Teresa Frisan

Toll-like Receptor 4 Resides in the Golgi Apparatus and Colocalizes with Internalized Lipopolysaccharide in Intestinal Epithelial Cells

Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage. It recognizes lipopolysaccharide (LPS) and mediates cellular activation and production of proinflammatory cytokines. Less is known about the distribution and role of TLR4 in epithelial cells that are continuously exposed to microbes and microbial products. Here we show that the murine small intestinal epithelial cell line m-ICcl2 is highly responsive to LPS and expresses both CD14 and TLR4. Transcription and surface membrane staining for CD14 were up-regulated upon LPS exposure. Surprisingly, TLR4 immunostaining revealed a strictly cytoplasmic paranuclear distribution. This paranuclear compartment could be identified as the Golgi apparatus. LPS added to the supernatant was internalized by m-ICcl2 cells and colocalized with TLR4. Continuous exposure to LPS led to a tolerant phenotype but did not alter TLR4 expression nor cellular distribution. Thus, intestinal epithelial cells might be able to provide the initial proinflammatory signal to attract professional immune cells to the side of infection. The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.

c- myc overexpression activates alternative pathways for intracellular proteolysis in lymphoma cells

Burkitts lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Here we show that BL cells are resistant to apoptosis and do not accumulate ubiquitin conjugates in response to otherwise toxic doses of inhibitors of the proteasome. Deubiquitinating enzymes and the cytosolic subtilisin-like protease tripeptidylpeptidase II are upregulated in BLs, and could be rapidly induced by the overexpression of c-myc in normal B cells carrying oestrogen-driven recombinant Epstein–Barr virus. Apoptosis was induced by inhibiting tripeptidylpeptidase II, suggesting that the activity of this protease may be required for the survival of BL cells. We thus show that there is a regulatory link between c-myc activation and changes in proteolysis that may affect malignant transformation.

The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA

Among bacterial protein toxins, the cytolethal distending toxins (CDTs) are unique in their ability to activate the DNA damage checkpoint responses, causing cell cycle arrest or apoptosis in intoxicated cells. We provide direct evidence that natural intoxication of cells with the Haemophilus ducreyi CDT (HdCDT) holotoxin induces DNA double‐strand breaks similarly to ionizing radiation. Upon DNA damage, epithelial cells and fibroblasts promote the formation of actin stress fibres via activation of the small GTPase RhoA. This phenomenon is not toxin specific, but is part of the ATM‐induced cellular responses to genotoxic stresses, including ionizing radiation. Activation of RhoA is associated with prolonged cell survival, as HdCDT‐treated epithelial cells expressing a dominant‐negative form of RhoA detach and consequently die faster than cells expressing a functional RhoA. Our data highlight several novel aspects of CDT biology: (i) we show that a member of the CDT family causes DNA double‐strand breaks in naturally intoxicated cells, acting as a true genotoxic agent; and (ii) we disclose the existence of a novel signalling pathway for intracellularly triggered activation of the RhoA GTPase via the ATM kinase in response to DNA damage, possibly required to prolong cell survival.

The Haemophilus ducreyi cytolethal distending toxin activates sensors of DNA damage and repair complexes in proliferating and non‐proliferating cells

Cytolethal distending toxins (CDTs) block proliferation of mammalian cells by activating DNA damage‐induced checkpoint responses. We demonstrate that the Haemophilus ducreyi CDT (HdCDT) induces phosphorylation of the histone H2AX as early as 1 h after intoxication and re‐localization of the DNA repair complex Mre11 in HeLa cells with kinetics similar to those observed upon ionizing radiation. Early phosphorylation of H2AX was dependent on a functional Ataxia Telangiectasia mutated (ATM) kinase. Microinjection of a His‐tagged HdCdtB subunit, homologous to the mammalian DNase I, was sufficient to induce re‐localization of the Mre11 complex 1 h post treatment. However, the enzymatic potency was much lower than that exerted by bovine DNase I, which caused marked chromatin changes at 106 times lower concentrations than HdCdtB. H2AX phosphorylation and Mre11 re‐localization were induced also in HdCDT‐treated, non‐proliferating dendritic cells (DCs) in a differentiation dependent manner, and resulted in cell death. The data highlight several novel aspects of CDTs biology. We demonstrate that the toxin activates DNA damage‐associated molecules in an ATM‐dependent manner, both in proliferating and non‐proliferating cells, acting as other DNA damaging agents. Induction of apoptotic death of immature DCs by HdCDT may represent a previously unknown mechanism of immune evasion by CDT‐producing microbes.

