Publications by authors named "Romano Strobelt"

5 Publications

  • Page 1 of 1

NQO1 Binds and Supports SIRT1 Function.

Front Pharmacol 2021 21;12:671929. Epub 2021 Jun 21.

Department of Molecular Genetics Weizmann Institute of Science, Rehovot, Israel.

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD dependent deacetylase may act as a rheostat in sensing mitochondrial stress.
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http://dx.doi.org/10.3389/fphar.2021.671929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255383PMC
June 2021

Deciphering the BSE-type specific cell and tissue tropisms of atypical (H and L) and classical BSE.

Prion 2019 01;13(1):160-172

Friedrich-Loeffler-Institut, Institute of Novel and Emerging Infectious Diseases , Greifswald , Germany.

After the discovery of two atypical bovine spongiform encephalopathy (BSE) forms in France and Italy designated H- and L-BSE, the question arose whether these new forms differed from classical BSE (C-BSE) in their pathogenesis. Samples collected from cattle in the clinical stage of BSE during an intracranial challenge study with L- and H-BSE were analysed using biochemical and histological methods as well as in a transgenic mouse bioassay. Our results generally confirmed what had been described for C-BSE to be true also for both atypical BSE forms, namely the restriction of the pathological prion protein (PrP) and BSE infectivity to the nervous system. However, analysis of samples collected under identical conditions from both atypical H- and L-BSE forms allowed us a more precise assessment of the grade of involvement of different tissues during the clinical end stage of disease as compared to C-BSE. One important feature is the involvement of the peripheral nervous and musculoskeletal tissues in both L-BSE and H-BSE affected cattle. We were, however, able to show that in H-BSE cases, the PrP depositions in the central and peripheral nervous system are dominated by a glial pattern, whereas a neuronal deposition pattern dominates in L-BSE cases, indicating differences in the cellular and topical tropism of both atypical BSE forms. As a consequence of this cell tropism, H-BSE seems to spread more rapidly from the CNS into the periphery via the glial cell system such as Schwann cells, as opposed to L-BSE which is mostly propagated via neuronal cells.
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http://dx.doi.org/10.1080/19336896.2019.1651180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746549PMC
January 2019

Sulfur mustard resistant keratinocytes obtained elevated glutathione levels and other changes in the antioxidative defense mechanism.

Toxicol Lett 2018 Sep 26;293:51-61. Epub 2017 Nov 26.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany; Universität der Bundeswehr München, Faculty of Human Sciences, Department for Sports Sciences, Werner-Heisenberg-Weg 39, 85577 Neubiberg, Germany. Electronic address:

Background: Sulfur mustard (SM) is a potent blistering chemical warfare agent, which was first used in 1917. Despite the Chemical Weapons Convention, a use was recently reported in Syria in 2015. This emphasizes the importance to develop countermeasures against chemical warfare agents. Despite intensive research, there is still no antidote or prophylaxis available against SM.

Methods: The newly developed SM-resistant keratinocyte cell line HaCaT/SM was used to identify new target structures for drug development, particularly the adaptations in protective measures against oxidative stress. For this purpose, glutathione (GSH) and NAD(P)H levels, the effect of glutathione S-transferase (GST) inhibition as well as activation and expression of Nrf2, GST, glutamate cysteine ligase (GCL) and glutathione-disulfide reductase (GSR) as well as multi-drug resistance (MDR) proteins 1, 3 and 5 were investigated.

Results: The HaCaT/SM cells showed not only a better survival after treatment with SM or cytostatic drugs, but also hydrogen peroxide (HO). They exhibit more GSH even after SM treatment. Nrf2 levels were significantly lower. Inhibition of GST led to significantly decreased, activation to slightly higher IC values after SM treatment and a lower expression of GST was observed. The cells also expressed less GCLC and GSR. Expression of MDR1, MDR3 and MDR5 was higher under control conditions, but less stimulated by SM treatment. An increased NADP/NADPH ratio as well as higher NAD levels were shown.

Conclusion: In summary, an improved response of the resistant cell line to oxidative stress was observed. The underlying mechanisms are elevated GSH levels as well as lower expression of Nrf2 and its targets GCLC and GST as well as GSR and MDR1, MDR3 and MDR5. GST is an especially interesting target because its inhibition already induced a significant SM sensitivity. SM resistance also caused redox equivalent level differences. Taken together, these findings provide further insight into the mechanism of SM resistance and may open a window for novel therapeutic targets in SM therapy.
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http://dx.doi.org/10.1016/j.toxlet.2017.11.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235149PMC
September 2018

Characterization of sulfur mustard resistant keratinocyte cell line HaCaT/SM.

Toxicol Lett 2016 Feb 9;244:49-55. Epub 2015 Oct 9.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany; Department of Molecular and Cellular Sports Medicine, German Sports University, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany. Electronic address:

Background: The cell line HaCaT/SM was derived from the human keratinocyte cell line HaCaT. HaCaT/SM cells display a high resistance against sulfur mustard (SM). Intention of the presented study was to determine the cellular and molecular differences between HaCaT/SM and HaCaT so as to evaluate which changes might be responsible for being resistant against SM.

Methods: Both cell lines HaCaT and HaCaT/SM were analyzed with respect to their cell growth, nuclei perimeter, clonogenicity and secretion profile. Moreover DNA alkylation pattern under presence of SM was investigated.

Results: In comparison to HaCaT, the HaCaT/SM showed a significant smaller nuclei perimeter. For DNA alkylation a significant difference was observed over time. The clonogenicity of HaCaT/SM was increased to 150%. The secretion profile of these cells demonstrated a strong increase of ANG, PDGF-AA, TIMP1, TIMP2, and a decrease of AREG, CCL5, CXC1, CXC2/3, CXCL6, CXCL7, CXCL8, CXCL10, MIF, Trappin-1.

Conclusion: The sulfur mustard (SM) resistant cell line HaCaT/SM demonstrates a wide range of significant differences to their origin cell line HaCaT. These differences might be responsible to provide resistance against SM and might also be useful to establish treatment concepts for humans after SM exposure.
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http://dx.doi.org/10.1016/j.toxlet.2015.10.001DOI Listing
February 2016

Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

Toxicol Lett 2016 Feb 14;244:56-71. Epub 2015 Sep 14.

University of Konstanz, Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany. Electronic address:

Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of N7-ETE-guanine DNA adducts, the excision rate of CEES-induced DNA adducts was not affected by PARP inhibition. Furthermore, while CEES induced moderate changes in cellular NAD(+) levels, annexin V/PI flow cytometry analysis revealed that these changes did not affect CEES-induced short-term cytotoxicity 24h after treatment. In contrast, PARP inhibition impaired cell proliferation and clonogenic survival, and potentiated micronuclei formation of HaCaT cells upon CEES treatment. Similarly, PARP inhibition affected clonogenic survival of cells treated with bi-functional mustards such as SM and HN2. In conclusion, we demonstrate that PARylation plays a functional role in mustard-induced cellular stress response with substance-specific differences. Since PARP inhibitors exhibit therapeutic potential to treat SM-related pathologies and to sensitize cancer cells for mustard-based chemotherapy, potential long-term effects of PARP inhibition on genomic stability and carcinogenesis should be carefully considered when pursuing such a strategy.
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http://dx.doi.org/10.1016/j.toxlet.2015.09.010DOI Listing
February 2016
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