Interleukin 6 signaling regulates promyelocytic leukemia protein gene expression in human normal and cancer cells

Tumor suppressor PML is induced under viral and genotoxic stresses by interferons and JAK-STAT signaling. However, the mechanism responsible for its cell type-specific regulation under non-stimulated conditions is poorly understood. To analyze the variation of PML expression, we utilized three human cell types, BJ fibroblasts and HeLa and U2OS cell lines, each with a distinct PML expression pattern. Analysis of JAK-STAT signaling in the three cell lines revealed differences in levels of activated STAT3 but not STAT1 correlating with PML mRNA and protein levels. RNAi-mediated knockdown of STAT3 decreased PML expression; both STAT3 level/activity and PML expression relied on IL6 secreted into culture media. We mapped the IL6-responsive sequence to an ISRE(-595/-628) element of the PML promoter. The PI3K/Akt/NFκB branch of IL6 signaling showed also cell-type dependence, being highest in BJ, intermediate in HeLa, and lowest in U2OS cells and correlated with IL6 secretion. RNAi-mediated knockdown of NEMO (NF-κ-B essential modulator), a key component of NFκB activation, suppressed NFκB targets LMP2 and IRF1 together with STAT3 and PML. Combined knockdown of STAT3 and NEMO did not further promote PML suppression, and it can be bypassed by exogenous IL6, indicating the NF-κB pathway acts upstream of JAK-STAT3 through induction of IL6. Our results indicate that the cell type-specific activity of IL6 signaling pathways governs PML expression under unperturbed growth conditions. As IL6 is induced in response to various viral and genotoxic stresses, this cytokine may regulate autocrine/paracrine induction of PML under these pathophysiological states as part of tissue adaptation to local stress.     

Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities

Homologous recombination (HR) is essential for faithful repair of DNA lesions yet must be kept in check, as unrestrained HR may compromise genome integrity and lead to premature aging or cancer. To limit unscheduled HR, cells possess DNA helicases capable of preventing excessive recombination. In this study, we show that the human Fbh1 (hFbh1) helicase accumulates at sites of DNA damage or replication stress in a manner dependent fully on its helicase activity and partially on its conserved F box. hFbh1 interacted with single-stranded DNA (ssDNA), the formation of which was required for hFbh1 recruitment to DNA lesions. Conversely, depletion of endogenous Fbh1 or ectopic expression of helicase-deficient hFbh1 attenuated ssDNA production after replication block. Although elevated levels of hFbh1 impaired Rad51 recruitment to ssDNA and suppressed HR, its small interfering RNA–mediated depletion increased the levels of chromatin-associated Rad51 and caused unscheduled sister chromatid exchange. Thus, by possessing both pro- and anti-recombinogenic potential, hFbh1 may cooperate with other DNA helicases in tightly controlling cellular HR activity.     

The Mycobacterium tuberculosis DosR Regulon Assists in Metabolic Homeostasis and Enables Rapid Recovery from Nonrespiring Dormancy

