Publications by authors named "Zdenek Hodny"

55 Publications

The human nucleoporin Tpr protects cells from RNA-mediated replication stress.

Nat Commun 2021 06 24;12(1):3937. Epub 2021 Jun 24.

IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milano, Italy.

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.
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http://dx.doi.org/10.1038/s41467-021-24224-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225803PMC
June 2021

The Effect of Chemical Structure of OEG Ligand Shells with Quaternary Ammonium Moiety on the Colloidal Stabilization, Cellular Uptake and Photothermal Stability of Gold Nanorods.

Int J Nanomedicine 2021 18;16:3407-3427. Epub 2021 May 18.

Department of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.

Purpose: Plasmonic photothermal cancer therapy by gold nanorods (GNRs) emerges as a promising tool for cancer treatment. The goal of this study was to design cationic oligoethylene glycol (OEG) compounds varying in hydrophobicity and molecular electrostatic potential as ligand shells of GNRs. Three series of ligands with different length of OEG chain (ethylene glycol units = 3, 4, 5) and variants of quaternary ammonium salts (QAS) as terminal functional group were synthesized and compared to a prototypical quaternary ammonium ligand with alkyl chain - (16-mercaptohexadecyl)trimethylammonium bromide (MTAB).

Methods: Step-by-step research approach starting with the preparation of compounds characterized by NMR and HRMS spectra, GNRs ligand exchange evaluation through characterization of cytotoxicity and GNRs cellular uptake was used. A method quantifying the reshaping of GNRs was applied to determine the effect of ligand structure on the heat transport from GNRs under fs-laser irradiation.

Results: Fourteen out of 18 synthesized OEG compounds successfully stabilized GNRs in the water. The colloidal stability of prepared GNRs in the cell culture medium decreased with the number of OEG units. In contrast, the cellular uptake of GNRs by HeLa cells increased with the length of OEG chain while the structure of the QAS group showed a minor role. Compared to MTAB, more hydrophilic OEG compounds exhibited nearly two order of magnitude lower cytotoxicity in free state and provided efficient cellular uptake of GNRs close to the level of MTAB. Regarding photothermal properties, OEG compounds evoked the photothermal reshaping of GNRs at lower peak fluence (14.8 mJ/cm) of femtosecond laser irradiation than the alkanethiol MTAB.

Conclusion: GNRs appear to be optimal for clinical applications with systemic administration of NPs not-requiring irradiation at high laser intensity such as drug delivery and photothermal therapy inducing apoptosis.
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http://dx.doi.org/10.2147/IJN.S304953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140906PMC
June 2021

Suprabasin-A Review.

Genes (Basel) 2021 01 18;12(1). Epub 2021 Jan 18.

Laboratory of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 14220 Prague, Czech Republic.

Among the ~22,000 human genes, very few remain that have unknown functions. One such example is suprabasin (). Originally described as a component of the cornified envelope, the function of stratified epithelia-expressed is unknown. Both the lack of knowledge about the gene role under physiological conditions and the emerging link of to various human diseases, including cancer, attract research interest. The association of expression with poor prognosis of patients suffering from oesophageal carcinoma, glioblastoma multiforme, and myelodysplastic syndromes suggests that may play a role in human tumourigenesis. Three isoforms code for the secreted proteins with putative function as signalling molecules, yet with poorly described effects. In this first review about , we summarised the current knowledge accumulated since its original description, and we discuss the potential mechanisms and roles of in both physiology and pathology.
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http://dx.doi.org/10.3390/genes12010108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831088PMC
January 2021

Design, synthesis, and evaluation of BP-1-102 analogs with modified hydrophobic fragments for STAT3 inhibition.

J Enzyme Inhib Med Chem 2021 Dec;36(1):410-424

Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech Republic.

Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed and synthesised with the aim to modify hydrophobic fragments of the molecules that are important for interaction with the STAT3 SH2 domain. The cytotoxic activity of the reference and novel compounds was evaluated using several human and two mouse cancer cell lines. BP-1-102 and its two analogs emerged as effective cytotoxic agents and were further tested in additional six human and two murine cancer cell lines, in all of which they manifested the cytotoxic effect in a micromolar range. Reference compound S3I-201.1066 was found ineffective in all tested cell lines, in contrast to formerly published data. The ability of selected BP-1-102 analogs to induce apoptosis and inhibition of STAT3 receptor-mediated phosphorylation was confirmed. The structure-activity relationship confirmed a demand for two hydrophobic substituents, i.e. the pentafluorophenyl moiety and another spatially bulky moiety, for effective cytotoxic activity and STAT3 inhibition.
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http://dx.doi.org/10.1080/14756366.2020.1871336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808747PMC
December 2021

Aberrantly elevated suprabasin in the bone marrow as a candidate biomarker of advanced disease state in myelodysplastic syndromes.

Mol Oncol 2020 10 11;14(10):2403-2419. Epub 2020 Aug 11.

Department of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.

