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Photodynamic therapy (PDT) is a low-invasive treatment method that can be used to treat VIN patients. A photosensitizer (PS) applied to a patient is activated with use of the appropriate wavelength of light, which in an oxygen environment leads to the formation of a reactive oxygen species (ROS) that destroys the tumor. However, cells can protect themselves against these cytotoxic products by increasing their antioxidant mechanisms and repair capacity. Changes in the cytoskeleton may also influence resistance to PDT. Our results revealed that PDT-resistant cells changed the amount of ROS. Cells resistant to PDT A-431 exhibited a decreased ROS level and showed higher viability after oxidizing agent treatment. Resistant Cal-39 cells exhibited a decreased O 2 - level but increased other ROS. This provides protection from PDT but not from other oxidizing agents. Moreover, PDT leads to alterations in the cytoskeleton that may result in an epithelial-mesenchymal transition (EMT) or increased adhesion. Both EMT and cell adhesion may activate signaling pathways involved in survival. This means that resistance to PDT in vulvar cancer may be at least in part a result of changes in ROS level and alterations in the cytoskeleton.

Photodynamic therapy (PDT) is a valuable treatment method for vulvar intraepithelial neoplasia (VIN). It allows for the treatment of a multifocal disease with minimal tissue destruction. 5-Aminolevulinic acid (5-ALA) is the most commonly used prodrug, which is converted in the heme pathway to protoporphyrin IX (PpIX), an actual photosensitizer (PS). Unfortunately, not all patients treated with PDT undergo complete remission. The main cause of their failure is resistance to anticancer therapy. In many cancers, resistance to various anticancer treatments is correlated with increased activity of the DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1). Enhanced activity of drug pumps may also affect the effectiveness of therapy. To investigate whether multidrug resistance mechanisms underlie PDT resistance in VIN, porphyrins were isolated from sensitive and resistant vulvar cancer cells and their culture media. APE1 activity was measured, and survival assay after PDT combined with APE1 inhibitor was performed. Our results revealed that resistant cells accumulated and effluxed less porphyrins than sensitive cells, and in response to PDT, resistant cells increased APE1 activity. Moreover, PDT combined with inhibition of APE1 significantly decreased the survival of PDT-resistant cells. This means that resistance to PDT in vulvar cancer may be the result of alterations in the heme synthesis pathway. Moreover, increased APE1 activity may be essential for the repair of PDT-mediated DNA damage, and inhibition of APE1 activity may increase the efficacy of PDT.