Cellular internalization of cytolethal distending toxin: a new end to a known pathway

The cytolethal distending toxins (CDTs) are unique in their ability to induce DNA damage, activate checkpoint responses and cause cell cycle arrest or apoptosis in intoxicated cells. However, little is known about their cellular internalization pathway. We demonstrate that binding of the Haemophilus ducreyi CDT (HdCDT) on the plasma membrane of sensitive cells was abolished by cholesterol extraction with methyl‐β‐cyclodextrin. The toxin was internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER), as assessed by N‐linked glycosylation. Further translocation from the ER did not require the ER‐associated degradation (ERAD) pathway, and was Derlin‐1 independent. The genotoxic activity of HdCDT was dependent on its internalization and its DNase activity, as induction of DNA double‐stranded breaks was prevented in Brefeldin A‐treated cells and in cells exposed to a catalytically inactive toxin. Our data contribute to a better understanding of the CDT mode of action and highlight two important aspects of the biology of this bacterial toxin family: (i) HdCDT translocation from the ER to the nucleus does not involve the classical pathways followed by other retrogradely transported toxins and (ii) toxin internalization is crucial for execution of its genotoxic activity.

MYC overexpression imposes a nonimmunogenic phenotype on Epstein–Barr virus-infected B cells

Lymphoblastoid cell lines, generated by immortalization of normal B cells by Epstein–Barr virus (EBV) in vitro, have strong antigen-presenting capacity, are sensitive to EBV-specific cytotoxic T cells, and are highly allostimulatory in mixed lymphocyte culture. By contrast, EBV-positive Burkitt lymphoma (BL) cells are poor antigen presenters, are not recognized by EBV-specific cytotoxic T cells, and are poorly allostimulatory, which raises the question of whether immunological pressure exerted during BL pathogenesis in vivo has selected for a ‘nonimmunogenic’ tumor phenotype. The present work addresses this question by examining the immunogenicity/antigenicity of cell lines, generated by conversion of a conditionally immortalized lymphoblastoid cell line to permanent growth independent of EBV-latent proteins by introduction of a constitutively active or tetracycline-regulated c-myc gene (A1 and P493–6 cells, respectively). Compared with its parental lymphoblastoid cell line, A1 cells showed many of the features of the nonimmunogenic BL phenotype, namely poor allostimulatory activity, poor antigen-presenting function associated with impaired proteasomal activity, down-regulation of peptide transporter, reduced HLA class I expression, and an inability to present endogenously expressed EBV-latent proteins to cytotoxic T cells. P493–6 cells, when grown in the presence of estrogen with the exogenous c-myc gene switched off, were strongly immunogenic. The cells had lost their immunogenic potential, however, when grown on a c-myc-driven proliferation program in the absence of estrogen. Deregulation of c-myc, a step central to the development of uncontrolled BL cell growth in vivo, can thus impose a nonimmunogenic phenotype on proliferating human B cells in the absence of any immune pressure.