Myelodysplastic syndromes (MDS) are preleukemic disorders characterized by clonal growth of mutant hematopoietic stem and progenitor cells. MDS are associated with proinflammatory signaling, dysregulated immune response, and cell death in the bone marrow (BM). Aging, autoinflammation and autoimmunity are crucial features of disease progression, concordant with promoting growth of malignant clones and accumulation of mutations. Suprabasin (SBSN), a recently proposed proto-oncogene of unknown function, physiologically expressed in stratified epithelia, is associated with poor prognosis of several human malignancies. Here, we showed that SBSN is expressed in the BM by myeloid cell subpopulations, including myeloid-derived suppressor cells, and is secreted into BM plasma and peripheral blood of MDS patients. The highest expression of SBSN was present in a patient group with poor prognosis. SBSN levels in the BM correlated positively with blast percentage and negatively with CCL2 chemokine levels and lymphocyte count. In vitro treatment of leukemic cells with interferon-gamma and demethylating agent 5-azacytidine (5-AC) induced SBSN expression. This indicated that aberrant cytokine levels in the BM and epigenetic landscape modifications in MDS patients may underlie ectopic expression of SBSN. Our findings suggest SBSN as a candidate biomarker of high-risk MDS with a possible role in disease progression and therapy resistance.
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http://dx.doi.org/10.1002/1878-0261.12768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530796PMC
October 2020

Isoform-resolved correlation analysis between mRNA abundance regulation and protein level degradation.

Mol Syst Biol 2020 03;16(3):e9170

Yale Cancer Biology Institute, Yale University, West Haven, CT, USA.

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post-translational turnover, we devised a strategy combining pulse stable isotope-labeled amino acids in cells (pSILAC), data-independent acquisition mass spectrometry (DIA-MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome-wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.
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http://dx.doi.org/10.15252/msb.20199170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073818PMC
March 2020

Dynamic PML protein nucleolar associations with persistent DNA damage lesions in response to nucleolar stress and senescence-inducing stimuli.

Aging (Albany NY) 2019 09 7;11(17):7206-7235. Epub 2019 Sep 7.

Department of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.

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.
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http://dx.doi.org/10.18632/aging.102248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756913PMC
September 2019

Highly hydrophilic cationic gold nanorods stabilized by novel quaternary ammonium surfactant with negligible cytotoxicity.

J Biophotonics 2019 12 23;12(12):e201900024. Epub 2019 Jul 23.

Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic.

The photothermal cancer therapy using cationic gold nanorods (GNRs) stabilized by quaternary ammonium salts (QAS) have a great potential to enhance conventional cancer treatment as it promises the effective eradication of cancer cells including cells resistant to radio- and chemo-therapy and the stimulation of anti-tumor immune response. However, as the cytotoxicity of the conventional alkanethiol-QAS compounds limits their utility in medicine, here we developed GNRs modified by novel highly hydrophilic cationic surfactant composed of the quaternary ammonium group and ethylene glycol chain N,N,N-trimethyl-3,6,9,12,15-pentaoxaheptadecyl-17-sulfanyl-1-ammonium bromide (POSAB) showing insignificant cytotoxicity in the free state. Surface modification of GNRs by POSAB allowed to prepare nanoparticles with good stability in water, high cellular uptake and localization in lysosomes that are a promising alternative to alkanethiol-stabilized GNRs especially for biomedical applications.
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http://dx.doi.org/10.1002/jbio.201900024DOI Listing
December 2019

Quantification of cellular protein and redox imbalance using SILAC-iodoTMT methodology.

Redox Biol 2019 06 21;24:101227. Epub 2019 May 21.

Department of Genome Integrity, Institute of Molecular Genetics of the ASCR, v. v. i., Videnska 1083, 142 20, Prague, Czech Republic. Electronic address:

Under normal conditions, the cellular redox status is maintained in a steady state by reduction and oxidation processes. These redox alterations in the cell are mainly sensed by protein thiol residues of cysteines thus regulating protein function. The imbalance in redox homeostasis may therefore regulate protein turnover either directly by redox modulating of transcription factors or indirectly by the degradation of damaged proteins due to oxidation. A new analytical method capable of simultaneously assessing cellular protein expression and cysteine oxidation would provide a valuable tool for the field of cysteine-targeted biology. Here, we show a workflow based on protein quantification using metabolic labeling and determination of cysteine oxidation using reporter ion quantification. We applied this approach to determine protein and redox changes in cells after 5-min, 60-min and 32-h exposure to HO, respectively. Based on the functional analysis of our data, we confirmed a biological relevance of this approach and its applicability for parallel mapping of cellular proteomes and redoxomes under diverse conditions. In addition, we revealed a specific pattern of redox changes in peroxiredoxins in a short time-interval cell exposure to HO. Overall, our present study offers an innovative, versatile experimental approach to the multifaceted assessment of cellular proteome and its redox status, with broad implications for biomedical research towards a better understanding of organismal physiology and diverse disease conditions.
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http://dx.doi.org/10.1016/j.redox.2019.101227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545335PMC
June 2019

PML nuclear bodies are recruited to persistent DNA damage lesions in an RNF168-53BP1 dependent manner and contribute to DNA repair.

DNA Repair (Amst) 2019 06 6;78:114-127. Epub 2019 Apr 6.