Photodynamic therapy (PDT) is a clinically approved cancer therapy of low invasiveness. The therapeutic procedure involves administering a photosensitizing drug (PS), which is then activated with monochromatic light of a specific wavelength. The photochemical reaction produces highly toxic oxygen species. The development of resistance to PDT in some cancer cells is its main limitation. Several mechanisms are known to be involved in the development of cellular defense against cytotoxic effects of PDT, including activation of antioxidant enzymes, drug efflux pumps, degradation of PS, and overexpression of protein chaperons. Another putative factor that plays an important role in the development of resistance of cancer cells to PDT seems to be DNA repair; however, it has not been well studied so far. To explore the role of DNA repair and other potential novel mechanisms associated with the resistance to PDT in the glioblastoma cells, cells stably resistant to PDT were isolated from PDT sensitive cells following repetitive PDT cycles. Duly characterization of isolated PDT-resistant glioblastoma revealed that the resistance to PDT might be a consequence of several mechanisms, including higher repair efficiency of oxidative DNA damage and repair of DNA breaks. Higher activity of APE1 endonuclease and increased expression and activation of DNA damage kinase ATM was demonstrated in the U-87 MGR cell line, suggesting and proving that they are good targets for sensitization of resistant cells to PDT., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyrimidinic (AP) endonuclease 1 (APE1/APEX1/HAP-1), referred also as a redox factor 1 (Ref-1), stimulates the DNA binding activities of the oxidized TFs such as AP-1 and NF-κB. Also, APE1 participates in the base excision repair (BER) and nucleotide incision repair (NIR) pathways to remove oxidative DNA base damage. At present, the molecular mechanism underlying the TF-stimulating/redox function of APE1 and its biological role remains disputed. Here, we provide evidence that, instead of direct cysteine reduction in TFs by APE1, APE1-catalyzed NIR and TF-stimulating activities may be based on transient cooperative binding of APE1 to DNA and induction of conformational changes in the helix. The structure of DNA duplex strongly influences NIR and TF-stimulating activities. Homologous plant AP endonucleases lacking conserved cysteine residues stimulate DNA binding of the p50 subunit of NF-κB. APE1 acts synergistically with low-molecular-weight reducing agents on TFs. Finally, APE1 stimulates DNA binding of the redox-insensitive p50-C62S mutant protein. Electron microscopy imaging of APE1 complexes with DNA revealed preferential polymerization of APE1 on the gapped and intrinsically curved DNA duplexes. Molecular modeling offers a structural explanation how full-length APE1 can oligomerize on DNA. In conclusion, we propose that DNA-directed APE1 oligomerization can be regarded as a substitute for diffusion of APE1 along the DNA contour to probe for anisotropic flexibility. APE1 oligomers exacerbate pre-existing distortions in DNA and enable both NIR activity and DNA binding by TFs regardless of their oxidation state., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The ability to preserve living cells or stem cells is critical for their use in cell therapy, especially for regenerative, reproductive, and transfusion medicine. This article addresses the low survival rates of cells and their loss of function during traditional freezing and banking (cells in a liquid medium with cryoprotectants). Aim: In this article, we developed multiple emulsions (water-in-oil-in-water type) for the effective encapsulation and cryopreservation of cells. In multiple emulsions, the oil drops, acting as a protective membrane, contain even smaller water droplets with encapsulated living cells, dispersed in the continuous water phase. Materials and Methods: The multiple emulsions with HEK293 cells encapsulated in the internal alginate droplets were successfully prepared in a Couette-Taylor flow biocontactor. The cryoprotectants (sucrose/dimethyl sulfoxide-DMSO) were located within the internal or external or both water phases of the emulsions. Encapsulated and non-encapsulated cells were frozen to -80°C (cooling rate: -1°C/min) and then transferred to liquid nitrogen (-196°C) for 24 hours. The standard rapid warming procedure was applied to thaw samples. Cell proliferation and viability were measured by using the AlamarBlue™ assay after recovery of cells. Results: The results showed that the viability of cells encapsulated in the internal droplets of multiple emulsions, and then cryopreserved, was significantly higher, up to 27.9%, than that observed for cells conventionally cryopreserved (non-encapsulated cells in water). Moreover, the effective cell-loaded multiple emulsions-based banking method allowed DMSO-toxic cryoprotectant-to be eliminated from the cryopreservation system. Conclusion: The proposed approach of the cryoprotection of cells encapsulated in multiple emulsions could minimize cell damage, degradation, and their loss during freezing - thawing processes.

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1 -/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl 4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1 -/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1 -/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats., (Copyright © 2018 Elsevier Inc. All rights reserved.)

