Publications by authors named "Dusana Majera"

11 Publications

  • Page 1 of 1

Effect of Sepatronium Bromide (YM-155) on DNA Double-Strand Breaks Repair in Cancer Cells.

Int J Mol Sci 2020 Dec 11;21(24). Epub 2020 Dec 11.

Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, 779 00 Olomouc, Czech Republic.

Survivin, as an antiapoptotic protein often overexpressed in cancer cells, is a logical target for potential cancer treatment. By overexpressing survivin, cancer cells can avoid apoptotic cell death and often become resistant to treatments, representing a significant obstacle in modern oncology. A survivin suppressor, an imidazolium-based compound known as YM-155, is nowadays studied as an attractive anticancer agent. Although survivin suppression by YM-155 is evident, researchers started to report that YM-155 is also an inducer of DNA damage introducing yet another anticancer mechanism of this drug. Moreover, the concentrations of YM-155 for DNA damage induction seems to be far lower than those needed for survivin inhibition. Understanding the molecular mechanism of action of YM-155 is of vital importance for modern personalized medicine involving the selection of responsive patients and possible treatment combinations. This review focuses mainly on the documented effects of YM-155 on DNA damage signaling pathways. It summarizes up to date literature, and it outlines the molecular mechanism of YM-155 action in the context of the DNA damage field.
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http://dx.doi.org/10.3390/ijms21249431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763167PMC
December 2020

Targeting the NPL4 Adaptor of p97/VCP Segregase by Disulfiram as an Emerging Cancer Vulnerability Evokes Replication Stress and DNA Damage while Silencing the ATR Pathway.

Cells 2020 02 18;9(2). Epub 2020 Feb 18.

Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, 77 147 Olomouc, Czech Republic.

Research on repurposing the old alcohol-aversion drug disulfiram (DSF) for cancer treatment has identified inhibition of NPL4, an adaptor of the p97/VCP segregase essential for turnover of proteins involved in multiple pathways, as an unsuspected cancer cell vulnerability. While we reported that NPL4 is targeted by the anticancer metabolite of DSF, the bis-diethyldithiocarbamate-copper complex (CuET), the exact, apparently multifaceted mechanism(s) through which the CuET-induced aggregation of NPL4 kills cancer cells remains to be fully elucidated. Given the pronounced sensitivity to CuET in tumor cell lines lacking the genome integrity caretaker proteins BRCA1 and BRCA2, here we investigated the impact of NPL4 targeting by CuET on DNA replication dynamics and DNA damage response pathways in human cancer cell models. Our results show that CuET treatment interferes with DNA replication, slows down replication fork progression and causes accumulation of single-stranded DNA (ssDNA). Such a replication stress (RS) scenario is associated with DNA damage, preferentially in the S phase, and activates the homologous recombination (HR) DNA repair pathway. At the same time, we find that cellular responses to the CuET-triggered RS are seriously impaired due to concomitant malfunction of the ATRIP-ATR-CHK1 signaling pathway that reflects an unorthodox checkpoint silencing mode through ATR (Ataxia telangiectasia and Rad3 related) kinase sequestration within the CuET-evoked NPL4 protein aggregates.
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http://dx.doi.org/10.3390/cells9020469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072750PMC
February 2020

Disulfiram's anti-cancer activity reflects targeting NPL4, not inhibition of aldehyde dehydrogenase.

Oncogene 2019 10 7;38(40):6711-6722. Epub 2019 Aug 7.

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

Aldehyde dehydrogenase (ALDH) is a proposed biomarker and possible target to eradicate cancer stem cells. ALDH inhibition as a treatment approach is supported by anti-cancer effects of the alcohol-abuse drug disulfiram (DSF, Antabuse). Given that metabolic products of DSF, rather than DSF itself inhibit ALDH in vivo, and that DSF's anti-cancer activity is potentiated by copper led us to investigate the relevance of ALDH as the suggested molecular cancer-relevant target of DSF. Here we show that DSF does not directly inhibit ALDH activity in diverse human cell types, while DSF's in vivo metabolite, S-methyl-N,N-diethylthiocarbamate-sulfoxide inhibits ALDH activity yet does not impair cancer cell viability. Our data indicate that the anti-cancer activity of DSF does not involve ALDH inhibition, and rather reflects the impact of DSF's copper-containing metabolite (CuET), that forms spontaneously in vivo and in cell culture media, and kills cells through aggregation of NPL4, a subunit of the p97/VCP segregase. We also show that the CuET-mediated, rather than any ALDH-inhibitory activity of DSF underlies the preferential cytotoxicity of DSF towards BRCA1- and BRCA2-deficient cells. These findings provide evidence clarifying the confusing literature about the anti-cancer mechanism of DSF, a drug currently tested in clinical trials for repositioning in oncology.
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http://dx.doi.org/10.1038/s41388-019-0915-2DOI Listing
October 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