Department of Genome Integrity, Institute of Molecular Genetics, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic; Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic; Genome Integrity Unit, Danish Cancer Society Research Center, Copenhagen, Denmark; Science for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 21, Stockholm, Sweden. Electronic address:

The bulk of DNA damage caused by ionizing radiation (IR) is generally repaired within hours, yet a subset of DNA lesions may persist even for long periods of time. Such persisting IR-induced foci (pIRIF) co-associate with PML nuclear bodies (PML-NBs) and are among the characteristics of cellular senescence. Here we addressed some fundamental questions concerning the nature and determinants of this co-association, the role of PML-NBs at such sites, and the reason for the persistence of DNA damage in human primary cells. We show that the persistent DNA lesions are devoid of homologous recombination (HR) proteins BRCA1 and Rad51. Our super-resolution microscopy-based analysis showed that PML-NBs are juxtaposed to and partially overlap with the pIRIFs. Notably, depletion of 53BP1 resulted in decreased intersection between PML-NBs and pIRIFs implicating the RNF168-53BP1 pathway in their interaction. To test whether the formation and persistence of IRIFs is PML-dependent and to investigate the role of PML in the context of DNA repair and senescence, we genetically deleted PML in human hTERT-RPE-1 cells. Unexpectedly, upon high-dose IR treatment, cells displayed similar DNA damage signalling, repair dynamics and kinetics of cellular senescence regardless of the presence or absence of PML. In contrast, the PML knock-out cells showed increased sensitivity to low doses of IR and DNA-damaging agents mitomycin C, cisplatin and camptothecin that all cause DNA lesions requiring repair by HR. These results, along with enhanced sensitivity of the PML knock-out cells to DNA-PK and PARP inhibitors implicate PML as a factor contributing to HR-mediated DNA repair.
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http://dx.doi.org/10.1016/j.dnarep.2019.04.001DOI Listing
June 2019

Interferon-regulated suprabasin is essential for stress-induced stem-like cell conversion and therapy resistance of human malignancies.

Mol Oncol 2019 07 10;13(7):1467-1489. Epub 2019 Jun 10.

Laboratory of Genome Integrity, Institute of Molecular Genetics of the ASCR, v. v. i., Prague, Czech Republic.

Radiation and chemotherapy represent standard-of-care cancer treatments. However, most patients eventually experience tumour recurrence, treatment failure and metastatic dissemination with fatal consequences. To elucidate the molecular mechanisms of resistance to radio- and chemotherapy, we exposed human cancer cell lines (HeLa, MCF-7 and DU145) to clinically relevant doses of 5-azacytidine or ionizing radiation and compared the transcript profiles of all surviving cell subpopulations, including low-adherent stem-like cells. Stress-mobilized low-adherent cell fractions differed from other survivors in terms of deregulation of hundreds of genes, including those involved in interferon response. Exposure of cancer cells to interferon-gamma but not interferon-beta resulted in the development of a heterogeneous, low-adherent fraction comprising not only apoptotic/necrotic cells but also live cells exhibiting active Notch signalling and expressing stem-cell markers. Chemical inhibition of mitogen-activated protein kinase/ERK kinase (MEK) or siRNA-mediated knockdown of extracellular signal-regulated kinase 1/2 (Erk1/2) and interferon responsible factor 1 (IRF1) prevented mobilization of the surviving low-adherent population, indicating that interferon-gamma-mediated loss of adhesion and anoikis resistance required an active Erk pathway interlinked with interferon signalling by transcription factor IRF1. Notably, a skin-specific protein suprabasin (SBSN), a recently identified oncoprotein, was among the top scoring genes upregulated in surviving low-adherent cancer cells induced by 5-azacytidine or irradiation. SBSN expression required the activity of the MEK/Erk pathway, and siRNA-mediated knockdown of SBSN suppressed the low-adherent fraction in irradiated, interferon-gamma- and 5-azacytidine-treated cells, respectively, implicating SBSN in genotoxic stress-induced phenotypic plasticity and stress resistance. Importantly, SBSN expression was observed in human clinical specimens of colon and ovarian carcinomas, as well as in circulating tumour cells and metastases of the 4T1 mouse model. The association of SBSN expression with progressive stages of cancer development indicates its role in cancer evolution and therapy resistance.
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http://dx.doi.org/10.1002/1878-0261.12480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599850PMC
July 2019

Targeting genotoxic and proteotoxic stress-response pathways in human prostate cancer by clinically available PARP inhibitors, vorinostat and disulfiram.

Prostate 2019 03 29;79(4):352-362. Epub 2018 Nov 29.

Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.

Background: Castration-resistant prostate cancer (PCa) represents a serious health challenge. Based on mechanistically-supported rationale we explored new therapeutic options based on clinically available drugs with anticancer effects, including inhibitors of PARP1 enzyme (PARPi), and histone deacetylases (vorinostat), respectively, and disulfiram (DSF, known as alcohol-abuse drug Antabuse) and its copper-chelating metabolite CuET that inhibit protein turnover.

Methods: Drugs and their combination with ionizing radiation (IR) were tested in various cytotoxicity assays in three human PCa cell lines including radio-resistant stem-cell like derived cells. Mechanistically, DNA damage repair, heat shock and unfolded protein response (UPR) pathways were assessed by immunofluorescence and immunoblotting.

Results: We observed enhanced sensitivity to PARPi/IR in PC3 cells consistent with lower homologous recombination (HR) repair. Vorinostat sensitized DU145 cells to PARPi/IR and decreased mutant p53. Vorinostat also impaired HR-mediated DNA repair, as determined by Rad51 foci formation and downregulation of TOPBP1 protein, and overcame radio-resistance of stem-cell like DU145-derived cells. All PCa models responded well to CuET or DSF combined with copper. We demonstrated that DSF interacts with copper in the culture media and forms adequate levels of CuET indicating that DSF/copper and CuET may be considered as comparable treatments. Both DSF/copper and CuET evoked hallmarks of UPR in PCa cells, documented by upregulation of ATF4, CHOP and phospho-eIF2α, with ensuing heat shock response encompassing activation of HSF1 and HSP70. Further enhancing the cytotoxicity of CuET, combination with an inhibitor of the anti-apoptotic protein survivin (YM155, currently undergoing clinical trials) promoted the UPR-induced toxicity, yielding synergistic effects of CuET and YM155.