Mycobacterium tuberculosis survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The M. tuberculosis DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for M. tuberculosis survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once M. tuberculosis exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for M. tuberculosis to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.Mycobacterium tuberculosis, a major human pathogen, infects nearly one-third of the people in the world and causes two million deaths per year (8). Most infections are latent, and a substantial number of new infections are transmitted by individuals in whom latent infections are being reactivated. Latency is a clinical term describing people that are infected with M. tuberculosis but lack symptoms of active disease. Traditionally, it has been thought that bacilli in latently infected individuals reside almost exclusively inside granulomas and mature tubercle lesions. Recent studies indicate that in latently infected individuals M. tuberculosis may also be found outside granulomas in places such as endothelial cells, fibroblasts, and adipose tissue (17, 28). The evidence for M. tuberculosis metabolic activity in vivo is more limited, but two studies by Lillebaek et al. are informative (24, 25). In these studies the researchers used detailed records of tuberculosis epidemiology and strain types in the fairly static population of Denmark. They found that strains isolated from patients thought to have reactivated disease (rather than a primary infection) were nearly identical to strains present 30 years earlier in the same geographic population. The near-identity of the strains and the fact that infections were attributed to reactivation suggest that bacteria in latently infected individuals experience little genetic change during years of latent infection. The researchers concluded that during latency, M. tuberculosis divides infrequently and is likely in a minimal metabolic state.One approach to study the M. tuberculosis metabolic state during latent infection is to use in vitro models that mimic conditions thought to exist in vivo. Such conditions include hypoxia produced in avascular calcified granulomas (40) and nitric oxide (NO) (27) or carbon monoxide (CO) (33) produced by activated immune cells. A widely used model is the “Wayne model” pioneered by Lawrence Wayne. In this model, a low-inoculum culture is sealed in a tube with stirring and allowed to slowly consume oxygen until the culture is anaerobic, resulting in a nonreplicating and apparently dormant state (45, 46). Another model used to look at dormant M. tuberculosis is a constant-hypoxia model that maintains a 0.2% oxygen tension in culture flasks (31).The common theme in these in vitro models used to obtain M. tuberculosis dormancy is inhibition of respiration. The DosR regulon is a set of at least 48 coregulated genes that are induced by three conditions that inhibit aerobic respiration: hypoxia, NO, and CO (42). Induction of the DosR regulon closely mirrors inhibition of respiration, indicating that control of the regulon is linked to the aerobic respiratory state of the bacilli (43). Several studies have shown that the DosR regulon is controlled by a three-component regulatory system composed of two sensor histidine kinases, DosS and DosT, and a response regulator, DosR (42). DosS and DosT both bind the respiration-impairing gases NO and CO (19, 20, 38), further supporting the hypothesis that the DosR regulon responds to, and is important during, conditions that do not allow aerobic respiration. Although the majority of the DosR-regulated genes have not been characterized, the timing of their induction combined with the conditions under which they respond suggests that they may play a role in adaptation of M. tuberculosis to its host environment. Consistent with this notion, DosR regulon genes are induced in the lungs of M. tuberculosis-infected mice (43), as well as in interferon-gamma-activated murine macrophages (34) and guinea pigs (37).Several studies have suggested that the DosR regulon plays a role in latent infection and in persistence in animal models that resemble human infection in some respects. Leyten et al. found that latently infected humans are more likely than humans with active infections to bear T cells specific for DosR regulon antigens (23), suggesting that the regulon is expressed during latency. Two recent studies confirmed that there is an immune response to DosR regulon antigens during latent infection (4, 36). Further evidence for clinical relevance in humans comes from a study showing that M. tuberculosis in sputum expresses the DosR regulon (15). The importance of this regulon for persistence in rabbit and guinea pig models was demonstrated by data showing a 2-log decrease in recovery of a DosR mutant 2 weeks (guinea pig) and 8 weeks (rabbit) after aerosol infection (11). A DosR mutant was also found to be significantly attenuated in guinea pig infection (26), further supporting the notion that the DosR regulon is required for persistence in vivo. It should be noted that in both studies showing the DosR phenotype (11, 26), full complementation and reversion to full virulence were not observed. However, it is now known that regulation of dosR expression is quite complex. Multiple regulatory sequences exist in and upstream of Rv3134c, the gene directly upstream of dosR (8). Failure to include such a regulatory sequence in a complemented strain would likely result in misregulation of dosR and poor complementation. Studies of DosR regulon mutants for murine infection have produced inconsistent findings that vary from hypervirulent (30) to attenuated (11) and not attenuated (3, 31). When animal models are compared, it is important to remember that M. tuberculosis-induced granulomas in primates, rabbits, and guinea pigs develop caseous necrosis and are hypoxic and/or anaerobic, while M. tuberculosis induced-granulomas in mice are neither hypoxic nor anaerobic (2, 21, 41). Furthermore, M. tuberculosis divides regularly in chronic murine infections (16), in contrast to the replication during latent infections, as demonstrated in the studies of Lillebaek et al. (24, 25). Such studies underscore the significant differences between models.A previous study with a DosR mutant in a closely related Mycobacterium bovis BCG strain showed that DosR expression is required for survival in an in vitro Wayne-like model of dormancy (5). Unexpectedly, two similar studies in M. tuberculosis did not show a strong survival defect for a DosR mutant (31, 43). The most recent study showed that there was only a modest survival defect in an H37Rv DosR mutant and concluded that the DosR regulon is a short-term phenomenon and is not responsible for the adaptation necessary to survive under primarily hypoxic conditions in vitro (31, 32).In this study we showed that the DosR regulon is required for M. tuberculosis survival during anaerobic dormancy. We also used a combination of genetic and biochemical approaches to demonstrate that this regulon is necessary to shift away from oxygen consumption, maintain ATP levels, and balance the redox state (NAD/NADH ratio) of the cell as oxygen becomes scarce. Furthermore, we showed that the DosR regulon is necessary for optimal transition of M. tuberculosis back to aerobic growth from an anaerobic or nitric oxide-induced nonrespiring state.     

Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling

Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX⁺ foci), enhanced senescence-associated β-galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the 'distended' morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.     

Mutant p53 can delay growth arrest and loss of CDK2 activity in senescing human fibroblasts without reducing p21(WAF1) expression

Functional wild-type p53 is required for human diploid fibroblasts (HDF) to enter an irreversible growth arrest known as replicative senescence. Experimentally, abrogation of p53 function by expression of human papillomavirus type 16 E6 or disruption of a key downstream effector p21 by homologous recombination both extended HDF life span. However, although sufficient to extend life span, p21 down-regulation is not necessary, because expression of a dominant-negative mutant p53 (143(ala)) extends life span without apparently decreasing p21 expression. Given the importance of p53 in cellular senescence and the general assumption that p21 may be the sole mediator of its action in this process, we have investigated how abrogation of p53 function can overcome senescence without lowering expression of p21. We have found up-regulated levels of the cyclin-dependent kinase 2 (cdk2) protein in HDF expressing 143(ala) mutant p53 as compared to senescent controls, together with an increase in p21-free cdk2 which, in conjunction with cyclin E, is able to form an active kinase which can phosphorylate the retinoblastoma protein. However, forced overexpression of cdk2 in near-senescent HDF failed to restore cdk2-associated kinase activity. Our data suggest that p53-mediated senescence depends on factor(s) other than p21 which modulate formation of cyclin E-cdk2 complexes.     

Human Tousled like kinases are targeted by an ATM- and Chk1-dependent DNA damage checkpoint

All eukaryotes respond to DNA damage by modulation of diverse cellular processes to preserve genomic integrity and ensure survival. Here we identify mammalian Tousled like kinases (Tlks) as a novel target of the DNA damage checkpoint. During S-phase progression, when Tlks are maximally active, generation of DNA double-strand breaks (DSBs) leads to rapid and transient inhibition of Tlk activity. Experiments with chemical inhibitors, genetic models and gene targeting through RNA interference demonstrate that this response to DSBs requires ATM and Chk1 function. Chk1 phosphorylates Tlk1 on serine 695 (S695) in vitro, and this UCN-01- and caffeine-sensitive site is phosphorylated in vivo in response to DNA damage. Substitution of S695 to alanine impaired efficient downregulation of Tlk1 after DNA damage. These findings identify an unprecedented functional co- operation between ATM and Chk1 in propagation of a checkpoint response during S phase and suggest that, through transient inhibition of Tlk kinases, the ATM-Chk1-Tlk pathway may regulate processes involved in chromatin assembly.     

DPI induces mitochondrial superoxide-mediated apoptosis

The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.     

Watching the DNA repair ensemble dance

Repair of damaged DNA is a dynamic process that requires careful orchestration of a multitude of enzymes, adaptor proteins, and chromatin constituents. In this issue of Cell, Lisby et al. (2004) provide a visual glimpse into how the diverse signaling and repair machines are organized in space and time around the deadliest genetic lesions--the DNA double-strand breaks.