Critical Involvement of the ATM-Dependent DNA Damage Response in the Apoptotic Demise of HIV-1-Elicited Syncytia

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53

Lack of Effective Anti-Apoptotic Activities Restricts Growth of Parachlamydiaceae in Insect Cells

The fundamental role of programmed cell death in host defense is highlighted by the multitude of anti-apoptotic strategies evolved by various microbes, including the well-known obligate intracellular bacterial pathogens Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae. As inhibition of apoptosis is assumed to be essential for a successful infection of humans by these chlamydiae, we analyzed the anti-apoptotic capacity of close relatives that occur as symbionts of amoebae and might represent emerging pathogens. While Simkania negevensis was able to efficiently replicate within insect cells, which served as model for metazoan-derived host cells, the Parachlamydiaceae (Parachlamydia acanthamoebae and Protochlamydia amoebophila) displayed limited intracellular growth, yet these bacteria induced typical features of apoptotic cell death, including formation of apoptotic bodies, nuclear condensation, internucleosomal DNA fragmentation, and effector caspase activity. Induction of apoptosis was dependent on bacterial activity, but not bacterial de novo protein synthesis, and was detectable already at very early stages of infection. Experimental inhibition of host cell death greatly enhanced parachlamydial replication, suggesting that lack of potent anti-apoptotic activities in Parachlamydiaceae may represent an important factor compromising their ability to successfully infect non-protozoan hosts. These findings highlight the importance of the evolution of anti-apoptotic traits for the success of chlamydiae as pathogens of humans and animals

HIV gp120 Induces, NF-κB Dependent, HIV Replication that Requires Procaspase 8

HIV envelope glycoprotein gp120 causes cellular activation resulting in anergy, apoptosis, proinflammatory cytokine production, and through an unknown mechanism, enhanced HIV replication.We describe that the signals which promote apoptosis are also responsible for the enhanced HIV replication. Specifically, we demonstrate that the caspase 8 cleavage fragment Caspase8p43, activates p50/p65 Nuclear Factor kappaB (NF-kappaB), in a manner which is inhibited by dominant negative IkappaBalpha. This caspase 8 dependent NF-kappaB activation occurs following stimulation with gp120, TNF, or CD3/CD28 crosslinking, but these treatments do not activate NF-kappaB in cells deficient in caspase 8. The Casp8p43 cleavage fragment also transactivates the HIV LTR through NF-kappaB, and the absence of caspase 8 following HIV infection greatly inhibits HIV replication.Gp120 induced caspase 8 dependent NF-kappaB activation is a novel pathway of HIV replication which increases understanding of the biology of T-cell death, as well as having implications for understanding treatment and prevention of HIV infection

IGF-I activates caspases 3/7, 8 and 9 but does not induce cell death in colorectal cancer cells

Background: Colorectal cancer is the third most common cancer in the western world. Chemotherapy is often ineffective to treat the advanced colorectal cancers due to the chemoresistance. A major contributor to chemo-resistance is tumour-derived inhibition or avoidance of apoptosis. Insulin-like growth factor I (IGF-I) has been known to play a prominent role in colorectal cancer development and progression. The role of IGF-I in cancer cell apoptosis is not completely understood.Methods: Using three colorectal cancer cell lines and one muscle cell line, associations between IGF-I and activities of caspase 3/7, 8 and 9 have been examined; the role of insulin-like growth factor I receptor (IGF-IR) in the caspase activation has been investigated.Results: The results show that exogenous IGF-I significantly increases activity of caspases 3/7, 8 and 9 in all cell lines used; blocking IGF-I receptor reduce IGF-I-induced caspase activation. Further studies demonstrate that IGF-I induced caspase activation does not result in cell death. This is the first report to show that while IGF-I activates caspases 3/7, 8 and 9 it does not cause colorectal cancer cell death.Conclusion: The study suggests that caspase activation is not synonymous with apoptosis and that activation of caspases may not necessarily induce cell death

Bid Regulates the Pathogenesis of Neurotropic Reovirus

Reovirus infection leads to apoptosis in both cultured cells and the murine central nervous system (CNS). NF-κB-driven transcription of proapoptotic cellular genes is required for the effector phase of the apoptotic response. Although both extrinsic death-receptor signaling pathways and intrinsic pathways involving mitochondrial injury are implicated in reovirus-induced apoptosis, mechanisms by which either of these pathways are activated and their relationship to NF-κB signaling following reovirus infection are unknown. The proapoptotic Bcl-2 family member, Bid, is activated by proteolytic cleavage following reovirus infection. To understand how reovirus integrates host signaling circuits to induce apoptosis, we examined proapoptotic signaling following infection of Bid-deficient cells. Although reovirus growth was not affected by the absence of Bid, cells lacking Bid failed to undergo apoptosis. Furthermore, we found that NF-κB activation is required for Bid cleavage and subsequent proapoptotic signaling. To examine the functional significance of Bid-dependent apoptosis in reovirus disease, we monitored fatal encephalitis caused by reovirus in the presence and absence of Bid. Survival of Bid-deficient mice was significantly enhanced in comparison to wild-type mice following either peroral or intracranial inoculation of reovirus. Decreased reovirus virulence in Bid-null mice was accompanied by a reduction in viral yield. These findings define a role for NF-κB-dependent cleavage of Bid in the cell death program initiated by viral infection and link Bid to viral virulence

