PML, a multifunctional protein, is crucial for forming PML-nuclear bodies involved in stress responses. Under specific conditions, PML associates with nucleolar caps formed after RNA polymerase I (RNAPI) inhibition, leading to PML-nucleolar associations (PNAs). This study investigates PNAs-inducing stimuli by exposing cells to various genotoxic stresses. We found that the most potent inducers of PNAs introduced topological stress and inhibited RNAPI. Doxorubicin, the most effective compound, induced double-strand breaks (DSBs) in the rDNA locus. PNAs co-localized with damaged rDNA, segregating it from active nucleoli. Cleaving the rDNA locus with I-PpoI confirmed rDNA damage as a genuine stimulus for PNAs. Inhibition of ATM, ATR kinases, and RAD51 reduced I-PpoI-induced PNAs, highlighting the importance of ATM/ATR-dependent nucleolar cap formation and homologous recombination (HR) in their triggering. I-PpoI-induced PNAs co-localized with rDNA DSBs positive for RPA32-pS33 but deficient in RAD51, indicating resected DNA unable to complete HR repair. Our findings suggest that PNAs form in response to persistent rDNA damage within the nucleolar cap, highlighting the interplay between PML/PNAs and rDNA alterations due to topological stress, RNAPI inhibition, and rDNA DSBs destined for HR. Cells with persistent PNAs undergo senescence, suggesting PNAs help avoid rDNA instability, with implications for tumorigenesis and aging.

• Keywords
• PML, aberrant DNA topology, cancer biology, cell biology, cellular senescence, genome maintenance, human, persistent rDNA damage,
• MeSH
• Cell Nucleolus * metabolism   MeSH
• DNA Breaks, Double-Stranded MeSH
• Humans MeSH
• DNA Damage MeSH
• Promyelocytic Leukemia Protein * metabolism   genetics   MeSH
• DNA, Ribosomal * genetics   metabolism   MeSH
• RNA Polymerase I metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• PML protein, human MeSH Browser
• Promyelocytic Leukemia Protein * MeSH
• DNA, Ribosomal * MeSH
• RNA Polymerase I MeSH

Diverse stress insults trigger interactions of PML with nucleolus, however, the function of these PML nucleolar associations (PNAs) remains unclear. Here we show that during induction of DNA damage-induced senescence in human non-cancerous cells, PML accumulates at the nucleolar periphery simultaneously with inactivation of RNA polymerase I (RNAP I) and nucleolar segregation. Using time-lapse and high-resolution microscopy, we followed the genesis, structural transitions and destiny of PNAs to show that: 1) the dynamic structural changes of the PML-nucleolar interaction are tightly associated with inactivation and reactivation of RNAP I-mediated transcription, respectively; 2) the PML-nucleolar compartment develops sequentially under stress and, upon stress termination, it culminates in either of two fates: disappearance or persistence; 3) all PNAs stages can associate with DNA damage markers; 4) the persistent, commonly long-lasting PML multi-protein nucleolar structures (PML-NDS) associate with markers of DNA damage, indicating a role of PNAs in persistent DNA damage response characteristic for senescent cells. Given the emerging evidence implicating PML in homologous recombination-directed DNA repair, we propose that PNAs contribute to sequestration and faithful repair of the highly unstable ribosomal DNA repeats, a fundamental process to maintain a precise balance between DNA repair mechanisms, with implications for genomic integrity and aging.

• Keywords
• DNA damage, nucleolar segregation, rDNA loci, super-resolution microscopy, time-lapse imaging,
• MeSH
• Cell Nucleolus metabolism   MeSH
• Doxorubicin MeSH
• Stress, Physiological MeSH
• Cells, Cultured MeSH
• Humans MeSH
• DNA Damage * MeSH
• Promyelocytic Leukemia Protein metabolism   MeSH
• Cellular Senescence * MeSH
• Imaging, Three-Dimensional MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Doxorubicin MeSH
• PML protein, human MeSH Browser
• Promyelocytic Leukemia Protein MeSH

