Abstract
DNA-protein crosslinks (DPCs), formed by the covalent conjugation of proteins to DNA, are toxic lesions that interfere with DNA metabolic processing and transcription. The development of an ...accurate biochemical assay for DPC isolation is a priority for the mechanistic understanding of their repair. Here, we propose the STAR assay for the direct quantification of DPCs, sensitive to physiologically relevant treatment conditions. Implementing the STAR assay revealed the formation of small cross-linked peptides on DNA, created by the proteolytic degradation of DPCs by SPRTN. The initial proteolytic degradation of DPCs is required for the downstream activation of DNA repair, which is mediated through the phosphorylation of H2Ax. This leads to the accumulation of DNA repair factors on chromatin and the subsequent complete removal of the cross-linked peptides. These results confirmed that the repair of DPCs is a two-step process, starting with proteolytic resection by SPRTN, followed by the repair of the underlying damage to the DNA.
Graphical Abstract
Graphical Abstract
The first step in DPC repair is SPRTN-mediated proteolysis. The remaining cross-linked peptides are finally resolved by the activation of DNA damage repair.
Histones are an essential part of nucleosomes that regulate chromatin structure and function. Histone exchanges and modifications represent a scaffold for DNA transcription, repair, and replication. ...Studying histones and histone code is an important and fast-developing branch of epigenetic science. Here we propose a fast, efficient, and versatile assay for nucleosomal histone isolation from mammalian cells, without the use of acids or high salt solutions which are common for other histone isolation techniques. All components used in the protocol are common and inexpensive laboratory chemicals. The protocol has been evaluated on six commonly used cell lines and two animal tissue samples. The mild extraction conditions preserve delicate histone epigenetic changes, allowing its downstream analyses. We have demonstrated the assays' successful application during changes in the transcriptional activity of histone genes, cell cycle transitions, and DNA-damaging conditions. Histone fractions, obtained by the protocol, can be used for further applications, such as electrophoresis, immunoblot, and mass spectrometry. Therefore, the new proposed nucleosomal histone isolation method is sensitive, specific, and suitable for downstream applications of various kinds.
Nerolidol is a naturally occurring sesquiterpene alcohol with multiple properties, including antioxidant, antibacterial, and antiparasitic activities. A few studies investigating the antitumor ...properties of nerolidol have shown positive results in both cell culture and mouse models. In this study, we investigated the antitumor mechanism of
-nerolidol in bladder carcinoma cell lines. The results of our experiments on two bladder carcinoma cell lines revealed that nerolidol inhibited cell proliferation and induced two distinct cell death pathways. We confirmed that
-nerolidol induces DNA damage and ER stress. A mechanistic study identified a common cAMP, Ca
, and MAPK axis involved in signal propagation and amplification, leading to ER stress. Inhibition of any part of this signaling cascade prevented both cell death pathways. The two cell death mechanisms can be distinguished by the involvement of caspases. The early occurring cell death pathway is characterized by membrane blebbing and cell swelling followed by membrane rupture, which can be prevented by the inhibition of caspase activation. In the late cell death pathway, which was found to be caspase-independent, cytoplasmic vacuolization and changes in cell shape were observed.
-Nerolidol shows promising antitumor activity through an unorthodox mechanism of action that could help target resistant forms of malignancies, such as bladder cancer.
Endoplasmic reticulum (ER) stress, prompted by the accumulation of misfolded or unfolded proteins, triggers the activation of the unfolded protein response (UPR) pathway to restore ER homeostasis. ...This stress response is implicated in the development of hepatocellular carcinoma (HCC). A biallelic mutation in SPRTN is currently the only known single-gene mutation implicated in the early onset of HCC. However, the exact mechanism linking SPRTN mutations to HCC remains unclear. In our study, we analyzed SPRTN and UPR in 21 human HCC tissue samples using RT-qPCR, immunoblot, and immunohistochemistry. We found alterations in the expression levels of SPRTN and UPR-related genes and proteins in HCC samples. The impact of SPRTN on the ER stress response was assessed in SPRTN-depleted HepG2 cells through RNA sequencing, pull-down assay, comet assay, and mitotic index calculation. We demonstrated that SPRTN interacts with the UPR sensor GRP78. Furthermore, we observed a decrease in SPRTN levels during ER stress, and increased sensitivity to ER stress in SPRTN-depleted cells. These findings suggest an essential role for SPRTN in the ER stress response and provide new insights into HCC pathogenesis. This newly discovered function of SPRTN could significantly enhance our understanding and treatment of HCC.