5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1-/- primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2'-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2'-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1 -/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1 -/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1 -/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1 -/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1 -/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1 -/∆ and aged WT mice. Chronic treatment of Ercc1 -/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The base excision repair (BER) pathway consists of sequential action of DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease necessary to remove a damaged base and generate a single-strand break in duplex DNA. Human multifunctional AP endonuclease 1 (APE1, a.k.a. APEX1, HAP-1, or Ref-1) plays essential roles in BER by acting downstream of DNA glycosylases to incise a DNA duplex at AP sites and remove 3'-blocking sugar moieties at DNA strand breaks. Human 8-oxoguanine-DNA glycosylase (OGG1), methyl-CpG-binding domain 4 (MBD4, a.k.a. MED1), and alkyl-N-purine-DNA glycosylase (ANPG, a.k.a. Aag or MPG) excise a variety of damaged bases from DNA. Here we demonstrated that the redox-deficient truncated APE1 protein lacking the first N-terminal 61 amino acid residues (APE1-NΔ61) cannot stimulate DNA glycosylase activities of OGG1, MBD4, and ANPG on duplex DNA substrates. Electron microscopy imaging of APE1-DNA complexes revealed oligomerization of APE1 along the DNA duplex and APE1-mediated DNA bridging followed by DNA aggregation. APE1 polymerizes on both undamaged and damaged DNA in cooperative mode. Association of APE1 with undamaged DNA may enable scanning for damage; however, this event reduces effective concentration of the enzyme and subsequently decreases APE1-catalyzed cleavage rates on long DNA substrates. We propose that APE1 oligomers on DNA induce helix distortions thereby enhancing molecular recognition of DNA lesions by DNA glycosylases via a conformational proofreading/selection mechanism. Thus, APE1-mediated structural deformations of the DNA helix stabilize the enzyme-substrate complex and promote dissociation of human DNA glycosylases from the AP site with a subsequent increase in their turnover rate., Significance Statement: The major human apurinic/apyrimidinic (AP) endonuclease, APE1, stimulates DNA glycosylases by increasing their turnover rate on duplex DNA substrates. At present, the mechanism of the stimulation remains unclear. We report that the redox domain of APE1 is necessary for the active mode of stimulation of DNA glycosylases. Electron microscopy revealed that full-length APE1 oligomerizes on DNA possibly via cooperative binding to DNA. Consequently, APE1 shows DNA length dependence with preferential repair of short DNA duplexes. We propose that APE1-catalyzed oligomerization along DNA induces helix distortions, which in turn enable conformational selection and stimulation of DNA glycosylases. This new biochemical property of APE1 sheds light on the mechanism of redox function and its role in DNA repair., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The main cause of death in mantle cell lymphoma (MCL) patients is relapse due to undetermined minimal residual disease (MRD) and therefore monitoring MRD is crucial for making the best treatment decisions. The gold standard method for MRD analysis is the quantitative polymerase chain reaction. The most commonly used molecular markers for measuring MRD in MCL are: t(11;14)(q13;p32) translocation or CCND1 expression and IGH rearrangement. Such markers can, however, be found in other B cell non-Hodgkin lymphomas. Recent studies demonstrate that SOX11 expression is highly specific for MCL and could be used as a marker for measuring MRD. Moreover, evidence shows that SOX11 level could be predictive for overall survival (OS) and progression-free survival (PFS). We have measured MRD level in follow-up samples from 27 patients diagnosed with MCL using the molecular markers: t(11;14), IGH rearrangement and SOX11 expression. We compared all markers by their sensitivity, utility and quantitative range. We also examined the predictive value of SOX11 expression for OS and PFS. SOX11 expression was found to have better specificity, quantitative range and utility than the t(11;14). The predictive value of SOX11 expression was confirmed. At diagnosis, patients with high SOX11 expression had shorter PFS than patients with low SOX11 expression (p = 0.04*); differences between OS being statistically insignificant. To our best knowledge this is a first study comparing SOX11 with t(11;14) and IGH rearrangement as markers of MRD level. Moreover, in this study we confirmed that SOX11 is useful in cases when other molecular markers cannot be used.

Background . Endotoxin (LPS), the component of Gram-negative bacteria, is responsible for sepsis and neonatal mortality, but low concentrations of LPS produced tissue protection in experimental studies. The effects of LPS applied to the suckling rats on the pancreas of adult animals have not been previously explored. We present the impact of neonatal endotoxemia on the pancreatic exocrine function and on the acute pancreatitis which has been investigated in the adult animals. Endotoxemia was induced in suckling rats by intraperitoneal application of LPS from Escherichia coli or Salmonella typhi . In the adult rats, pretreated in the early period of life with LPS, histological manifestations of acute pancreatitis have been reduced. Pancreatic weight and plasma lipase activity were decreased, and SOD concentration was reversed and accompanied by a significant reduction of lipid peroxidation products (MDA + 4 HNE) in the pancreatic tissue. In the pancreatic acini, the significant increases in protein signals for toll-like receptor 4 and for heat shock protein 60 were found. Signal for the CCK1 receptor was reduced and pancreatic secretory responses to caerulein were diminished, whereas basal enzyme secretion was unaffected. These pioneer studies have shown that exposition of suckling rats to endotoxin has an impact on the pancreas in the adult organism.