Conclusions: We propose that targeting genotoxic and proteotoxic stress responses by combinations of available drugs could inspire innovative strategies to treat castration-resistant PCa.
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http://dx.doi.org/10.1002/pros.23741DOI Listing
March 2019

Distinct phenotypes and 'bystander' effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines.

Int J Oncol 2018 Nov 5;53(5):1997-2009. Epub 2018 Sep 5.

Laboratory of Immunological and Tumour Models, Institute of Molecular Genetics of the Czech Academy of Sciences, v.v.i., Prague 4 142 20, Czech Republic.

Cellular senescence is the process of the permanent proliferative arrest of cells in response to various inducers. It is accompanied by typical morphological changes, in addition to the secretion of bioactive molecules, including proinflammatory cytokines and chemokines [known as the senescence-associated secretory phenotype (SASP)]. Thus, senescent cells may affect their local environment and induce a so-called 'bystander' senescence through the state of SASP. The phenotypes of senescent cells are determined by the type of agent inducing cellular stress and the cell lineages. To characterise the phenotypes of senescent cancer cells, two murine cell lines were employed in the present study: TC-1 and B16F10 (B16) cells. Two distinct senescence inductors were used: Chemotherapeutic agent docetaxel (DTX) and a combination of immunomodulatory cytokines, including interferon γ (IFNγ) and tumour necrosis factor α (TNFα). It was demonstrated that DTX induced senescence in TC-1 and B16 tumour cell lines, which was demonstrated by growth arrest, positive β-galactosidase staining, increased p21Waf1 (p21) expression and the typical SASP capable of inducing a 'bystander' senescence. By contrast, treatment with a combination of T helper cell 1 cytokines, IFNγ and TNFα, induced proliferation arrest only in B16 cells. Despite the presence of certain characteristic features resembling senescent cells (proliferation arrest, morphological changes and increased p21 expression), these cells were able to form tumours in vivo and started to proliferate upon cytokine withdrawal. In addition, B16 cells were not able to induce a 'bystander' senescence. In summary, the present study described cell line- and treatment-associated differences in the phenotypes of senescent cells that may be relevant in optimization of cancer chemo- and immunotherapy.
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http://dx.doi.org/10.3892/ijo.2018.4553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192732PMC
November 2018

Selective elimination of senescent cells by mitochondrial targeting is regulated by ANT2.

Cell Death Differ 2019 01 21;26(2):276-290. Epub 2018 May 21.

Laboratory of Molecular Therapy, Institute of Biotechnology, Czech Academy of Sciences, Prague, 252 50, Czech Republic.

Cellular senescence is a form of cell cycle arrest that limits the proliferative potential of cells, including tumour cells. However, inability of immune cells to subsequently eliminate senescent cells from the organism may lead to tissue damage, inflammation, enhanced carcinogenesis and development of age-related diseases. We found that the anticancer agent mitochondria-targeted tamoxifen (MitoTam), unlike conventional anticancer agents, kills cancer cells without inducing senescence in vitro and in vivo. Surprisingly, it also selectively eliminates both malignant and non-cancerous senescent cells. In naturally aged mice treated with MitoTam for 4 weeks, we observed a significant decrease of senescence markers in all tested organs compared to non-treated animals. Mechanistically, we found that the susceptibility of senescent cells to MitoTam is linked to a very low expression level of adenine nucleotide translocase-2 (ANT2), inherent to the senescent phenotype. Restoration of ANT2 in senescent cells resulted in resistance to MitoTam, while its downregulation in non-senescent cells promoted their MitoTam-triggered elimination. Our study documents a novel, translationally intriguing role for an anticancer agent targeting mitochondria, that may result in a new strategy for the treatment of age-related diseases and senescence-associated pathologies.
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http://dx.doi.org/10.1038/s41418-018-0118-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329828PMC
January 2019

Induction, regulation and roles of neural adhesion molecule L1CAM in cellular senescence.

Aging (Albany NY) 2018 03;10(3):434-462

Department of Genome Integrity, Institute of Molecular Genetics of the ASCR, Prague 14220, Czech Republic.

Aging involves tissue accumulation of senescent cells (SC) whose elimination through senolytic approaches may evoke organismal rejuvenation. SC also contribute to aging-associated pathologies including cancer, hence it is imperative to better identify and target SC. Here, we aimed to identify new cell-surface proteins differentially expressed on human SC. Besides previously reported proteins enriched on SC, we identified 78 proteins enriched and 73 proteins underrepresented in replicatively senescent BJ fibroblasts, including L1CAM, whose expression is normally restricted to the neural system and kidneys. L1CAM was: 1) induced in premature forms of cellular senescence triggered chemically and by gamma-radiation, but not in Ras-induced senescence; 2) induced upon inhibition of cyclin-dependent kinases by p16; 3) induced by TGFbeta and suppressed by RAS/MAPK(Erk) signaling (the latter explaining the lack of L1CAM induction in RAS-induced senescence); and 4) induced upon downregulation of growth-associated gene ANT2, growth in low-glucose medium or inhibition of the mevalonate pathway. These data indicate that L1CAM is controlled by a number of cell growth- and metabolism-related pathways during SC development. Functionally, SC with enhanced surface L1CAM showed increased adhesion to extracellular matrix and migrated faster. Our results provide mechanistic insights into senescence of human cells, with implications for future senolytic strategies.
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http://dx.doi.org/10.18632/aging.101404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892697PMC
March 2018

NQO1*2 polymorphism predicts overall survival in MDS patients.