Automatic Morphological Subtyping Reveals New Roles of Caspases in Mitochondrial Dynamics

Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO) cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology

Expression profiles of switch-like genes accurately classify tissue and infectious disease phenotypes in model-based classification

<p>Abstract</p> <p>Background</p> <p>Large-scale compilation of gene expression microarray datasets across diverse biological phenotypes provided a means of gathering a priori knowledge in the form of identification and annotation of bimodal genes in the human and mouse genomes. These switch-like genes consist of 15% of known human genes, and are enriched with genes coding for extracellular and membrane proteins. It is of interest to determine the prediction potential of bimodal genes for class discovery in large-scale datasets.</p> <p>Results</p> <p>Use of a model-based clustering algorithm accurately classified more than 400 microarray samples into 19 different tissue types on the basis of bimodal gene expression. Bimodal expression patterns were also highly effective in differentiating between infectious diseases in model-based clustering of microarray data. Supervised classification with feature selection restricted to switch-like genes also recognized tissue specific and infectious disease specific signatures in independent test datasets reserved for validation. Determination of "on" and "off" states of switch-like genes in various tissues and diseases allowed for the identification of activated/deactivated pathways. Activated switch-like genes in neural, skeletal muscle and cardiac muscle tissue tend to have tissue-specific roles. A majority of activated genes in infectious disease are involved in processes related to the immune response.</p> <p>Conclusion</p> <p>Switch-like bimodal gene sets capture genome-wide signatures from microarray data in health and infectious disease. A subset of bimodal genes coding for extracellular and membrane proteins are associated with tissue specificity, indicating a potential role for them as biomarkers provided that expression is altered in the onset of disease. Furthermore, we provide evidence that bimodal genes are involved in temporally and spatially active mechanisms including tissue-specific functions and response of the immune system to invading pathogens.</p

Identification of Novel Single Nucleotide Polymorphisms in Inflammatory Genes as Risk Factors Associated with Trachomatous Trichiasis

infection, the primary cause of trachoma. Despite control programs that include mass antibiotic treatment, reinfection and recurrence of trachoma are common after treatment cessation. Furthermore, a subset of infected individuals develop inflammation and are at greater risk for developing the severe sequela of trachoma known as trachomatous trichiasis (TT). While there are a number of environmental and behavioral risk factors for trachoma, genetic factors that influence inflammation and TT risk remain ill defined. = 0.001] with the combination of TNFA (-308A), LTA (252A), VCAM1 (-1594C), SCYA 11 (23T) minor allele, and the combination of TNFA (-308A), IL9 (113M), IL1B (5′UTR-T), and VCAM1 (-1594C). However, TT risk increased 13.5 times [odds ratio = 13.5 (95% confidence interval 3.3–22), p = 0.001] with the combination of TNFA (-308G), VDR (intron G), IL4R (50V), and ICAM1 (56M) minor allele.Evaluating genetic risk factors for trachoma will advance our understanding of disease pathogenesis, and should be considered in the context of designing global control programs

HtrA2/Omi Terminates Cytomegalovirus Infection and Is Controlled by the Viral Mitochondrial Inhibitor of Apoptosis (vMIA)

Viruses encode suppressors of cell death to block intrinsic and extrinsic host-initiated death pathways that reduce viral yield as well as control the termination of infection. Cytomegalovirus (CMV) infection terminates by a caspase-independent cell fragmentation process after an extended period of continuous virus production. The viral mitochondria-localized inhibitor of apoptosis (vMIA; a product of the UL37x1 gene) controls this fragmentation process. UL37x1 mutant virus-infected cells fragment three to four days earlier than cells infected with wt virus. Here, we demonstrate that infected cell death is dependent on serine proteases. We identify mitochondrial serine protease HtrA2/Omi as the initiator of this caspase-independent death pathway. Infected fibroblasts develop susceptibility to death as levels of mitochondria-resident HtrA2/Omi protease increase. Cell death is suppressed by the serine protease inhibitor TLCK as well as by the HtrA2-specific inhibitor UCF-101. Experimental overexpression of HtrA2/Omi, but not a catalytic site mutant of the enzyme, sensitizes infected cells to death that can be blocked by vMIA or protease inhibitors. Uninfected cells are completely resistant to HtrA2/Omi induced death. Thus, in addition to suppression of apoptosis and autophagy, vMIA naturally controls a novel serine protease-dependent CMV-infected cell-specific programmed cell death (cmvPCD) pathway that terminates the CMV replication cycle