Aim: Developing pH-responsive multiple emulsion platforms for effective glioblastoma multiforme therapy with reduced toxicity, a drug release study and modeling., Materials & Methods: Cancer cell line: U87 MG, multiple emulsions with pH-responsive biopolymer and encapsulated doxorubicin (DOX); preparation of multiple emulsions in a Couette-Taylor flow biocontactor, in vitro release study of DOX (fluorescence intensity analysis), in vitro cytotoxicity study (alamarBlue cell viability assay) and numerical simulation of DOX release rates., Results: The multiple emulsions offered a high DOX encapsulation efficiency (97.4 ± 1%) and pH modulated release rates of a drug. Multiple emulsions with a low concentration of DOX (0.02 μM) exhibited broadly advanced cell (U87 MG) cytotoxicity than free DOX solution used at the same concentration., Conclusion: Emulsion platforms could be explored for potential delivery of chemotherapeutics in glioblastoma multiforme therapy.

Exocyclic adducts to DNA bases are formed as a consequence of exposure to certain environmental carcinogens as well as inflammation and lipid peroxidation (LPO). Complex family of LPO products gives rise to a variety of DNA adducts, which can be grouped in two classes: (i) small etheno-type adducts of strong mutagenic potential, and (ii) bulky, propano-type adducts, which block replication and transcription, and are lethal lesions. Etheno-DNA adducts are removed from the DNA by base excision repair (BER), AlkB and nucleotide incision repair enzymes (NIR), while substituted propano-type lesions by nucleotide excision repair (NER) and homologous recombination (HR). Changes of the level and activity of several enzymes removing exocyclic adducts from the DNA was reported during carcinogenesis. Also several beyond repair functions of these enzymes, which participate in regulation of cell proliferation and growth, as well as RNA processing was recently described. In addition, adducts of LPO products to proteins was reported during aging and age-related diseases. The paper summarizes pathways for exocyclic adducts removal and describes how proteins involved in repair of these adducts can modify pathological states of the organism., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Oxidative DNA damage may be a risk factor for development of various pathologies, including malignancy. We studied inflammation triggered modulation of repair activity in the intestines of three weeks old rats injected i.p. with E.coli or S. typhimurium lipopolysaccharides (LPS) at doses of 1, 5 or 10 mg/kg. Subsequent formation in these animals of colonic preneoplastic lesions, aberrant crypt foci (ACF) was also investigated. Five days after LPS administration no differences were observed in repair rate of 1,N(6)-ethenoadenine (εA), 3,N(4)-ethenocytosine (εC) and 8-oxoguanine (8-oxoG) in intestines of these rats, as measured by the nicking assay. However a significant increase in all three repair activities was found within one and two months after S. typhimurium LPS treatment. E. coli LPS significantly increased only the 8-oxoG repair. S. typhimurium LPS stimulated mRNA transcription of pro-inflammatory proteins, lipooxygenase-12 and cyclooxygenase-2, as well as some DNA repair enzymes like AP-endonuclease (Ape1) and εC-glycosylase (Tdg). mRNA level of DNA glycosylases excising εA (MPG) and 8-oxoG (OGG1) was also increased by LPS treatment, but only at the highest dose. Transcription of all enzymes increased for up to 30 days after LPS, and subsequently decreased to the level observed before treatment, with the exception of APE1, which remained elevated even two months after LPS administration. Thus, the repair efficiency of εA, εC and 8-oxoG depends on the availability of APE1, which increases OGG1 and TDG turnover on damaged DNA, and presumably stimulates MPG. One and two months after administration of E. coli or S. typhimurium LPS, the number of aberrant crypt foci in rat colons increased in a dose and time dependent manner. Thus, inflammation stimulates the repair capacity for εA, εC and 8-oxoG, but simultaneously triggers the appearance of preneoplastic changes in the colons. This may be due to increased oxidative stress and imbalance in DNA repair.

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins., (Copyright © 2015 Elsevier B.V. All rights reserved.)