Br J Haematol 2019 01 24;184(2):305-308. Epub 2018 Jan 24.

1st Department of Medicine - Department of Haematology, First Faculty of Medicine, Charles University in Prague and General University Hospital, Prague, Czech Republic.

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http://dx.doi.org/10.1111/bjh.15088DOI Listing
January 2019

Biological safety and tissue distribution of (16-mercaptohexadecyl)trimethylammonium bromide-modified cationic gold nanorods.

Biomaterials 2018 Feb 1;154:275-290. Epub 2017 Nov 1.

Department of Genome Integrity, Institute of Molecular Genetics of the CAS, v.v.i., Prague, Czech Republic. Electronic address:

The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications. Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (GNRs). Interestingly, even after cellular accumulation of high amounts of GNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton, or in cell migration could be detected in vitro. In vivo, after intravenous administration, the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of GNRs administered as 10 or 50 μg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of GNRs by living systems.
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http://dx.doi.org/10.1016/j.biomaterials.2017.10.044DOI Listing
February 2018

Dynamic alterations of bone marrow cytokine landscape of myelodysplastic syndromes patients treated with 5-azacytidine.

Oncoimmunology 2016;5(10):e1183860. Epub 2016 May 13.

1st Department of Medicine, First Faculty of Medicine, Charles University in Prague and General University Hospital , Prague, Czech Republic.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis frequently progressing into acute myeloid leukemia (AML), with emerging evidence implicating aberrant bone marrow (BM) microenvironment and inflammation-related changes. 5-azacytidine (5-AC) represents standard MDS treatment. Besides inhibiting DNA/RNA methylation, 5-AC has been shown to induce DNA damage and apoptosis . To provide insights into effects, we assessed the proinflammatory cytokines alterations during MDS progression, cytokine changes after 5-AC, and contribution of inflammatory comorbidities to the cytokine changes in MDS patients. We found that IL8, IP10/CXCL10, MCP1/CCL2 and IL27 were significantly elevated and IL12p70 decreased in BM of MDS low-risk, high-risk and AML patients compared to healthy donors. Repeated sampling of the high-risk MDS patients undergoing 5-AC therapy revealed that the levels of IL8, IL27 and MCP1 in BM plasma were progressively increasing in agreement with experiments using several cancer cell lines. Moreover, the presence of inflammatory diseases correlated with higher levels of IL8 and MCP1 in low-risk but not in high-risk MDS. Overall, all forms of MDS feature a deregulated proinflammatory cytokine landscape in the BM and such alterations are further augmented by therapy of MDS patients with 5-AC.
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http://dx.doi.org/10.1080/2162402X.2016.1183860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087287PMC
May 2016

Two-Step Mechanism of Cellular Uptake of Cationic Gold Nanoparticles Modified by (16-Mercaptohexadecyl)trimethylammonium Bromide.

Bioconjug Chem 2016 Oct 26;27(10):2558-2574. Epub 2016 Sep 26.

Department of Genome Integrity, Institute of Molecular Genetics of the CAS, v.v.i. , CZ-142 20 Prague 4, Czech Republic.

Cationic colloidal gold nanorods (GNRs) have a great potential as a theranostic tool for diverse medical applications. GNRs' properties such as cellular internalization and stability are determined by physicochemical characteristics of their surface coating. GNRs modified by (16-mercaptohexadecyl)trimethylammonium bromide (MTAB), GNRs, show excellent cellular uptake. Despite their promise for biomedicine, however, relatively little is known about the cellular pathways that facilitate the uptake of GNRs, their subcellular fate and intracellular persistence. Here we studied the mechanism of cellular internalization and long-term fate of GNRs coated with MTAB, for which the synthesis was optimized to give higher yield, in various human cell types including normal diploid versus cancerous, and dividing versus nondividing (senescent) cells. The process of GNRs internalization into their final destination in lysosomes proceeds in two steps: (1) fast passive adhesion to cell membrane mediated by sulfated proteoglycans occurring within minutes and (2) slower active transmembrane and intracellular transport of individual nanorods via clathrin-mediated endocytosis and of aggregated nanorods via macropinocytosis. The expression of sulfated proteoglycans was the major factor determining the extent of uptake by the respective cell types. Upon uptake into proliferating cells, GNRs were diluted equally and relatively rapidly into daughter cells; however, in nondividing/senescent cells the loss of GNRs was gradual and very modest, attributable mainly to exocytosis. Exocytosed GNRs can again be internalized. These findings broaden our knowledge about cellular uptake of gold nanorods, a crucial prerequisite for future successful engineering of nanoparticles for biomedical applications such as photothermal cancer therapy or elimination of senescent cells as part of the emerging rejuvenation approach.
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http://dx.doi.org/10.1021/acs.bioconjchem.6b00491DOI Listing
October 2016

Tumor growth accelerated by chemotherapy-induced senescent cells is suppressed by treatment with IL-12 producing cellular vaccines.