The Biology of the Cytolethal Distending Toxins

The cytolethal distending toxins (CDTs), produced by a variety of Gram-negative pathogenic bacteria, are the first bacterial genotoxins described, since they cause DNA damage in the target cells. CDT is an A-B2 toxin, where the CdtA and CdtC subunits are required to mediate the binding on the surface of the target cells, allowing internalization of the active CdtB subunit, which is functionally homologous to the mammalian deoxyribonuclease I. The nature of the surface receptor is still poorly characterized, however binding of CDT requires intact lipid rafts, and its internalization occurs via dynamin-dependent endocytosis. The toxin is retrograde transported through the Golgi complex and the endoplasmic reticulum, and subsequently translocated into the nuclear compartment, where it exerts the toxic activity. Cellular intoxication induces DNA damage and activation of the DNA damage responses, which results in arrest of the target cells in the G1 and/or G2 phases of the cell cycle and activation of DNA repair mechanisms. Cells that fail to repair the damage will senesce or undergo apoptosis. This review will focus on the well-characterized aspects of the CDT biology and discuss the questions that still remain unanswered.

The cytolethal distending toxins induce DNA damage and cell cycle arrest

The cytolethal distending toxins (CDTs) are a newly discovered family of bacterial protein toxins with the unique ability to interfere with the cell cycle, causing irreversible cell cycle arrest and consequently death of the target cells. CDTs are encoded by three linked genes (cdtA, cdtB and cdtC) and are produced by a variety of Gram negative bacteria. The mechanism of action of this toxin family only now begins to be elucidated. CDTs are internalized by endocytosis and require an intact Golgi complex to exert their cytotoxic activity. The CdtB component was shown to have functional homology with the mammalian deoxyribonuclease I (DNase I) and the induction of cell cycle arrest in mammalian cells mimicked that induced by DNA damaging agents, suggesting that DNA is the cellular target. Still there are many issues that need to be clarified, such as identification of the function(s) of CdtA and CdtC, characterization of the receptor(s), understanding of the final steps of the internalization pathway and localization of the active component. This review focuses mainly on the effect of CDTs on mammalian cells, highlighting the questions that remain to be answered regarding their molecular mode of action.

Chronic exposure to the cytolethal distending toxins of Gram-negative bacteria promotes genomic instability and altered DNA damage response

Epidemiological evidence links chronic bacterial infections to the increased incidence of certain types of cancer but the molecular mechanisms by which bacteria contribute to tumour initiation and progression are still poorly characterized. Here we show that chronic exposure to the genotoxin cytolethal distending toxin (CDT) of Gram‐negative bacteria promotes genomic instability and acquisition of phenotypic properties of malignancy in fibroblasts and colon epithelial cells. Cells grown for more than 30 weeks in the presence of sublethal doses of CDT showed increased mutation frequency, and accumulation of chromatin and chromosomal aberrations in the absence of significant alterations of cell cycle distribution, decreased viability or senescence. Cell survival was dependent on sustained activity of the p38 MAP kinase. The ongoing genomic instability was associated with impaired activation of the DNA damage response and failure to efficiently activate cell cycle checkpoints upon exposure to genotoxic stress. Independently selected sublines showedenhanced anchorage‐independent growth as assessed by the formation of colonies in semisolid agarose. These findings support the notion that chronic infection by CDT‐producing bacteria may promote malignant transformation, and point to the impairment of cellular control mechanisms associated with the detection and repair of DNA damage as critical events in the process.

Myc Is Required for Activation of the ATM-Dependent Checkpoints in Response to DNA Damage

Background The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown. Principal Findings In this study, we demonstrate that expression of Myc is required to activate the Ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses in rat cell lines exposed to ionizing radiation (IR) or the bacterial cytolethal distending toxin (CDT). Phosphorylation of the ATM kinase and its downstream effectors, such as histone H2AX, were impaired in the myc null cell line HO15.19, compared to the myc positive TGR-1 and HOmyc3 cells. Nuclear foci formation of the Nijmegen Breakage Syndrome (Nbs) 1 protein, essential for efficient ATM activation, was also reduced in absence of myc. Knock down of the endogenous levels of MYC by siRNA in the human cell line HCT116 resulted in decreased ATM and CHK2 phosphorylation in response to irradiation. Conversely, cell death induced by UV irradiation, known to activate the ATR-dependent checkpoint, was similar in all the cell lines, independently of the myc status. Conclusion These data demonstrate that MYC contributes to the activation of the ATM-dependent checkpoint responses, leading to cell death in response to specific genotoxic stimuli.