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

Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.

Nature 2017 12 6;552(7684):194-199. Epub 2017 Dec 6.

Danish Cancer Society Research Center, DK-2100 Copenhagen, Denmark.

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.
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http://dx.doi.org/10.1038/nature25016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730499PMC
December 2017

DNA damage signalling barrier, oxidative stress and treatment-relevant DNA repair factor alterations during progression of human prostate cancer.

Mol Oncol 2016 06 3;10(6):879-94. Epub 2016 Mar 3.

Danish Cancer Society Research Center, Copenhagen, Denmark; Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden; Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic. Electronic address:

The DNA damage checkpoints provide an anti-cancer barrier in diverse tumour types, however this concept has remained unexplored in prostate cancer (CaP). Furthermore, targeting DNA repair defects by PARP1 inhibitors (PARPi) as a cancer treatment strategy is emerging yet requires suitable predictive biomarkers. To address these issues, we performed immunohistochemical analysis of multiple markers of DNA damage signalling, oxidative stress, DNA repair and cell cycle control pathways during progression of human prostate disease from benign hyperplasia, through intraepithelial neoplasia to CaP, complemented by genetic analyses of TMPRSS2-ERG rearrangement and NQO1, an anti-oxidant factor and p53 protector. The DNA damage checkpoint barrier (γH2AX, pATM, p53) mechanism was activated during CaP tumorigenesis, albeit less and with delayed culmination compared to other cancers, possibly reflecting lower replication stress (slow proliferation despite cases of Rb loss and cyclin D1 overexpression) and progressive loss of ATM activator NKX3.1. Oxidative stress (8-oxoguanine lesions) and NQO1 increased during disease progression. NQO1 genotypes of 390 men did not indicate predisposition to CaP, yet loss of NQO1 in CaP suggested potential progression-opposing tumour suppressor role. TMPRSS2-ERG rearrangement and PTEN loss, events sensitizing to PARPi, occurred frequently along with heterogeneous loss of DNA repair factors 53BP1, JMJD1C and Rev7 (all studied here for the first time in CaP) whose defects may cause resistance to PARPi. Overall, our results reveal an unorthodox DNA damage checkpoint barrier scenario in CaP tumorigenesis, and provide novel insights into oxidative stress and DNA repair, with implications for biomarker guidance of future targeted therapy of CaP.
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http://dx.doi.org/10.1016/j.molonc.2016.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423169PMC
June 2016

Cells and Stripes: A novel quantitative photo-manipulation technique.

Sci Rep 2016 Jan 18;6:19567. Epub 2016 Jan 18.

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

Laser micro-irradiation is a technology widely used in the DNA damage response, checkpoint signaling, chromatin remodeling and related research fields, to assess chromatin modifications and recruitment of diverse DNA damage sensors, mediators and repair proteins to sites of DNA lesions. While this approach has aided numerous discoveries related to cell biology, maintenance of genome integrity, aging and cancer, it has so far been limited by a tedious manual definition of laser-irradiated subcellular regions, with the ensuing restriction to only a small number of cells treated and analyzed in a single experiment. Here, we present an improved and versatile alternative to the micro-irradiation approach: Quantitative analysis of photo-manipulated samples using innovative settings of standard laser-scanning microscopes. Up to 200 cells are simultaneously exposed to a laser beam in a defined pattern of collinear rays. The induced striation pattern is then automatically evaluated by a simple algorithm, which provides a quantitative assessment of various laser-induced phenotypes in live or fixed cells. Overall, this new approach represents a more robust alternative to existing techniques, and provides a versatile tool for a wide range of applications in biomedicine.
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http://dx.doi.org/10.1038/srep19567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726120PMC
January 2016

Expression, purification and assembly of soluble multimeric MHC class II-invariant chain complexes.