Many cancers arise at sites of infection and inflammation. Cellular senescence, a permanent state of cell cycle arrest that provides a barrier against tumorigenesis, is accompanied by elevated proinflammatory cytokines such as IL1, IL6, IL8 and TNFα. Here we demonstrate that media conditioned by cells undergoing any of the three main forms of senescence, i.e. replicative, oncogene- and drug-induced, contain high levels of IL1, IL6, and TGFb capable of inducing reactive oxygen species (ROS)-mediated DNA damage response (DDR). Persistent cytokine signaling and activated DDR evoke senescence in normal bystander cells, accompanied by activation of the JAK/STAT, TGFβ/SMAD and IL1/NFκB signaling pathways. Whereas inhibition of IL6/STAT signaling had no effect on DDR induction in bystander cells, inhibition of either TGFβ/SMAD or IL1/NFκB pathway resulted in decreased ROS production and reduced DDR in bystander cells. Simultaneous inhibition of both TGFβ/SMAD and IL1/NFκB pathways completely suppressed DDR indicating that IL1 and TGFβ cooperate to induce and/or maintain bystander senescence. Furthermore, the observed IL1- and TGFβ-induced expression of NAPDH oxidase Nox4 indicates a mechanistic link between the senescence-associated secretory phenotype (SASP) and DNA damage signaling as a feature shared by development of all major forms of paracrine bystander senescence.

• MeSH
• Cell Line MeSH
• Bystander Effect drug effects   MeSH
• Etoposide pharmacology   MeSH
• Genes, ras * MeSH
• Interleukin-1 metabolism   MeSH
• Interleukin-6 metabolism   MeSH
• Janus Kinases metabolism   MeSH
• Culture Media, Conditioned metabolism   MeSH
• Humans MeSH
• NADPH Oxidase 4 MeSH
• NADPH Oxidases metabolism   MeSH
• NF-kappa B metabolism   MeSH
• Oxidative Stress drug effects   MeSH
• Paracrine Communication drug effects   MeSH
• DNA Damage * MeSH
• Cell Proliferation * MeSH
• Smad Proteins metabolism   MeSH
• RNA Interference MeSH
• Signal Transduction drug effects   MeSH
• Cellular Senescence drug effects   MeSH
• Transfection MeSH
• Transforming Growth Factor beta metabolism   MeSH
• STAT Transcription Factors metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Etoposide MeSH
• IL6 protein, human MeSH Browser
• Interleukin-1 MeSH
• Interleukin-6 MeSH
• Janus Kinases MeSH
• Culture Media, Conditioned MeSH
• NADPH Oxidase 4 MeSH
• NADPH Oxidases MeSH
• NF-kappa B MeSH
• NOX4 protein, human MeSH Browser
• Smad Proteins MeSH
• Transforming Growth Factor beta MeSH
• STAT Transcription Factors MeSH

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.

• MeSH
• Leukemia, Promyelocytic, Acute metabolism   pathology   MeSH
• Chromatin Immunoprecipitation MeSH
• DNA Primers MeSH
• Fluorescent Antibody Technique, Indirect MeSH
• HeLa Cells MeSH
• Interleukin-6 metabolism   MeSH
• Nuclear Proteins metabolism   MeSH
• Janus Kinases metabolism   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Humans MeSH
• Tumor Suppressor Proteins metabolism   MeSH
• Promyelocytic Leukemia Protein MeSH
• Base Sequence MeSH
• Signal Transduction * MeSH
• STAT Transcription Factors metabolism   MeSH
• Transcription Factors metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• DNA Primers MeSH
• Interleukin-6 MeSH
• Nuclear Proteins MeSH
• Janus Kinases MeSH
• Tumor Suppressor Proteins MeSH
• PML protein, human MeSH Browser
• Promyelocytic Leukemia Protein MeSH
• STAT Transcription Factors MeSH
• Transcription Factors MeSH

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/gammaH2AX(+) foci), enhanced senescence-associated beta-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.

• MeSH
• Bacterial Toxins metabolism   pharmacology   MeSH
• Cell Cycle drug effects   MeSH
• Cyclin-Dependent Kinases antagonists & inhibitors   metabolism   MeSH
• Cytokines metabolism   MeSH
• Phenotype MeSH
• Haemophilus ducreyi metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor * drug effects   physiology   MeSH
• DNA Damage * MeSH
• Signal Transduction physiology   MeSH
• Cellular Senescence physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Toxins MeSH
• Cyclin-Dependent Kinases MeSH
• Cytokines MeSH
• cytolethal distending toxin MeSH Browser