays that give crude information on cell viability but do not necessarily discriminate between apoptotic or necrotic modes of cell death. The chapter describes cell death assays that are based on the observation that apoptosis is accompanied by DNA fragmentation, either into the classical “ladder” pattern of 200 bp integer multiples, 50kb fragments, or single-stranded DNA cleavage. Assays used to measure this fragmentation include gel electrophoresis of total DNA, quantification of the release of radioactively labeled DNA, examination of cell cycle profile, and in situ nick translation. Because apoptotic cells are morphologically highly distinctive and are easily distinguishable from both viable and necrotic cells, most cell death assays should be coupled with direct morphological evaluation of the cell population under study to define with certainty the mode of cell death that is occurring.

FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway

Fas receptor-induced apoptosis plays critical roles in immune homeostasis. However, most of the signal transduction events distal to Fas ligation have not been elucidated. Here, we show that Ras is activated following ligation of Fas on lymphoid lines. The activation of Ras is a critical component of this apoptotic pathway, since inhibition of Ras by neutralizing antibody or a dominant-negative Ras mutant interfered with Fas-induced apoptosis. Furthermore, ligation of Fas also resulted in stimulation of the sphingomyelin signalling pathway to produce ceramides, which, in turn, are capable of inducing both Ras activation and apoptosis. This suggests that ceramides acts as second messengers in Fas signaling via Ras. Thus, ligation of the Fas molecule on lymphocyte lines induces activation of Ras via the action of ceramide, and this activation is necessary, but not sufficient, for subsequent apoptosis.

Regulation of the Fas Apoptotic Cell Death Pathway by Abl

Relatively little is known about oncogene involvement in the regulation of Fas-mediated apoptosis. Inhibition of Fas-induced cell death by the bcl-2 oncogene has been demonstrated to be only partial. In light of a growing body of evidence for the Abl kinase as a negative regulator of cell death, we sought to determine whether Abl expression could protect against Fas-mediated cell death. To address this question, we utilized two separate strategies. In the first, we expressed human Fas in K562, a chronic myelogenous leukemia cell line, which constitutively expresses bcr-abl and examined the effects of Fas ligation in these cells. Fas-positive K562 transformants (K562.Fas) were found to be protected against Fas-mediated cell death. However, down-regulation of Bcr-Abl protein levels in K562.Fas cells using antisense oligonucleotides targeted to bcr-abl mRNA rendered these cells highly susceptible to Fas-induced death. In the second approach we utilized a Fas-positive HL-60 cell line, which we transfected with a temperature-sensitive mutant of v-Abl. HL-60.v-Abl transfectants were found to be protected from Fas-induced apoptosis at the permissive but not the restrictive temperature for the Abl kinase. Taken together, these observations identify the Abl kinase as a negative regulator of Fas-mediated cell death. Since Abl was also found to block apoptosis mediated by ceramide, a recently proposed downstream effector of the apoptotic pathway initiated by Fas, we propose that Abl exerts its protective effects downstream of the early Fas-initiated signaling events.

Regulation of Joint Destruction and Inflammation by p53 in Collagen-Induced Arthritis

The role of the tumor suppressor p53 as a key regulator of inflammation was examined in murine collagen-induced arthritis (CIA), a model of rheumatoid arthritis. Wild-type DBA/1 mice develop progressive arthritis in this model, in which p53 expression and apoptosis are evident in the synovial cells. In contrast, the joints of p53(-/-) DBA/1 animals with CIA showed increased severity of arthritis using clinical and histological scoring methods with almost no apoptosis. Consistent with this, collagenase-3 expression and cytokine production (interleukin-1 and interleukin-6) in the joints of p53(-/-) mice with CIA were significantly greater than in wild-type mice. Anti-collagen antibody titers, however, were not different. Therefore, p53 expression occurs during inflammation and acts to suppress local inflammatory responses. Because mutations in p53 have been described in the synovial membrane of rheumatoid arthritis patients, the loss of p53 function in synoviocytes or other cells in the joint because of dominant-negative mutations might contribute to invasion and destruction of the joint in this disease.

Promotion and Inhibition of Activation-Induced Apoptosis in T-Cell Hybridomas by Oncogenes and Related Signals

The Two Signal: Death/Survival Model suggests that cellular proliferation and physiological cell death should be intimately associated such that, in the absence of external influences, a normal cell departing from rest will have an equal probability of undergoing either process. The c-Myc protooncogene product has been implicated in cell cycle progression and in the control of gene expression, and more recently c-Myc has also been seen to promote apoptotic cell death. As predicted from the model, c-Myc-induced apoptosis is inhibited by growth factors or other anti-apoptotic signals including those provided by some oncogenes. Here, we discuss experiments that test the Two Signal: Death/Survival Model in the phenomenon of activation-induced apoptosis in T-cell hybridomas. Ligation of the antigen receptor on these cells leads to activation, resulting in cytokine production and apoptosis. Inhibition of c-Myc expression by addition of antisense oligodeoxynucleotides or transforming growth factor beta inhibits this form of apoptosis. Because c-Myc is known to bind to several cellular proteins, including Max, we further examined the effects of expression of a dominant negative Max on activation-induced apoptosis. We found that this Max mutant, which interferes with the function of the Myc/Max heterodimer, inhibits the induction of apoptosis by antigen receptor ligation. Thus, both Myc and Max play roles in activation-induced apoptosis, presumably via control of gene expression. Further, as predicted, signals generated from growth factor receptors or the v-Abl oncogene interfere with activation-induced apoptosis. In contrast, the anti-apoptotic effects of Bcl-2 are not active in this form of apoptosis. Finally, a role for Fas/Fas-ligand interactions in activation-induced apoptosis is considered.

MYC, FAS, Apoptosis, and Immune Tolerance

The problem of immunological tolerance, the apparent ability of the immune system to discriminate between self and “nonself” is almost as old as the field of immunology. Arguably, Ehrlich’s description of the phenomenon and the ensuing belief that it is a fundamental attribute of the immune system delayed the recognition of the existence of autoimmune disease until the 1950’s (1). However, once it was realized that the immune system could respond to self molecules, the idea that self/nonself discrimination was “learned” (and therefore potentially fallible) came into vogue. It is therefore likely that this recognition prompted the appearance of models of immune tolerance, and served as the impetus for the development of the clonal selection theory (2,3). The application of this theory to explain tolerance through the deletion of potentially autoreactive cells as proposed by Burnet (3,4) persist in almost unchanged form today.