Oncotarget 2016 Aug;7(34):54952-54964

Immunology Unit, Czech Centre for Phenogenomics, BIOCEV and Department of Transgenic Models of Diseases, Institute of Molecular Genetics of the ASCR, v.v.i., Prague 14220, Czech Republic.

Standard-of-care chemo- or radio-therapy can induce, besides tumor cell death, also tumor cell senescence. While senescence is considered to be a principal barrier against tumorigenesis, senescent cells can survive in the organism for protracted periods of time and they can promote tumor development. Based on this emerging concept, we hypothesized that elimination of such potentially cancer-promoting senescent cells could offer a therapeutic benefit. To assess this possibility, here we first show that tumor growth of proliferating mouse TC-1 HPV-16-associated cancer cells in syngeneic mice becomes accelerated by co-administration of TC-1 or TRAMP-C2 prostate cancer cells made senescent by pre-treatment with the anti-cancer drug docetaxel, or lethally irradiated. Phenotypic analyses of tumor-explanted cells indicated that the observed acceleration of tumor growth was attributable to a protumorigenic environment created by the co-injected senescent and proliferating cancer cells rather than to escape of the docetaxel-treated cells from senescence. Notably, accelerated tumor growth was effectively inhibited by cell immunotherapy using irradiated TC-1 cells engineered to produce interleukin IL-12. Collectively, our data document that immunotherapy, such as the IL-12 treatment, can provide an effective strategy for elimination of the detrimental effects caused by bystander senescent tumor cells in vivo.
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http://dx.doi.org/10.18632/oncotarget.10712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342393PMC
August 2016

Interferon gamma/NADPH oxidase defense system in immunity and cancer.

Oncoimmunology 2016 Feb 1;5(2):e1080416. Epub 2015 Sep 1.

Department of Genome Integrity of Institute of Molecular Genetics of the ASCR, v.v.i., Prague, Czech Republic; Danish Cancer Society Research Center, Copenhagen, Denmark.

As a part of cellular pathogen defense, IFNγ triggers induction of NADPH oxidase NOX2, which produces superoxide into phagosomes of immune cells. Recent data show that a similar mechanism can also operate in IFNγ-mediated anticancer control. IFNγ is capable of inducing expression of constitutively active NADPH oxidase NOX4 in tumor cells leading to generation of reactive oxygen species (ROS) damaging DNA, activation of DNA damage response and cell cycle arrest/premature cellular senescence.
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http://dx.doi.org/10.1080/2162402X.2015.1080416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801460PMC
February 2016

Small Molecules Targeting Ataxia Telangiectasia and Rad3-Related (ATR) Kinase: An Emerging way to Enhance Existing Cancer Therapy.

Curr Cancer Drug Targets 2016 ;16(3):200-8

University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic.

The main aim of current cancer research is to find a way to selectively affect the tumor cells, while leaving normal cells intact. Ataxia telangiectasia and Rad3-related kinase (ATR), a member of the phosphatidylinositol-3-related protein kinases (PIKK), represents a candidate target for achieving this goal. ATR kinase is one of the main kinases of the DNA damage response signaling pathway and responds to DNA damage caused by replication stress and various genotoxic agents (i.e. chemotherapy, ionizing radiation, ultraviolet light). ATR activation triggers cell cycle checkpoints, DNA repair and apoptosis, but also resistance of tumor cells to DNA damaging agents, through stress support under replication stress. Thus, the inhibition of ATR leads to increased effectiveness of cancer therapy and in addition enables highly selective targeting of cancer cells through synthetic lethal interactions. Despite this great potential, only a few potent and selective inhibitors of ATR kinase have been developed to date. However, those which have been developed provide great promise, and are under evaluation in many current preclinical and clinical trials. The purpose of this review is to summarize the potential of ATR inhibitors and the medicinal chemistry efforts which resulted in their identification.
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http://dx.doi.org/10.2174/156800961603160206122927DOI Listing
November 2016

Cat scratches, not bites, are associated with unipolar depression--cross-sectional study.

Parasit Vectors 2016 Jan 5;9. Epub 2016 Jan 5.

Department of Genome Integrity, Institute of Molecular Genetics ASCR, v.v.i, Prague, Czech Republic.

Background: A recent study performed on 1.3 million patients showed a strong association between being bitten by a cat and probability of being diagnosed with depression. Authors suggested that infection with cat parasite Toxoplasma could be the reason for this association.

Method: A cross sectional internet study on a non-clinical population of 5,535 subjects was undertaken.

Results: The subjects that reported having been bitten by a dog and a cat or scratched by a cat have higher Beck depression score. They were more likely to have visited psychiatrists, psychotherapists and neurologists in past two years, to have been previously diagnosed with depression (but not with bipolar disorder). Multivariate analysis of models with cat biting, cat scratching, toxoplasmosis, the number of cats at home, and the age of subjects as independent variables showed that only cat scratching had positive effect on depression (p = 0.004). Cat biting and toxoplasmosis had no effect on the depression, and the number of cats at home had a negative effect on depression (p = 0.021).

Conclusions: Absence of association between toxoplasmosis and depression and five times stronger association of depression with cat scratching than with cat biting suggests that the pathogen responsible for mood disorders in animals-injured subjects is probably not the protozoon Toxoplasma gondii but another organism; possibly the agent of cat-scratched disease - the bacteria Bartonella henselae.
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http://dx.doi.org/10.1186/s13071-015-1290-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700762PMC
January 2016

Complementary genetic screens identify the E3 ubiquitin ligase CBLC, as a modifier of PARP inhibitor sensitivity.