FEBS Lett 2012 May 27;586(9):1318-24. Epub 2012 Mar 27.

Department of Biochemistry, Molecular and Structural Biology and Centre for Protein and Structure Production, Jozef Stefan Institute, Ljubljana, Slovenia.

Major histocompatibility class (MHC) II molecules are essential for running adaptive immune response. They are produced in the ER and targeted to late endosomes with the help of invariant chain (Ii) trimers. Ii trimerization may be induced by the Ii TM domain. To enable mechanistic and structural studies of MHC class II-Ii assembly, soluble forms of the complexes were expressed. We show that Ii trimerizes in the absence of the transmembrane part, prior to binding of α/β chains. The biochemical analysis supports the suggestion that the MHC class II-Ii complexes are not necessarily trimers of trimers, but that the Ii trimer can also be occupied by one or two MHC class II complexes.
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http://dx.doi.org/10.1016/j.febslet.2012.03.038DOI Listing
May 2012

Stefin A displaces the occluding loop of cathepsin B only by as much as required to bind to the active site cleft.

FEBS J 2010 Oct 22;277(20):4338-45. Epub 2010 Sep 22.

Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia.

Cathepsin B (EC 3.4.22.1) is one of the most versatile human cysteine cathepsins. It is important for intracellular protein degradation under normal conditions and is involved in a number of pathological processes. The occluding loop makes cathepsin B unique among cysteine cathepsins. This ∼ 20 residue long insertion imbedded into the papain-like protease scaffold restricts access to the active site cleft and endows cathepsin B with its carboxydipeptidase activity. Nevertheless, the enzyme also exhibits endopeptidase activity and is inhibited by stefins and cystatins. To clarify the structural properties of the occluding loop upon the binding of stefins, we determined the crystal structure of the complex between wild-type human stefin A and wild-type human cathepsin B at 2.6 Å resolution. The papain-like part of cathepsin B structure remains unmodified, whereas the occluding loop residues are displaced. The part enclosed by the disulfide bridge containing histidines 110 and 111 (i.e. the 'lasso' part) is rotated by ∼ 45° away from its original position. A comparison of the structure of the unliganded cathepsin B with the structure of the proenzyme, its complexes with chagasin and stefin A shows that the magnitude of the shift of the occluding loop is related to the size of the binding region. It is smallest in the procathepsin structures and increases in the series of complexes with stefin A and chagasin, although it has no impact on the binding constant. Hence, cathepsin B can dock inhibitors and certain substrates regardless of the size of the binding region.
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http://dx.doi.org/10.1111/j.1742-4658.2010.07824.xDOI Listing
October 2010

Expression and purification of recombinant NFI proteins for functional analysis.

Gen Physiol Biophys 2009 Dec;28(4):331-9

Laboratory of Molecular Biology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovakia.

Nuclear factor I (NFI) is a transcription factor playing wide role in signal transduction pathways and developmental processes in higher eukaryotes. In order to produce recombinant NFI proteins for functional and structural studies, full length cDNAs of individual isoforms were subcloned into pETM30 vector and expressed in Escherichia coli. Although the fusion proteins containing both glutathione S-transferase (GST) and His6 tags at the N-terminus could be overexpressed in detectable amounts, they were found mainly, if not exclusively, in insoluble form. Purification yield was improved by modification of cell disruption procedure and by the use of detergent Tween 20. The final purification strategy represents a triple-helix affinity chromatography consisting of prepurification of bacterial lysate on Heparin-Sepharose with subsequent immobilized metal affinity and glutathione affinity chromatography. Heparin chromatography was crucial for obtaining active NFI proteins, whereas the other steps significantly improved the purity of isolated proteins. As demonstrated by EMSA and DNase I protection assay, the recombinant proteins were able to recognize their cognate DNA sequences.
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http://dx.doi.org/10.4149/gpb_2009_04_331DOI Listing
December 2009
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