Oncotarget 2015 May;6(13):10746-58

The CRUK Gene Function Laboratory and Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK.

Based on a series of basic, preclinical and clinical studies, the Poly (ADP-ribose) Polymerase 1 (PARP1) inhibitor, olaparib, has recently been approved for use in ovarian cancer patients with BRCA1 or BRCA2 mutations. By identifying novel predictive biomarkers of tumour cell sensitivity to olaparib, it is possible that the utility of PARP inhibitors could be extended beyond this patient subgroup. Many of the known genetic determinants of PARP inhibitor response have key roles in DNA damage response (DDR) pathways. Although protein ubiquitylation is known to play an important role in regulating the DDR, the exact mechanisms by which this occurs are not fully understood. Using two parallel RNA interference-based screening approaches, we identified the E3 ubiquitin ligase, CBLC, as a candidate biomarker of response to olaparib. We validated this observation by demonstrating that silencing of CBLC causes increased sensitivity to olaparib in breast cancer cell line models and that defective homologous recombination (HR) DNA repair is the likely cause. This data provides an example of how defects in the ubiquitin machinery have the potential to influence the response of tumour cells to PARP inhibitors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4484416PMC
http://dx.doi.org/10.18632/oncotarget.3628DOI Listing
May 2015

DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase.

Cell Cycle 2015 ;14(3):375-87

a Department of Cell Signaling and Apoptosis.

Death domain-associated protein 6 (DAXX) is a histone chaperone, putative regulator of apoptosis and transcription, and candidate modulator of p53-mediated gene expression following DNA damage. DAXX becomes phosphorylated upon DNA damage, however regulation of this modification, and its relationship to p53 remain unclear. Here we show that in human cells exposed to ionizing radiation or genotoxic drugs etoposide and neocarzinostatin, DAXX became rapidly phosphorylated in an ATM kinase-dependent manner. Our deletion and site-directed mutagenesis experiments identified Serine 564 (S564) as the dominant ATM-targeted site of DAXX, and immunofluorescence experiments revealed localization of S564-phosphorylated DAXX to PML nuclear bodies. Furthermore, using a panel of human cell types, we identified the p53-regulated Wip1 protein phosphatase as a key negative regulator of DAXX phosphorylation at S564, both in vitro and in cells. Consistent with the emerging oncogenic role of Wip1, its DAXX-dephosphorylating impact was most apparent in cancer cell lines harboring gain-of-function mutant and/or overexpressed Wip1. Unexpectedly, while Wip1 depletion increased DAXX phosphorylation both before and after DNA damage and increased p53 stability and transcriptional activity, knock-down of DAXX impacted neither p53 stabilization nor p53-mediated expression of Gadd45a, Noxa, Mdm2, p21, Puma, Sesn2, Tigar or Wip1. Consistently, analyses of cells with genetic, TALEN-mediated DAXX deletion corroborated the notion that neither phosphorylated nor non-phosphorylated DAXX is required for p53-mediated gene expression upon DNA damage. Overall, we identify ATM kinase and Wip1 phosphatase as opposing regulators of DAXX-S564 phosphorylation, and propose that the role of DAXX phosphorylation and DAXX itself are independent of p53-mediated gene expression.
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http://dx.doi.org/10.4161/15384101.2014.988019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4353233PMC
December 2015

Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress.

Mol Oncol 2015 Mar 15;9(3):601-16. Epub 2014 Nov 15.

Danish Cancer Society Research Center, DK-2100 Copenhagen, Denmark; Department of Genome Integrity, Institute of Molecular Genetics, v.v.i., Academy of Sciences of the Czech Republic, CZ-142 20 Prague, Czech Republic; Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, CZ-775 15 Olomouc, Czech Republic. Electronic address:

Both Myc and Ras oncogenes impact cellular metabolism, deregulate redox homeostasis and trigger DNA replication stress (RS) that compromises genomic integrity. However, how are such oncogene-induced effects evoked and temporally related, to what extent are these kinetic parameters shared by Myc and Ras, and how are these cellular changes linked with oncogene-induced cellular senescence in different cell context(s) remain poorly understood. Here, we addressed the above-mentioned open questions by multifaceted comparative analyses of human cellular models with inducible expression of c-Myc and H-RasV12 (Ras), two commonly deregulated oncoproteins operating in a functionally connected signaling network. Our study of DNA replication parameters using the DNA fiber approach and time-course assessment of perturbations in glycolytic flux, oxygen consumption and production of reactive oxygen species (ROS) revealed the following results. First, overabundance of nuclear Myc triggered RS promptly, already after one day of Myc induction, causing slow replication fork progression and fork asymmetry, even before any metabolic changes occurred. In contrast, Ras overexpression initially induced a burst of cell proliferation and increased the speed of replication fork progression. However, after several days of induction Ras caused bioenergetic metabolic changes that correlated with slower DNA replication fork progression and the ensuing cell cycle arrest, gradually leading to senescence. Second, the observed oncogene-induced RS and metabolic alterations were cell-type/context dependent, as shown by comparative analyses of normal human BJ fibroblasts versus U2-OS sarcoma cells. Third, the energy metabolic reprogramming triggered by Ras was more robust compared to impact of Myc. Fourth, the detected oncogene-induced oxidative stress was due to ROS (superoxide) of non-mitochondrial origin and mitochondrial OXPHOS was reduced (Crabtree effect). Overall, our study provides novel insights into oncogene-evoked metabolic reprogramming, replication and oxidative stress, with implications for mechanisms of tumorigenesis and potential targeting of oncogene addiction.
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http://dx.doi.org/10.1016/j.molonc.2014.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528704PMC
March 2015

Phosphatidylinositol 3-Kinase (PI3K) and phosphatidylinositol 3-kinase-related kinase (PIKK) inhibitors: importance of the morpholine ring.

J Med Chem 2015 Jan 5;58(1):41-71. Epub 2014 Dec 5.

Biomedical Research Center, University Hospital Hradec Kralove , Sokolska 81, 500 05 Hradec Kralove, Czech Republic.

Phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 3-kinase-related protein kinases (PIKKs) are two related families of kinases that play key roles in regulation of cell proliferation, metabolism, migration, survival, and responses to diverse stresses including DNA damage. To design novel efficient strategies for treatment of cancer and other diseases, these kinases have been extensively studied. Despite their different nature, these two kinase families have related origin and share very similar kinase domains. Therefore, chemical inhibitors of these kinases usually carry analogous structural motifs. The most common feature of these inhibitors is a critical hydrogen bond to morpholine oxygen, initially present in the early nonspecific PI3K and PIKK inhibitor 3 (LY294002), which served as a valuable chemical tool for development of many additional PI3K and PIKK inhibitors. While several PI3K pathway inhibitors have recently shown promising clinical responses, inhibitors of the DNA damage-related PIKKs remain thus far largely in preclinical development.
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http://dx.doi.org/10.1021/jm501026zDOI Listing
January 2015

The development of ataxia telangiectasia mutated kinase inhibitors.

Mini Rev Med Chem 2014 Oct 13. Epub 2014 Oct 13.

University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic..

Radiation and genotoxic drugs are two of the cornerstones of current cancer treatment strategy. However, this type of therapy often suffers from radio- or chemo-resistance caused by DNA repair mechanisms. With the aim of increasing the efficacy of these treatments, there has been great interest in studying DNA damage responses (DDR). Among the plethora of signal and effector proteins involved in DDR, three related kinases ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related) and DNA-PK (DNA-dependent protein kinase) play the main roles in initiation and regulation of signaling pathways in response to DNA double and single strand breaks (DSB and SSB). ATM inhibitors, as well as those of ATR and DNA-PK, provide an opportunity to sensitize cancer cells to therapy. Moreover, they can lead to selective killing of cancer cells, exploiting a concept known as synthetic lethality. However, only a very few selective inhibitors have been identified to this date. This mini-review is focused both on the development of selective inhibitors of ATM and other inhibitors which have ATM as one of their targets.
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October 2014

TGF-β/NF1/Smad4-mediated suppression of ANT2 contributes to oxidative stress in cellular senescence.

Cell Signal 2014 Dec 16;26(12):2903-11. Epub 2014 Sep 16.

Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovak Republic. Electronic address:

Oxidative stress and persistent activation of DNA damage response (DDR) are causally involved in the development of cellular senescence, a phenomenon implicated in fundamental (patho)physiological processes such as aging, fetal development and tumorigenesis. Here, we report that adenine nucleotide translocase-2 (ANT2) is consistently down-regulated in all three major forms of cellular senescence: replicative, oncogene-induced and drug-induced, in both normal and cancerous human cells. We previously reported formation of novel NF1/Smad transcription repressor complexes in growth-arrested fibroblasts. Here we show that such complexes form in senescent cells. Mechanistically, binding of the NF1/Smad complexes to the NF1-dependent repressor elements in the ANT2 gene promoter repressed ANT2 expression. Etoposide-induced formation of these complexes and repression of ANT2 were relatively late events co-incident with production and secretion of, and dependent on, TGF-β. siRNA-mediated knock-down of ANT2 in proliferating cells resulted in increased levels of reactive oxygen species (ROS) and activation of the DDR. Knock-down of ANT2, together with etoposide treatment, further intensified ROS production and DNA damage signaling, leading to enhanced apoptosis. Together, our data show that TGF-β-mediated suppression of ANT2 through NF1/Smad4 complexes contributes to oxidative stress and DNA damage during induction of cellular senescence.
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http://dx.doi.org/10.1016/j.cellsig.2014.08.029DOI Listing
December 2014

The development of ataxia telangiectasia mutated kinase inhibitors.

Mini Rev Med Chem 2014 ;14(10):805-11

University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic.

Radiation and genotoxic drugs are two of the cornerstones of current cancer treatment strategy. However, this type of therapy often suffers from radio- or chemo-resistance caused by DNA repair mechanisms. With the aim of increasing the efficacy of these treatments, there has been great interest in studying DNA damage responses (DDR). Among the plethora of signal and effector proteins involved in DDR, three related kinases ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related) and DNA-PK (DNA-dependent protein kinase) play the main roles in initiation and regulation of signaling pathways in response to DNA double and single strand breaks (DSB and SSB). ATM inhibitors, as well as those of ATR and DNA-PK, provide an opportunity to sensitize cancer cells to therapy. Moreover, they can lead to selective killing of cancer cells, exploiting a concept known as synthetic lethality. However, only a very few selective inhibitors have been identified to this date. This mini-review is focused both on the development of selective inhibitors of ATM and other inhibitors which have ATM as one of their targets.
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June 2015
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