We have previously shown in a rat model that a single bout of high-intensity aerobic exercise 20h before a simulated dive
reduces bubble formation and after the dive protects from lethal ...decompression sickness. The present study investigated the
importance of these findings in man. Twelve healthy male divers were compressed in a hyperbaric chamber to 280kPa at a rate
of 100kPamin â1 breathing air and remaining at pressure for 80min. The ascent rate was 9mmin â1 with a 7min stop at 130kPa. Each diver underwent two randomly assigned simulated dives, with or without preceding exercise.
A single interval exercise performed 24h before the dive consisted of treadmill running at 90% of maximum heart rate for 3min,
followed by exercise at 50% of maximum heart rate for 2min; this was repeated eight times for a total exercise period of 40min.
Venous gas bubbles were monitored with an ultrasonic scanner every 20min for 80min after reaching surface pressure. The study
demonstrated that a single bout of strenuous exercise 24h before a dive to 18 m of seawater significantly reduced the average
number of bubbles in the pulmonary artery from 0.98 to 0.22 bubbles cm â2 ( P = 0.006) compared to dives without preceding exercise. The maximum bubble grade was decreased from 3 to 1.5 ( P = 0.002) by pre-dive exercise, thereby increasing safety. This is the first report to indicate that pre-dive exercise may
form the basis for a new way of preventing serious decompression sickness.
Ruijs-Aalfs syndrome is a segmental progeroid syndrome resulting from mutations in the
gene. Cells derived from patients with SPRTN mutations elicit genomic instability and people afflicted with this ...syndrome developed hepatocellular carcinoma. Here we describe the molecular mechanism by which SPRTN contributes to genome stability and normal cellular homeostasis. We show that SPRTN is a DNA-dependent mammalian protease required for resolving cytotoxic DNA-protein crosslinks (DPCs)- a function that had only been attributed to the metalloprotease Wss1 in budding yeast. We provide genetic evidence that SPRTN and Wss1 function distinctly in vivo to resolve DPCs. Upon DNA and ubiquitin binding, SPRTN can elicit proteolytic activity; cleaving DPC substrates and itself.
null cells or cells derived from patients with Ruijs-Aalfs syndrome are impaired in the resolution of covalent DPCs in vivo. Collectively, SPRTN is a mammalian protease required for resolving DNA-protein crosslinks in vivo whose function is compromised in Ruijs-Aalfs syndrome patients.
Purpose
Given that the tumor-promoting inflammation has been previously established in squamous cell carcinoma of the bladder but its contribution to development of urothelial carcinoma (UC) still ...remains elusive, our aim was to study changes in expression and activity of inflammation-mediating NF-κB and STAT3 transcription factors in human urothelial bladder carcinoma as well as expression of their target genes cyclin D1, VEGFA and TGFβ1.
Methods
Gene expression of STAT3, NF-κB, TGFβ1, cyclin D1 and VEGFA was measured by quantitative real-time polymerase chain reaction in both tumor and healthy bladder tissue from 36 patients with UC of the bladder. Activation of STAT3 and NF-κB was assessed with immunohistochemistry and immunoblot.
Results
Urothelial bladder carcinoma displayed elevated expression as well as activation of NF-κB (
P
= 5.38e−10) and STAT3 (
P
= 0.002) transcription factors. Furthermore, elevated level of expression was observed for cyclin D1, VEGFA and TGFβ1 (
P
= 9.71e−09,
P
= 9.71e−09,
P
= 5.38e−10). Preliminary statistical analysis indicated that the level of upregulation of STAT3 or NF-κB was probably not dependent upon the grade (
P
= 0.984 and 0.803, respectively) and invasiveness of the tumor (0.399 and 0.949), nor to the gender (0.780 and 0.536) and age (0.660 and 0.816) of the patients.
Conclusions
NF-κB and STAT3 signaling pathways, as main inflammatory mediators, are found to be activated in urothelial bladder carcinoma indicating that chronic inflammatory processes are accompanying development of this tumor type. Future studies will have to determine possible causative role of inflammatory processes in development of urothelial bladder carcinomas.
The aim of this study was to determine the antitumor activity of nerolidol, a naturally occurring sesquiterpene alcohol, in two bladder carcinoma cell lines and to elucidate the possible cell death ...pathway. Bladder carcinoma is one of the most common malignancies worldwide, with a high recurrence rate and low survival. In our study, we observed multiple cellular mechanisms of nerolidol with two distinct modes of cell death. Its specific induction of cell death, which does not depend on apoptotic factors, is a potential therapeutic approach to overcome the inherent resistance of tumors to apoptosis. Nerolidol is a naturally occurring sesquiterpene alcohol with multiple properties, including antioxidant, antibacterial, and antiparasitic activities. A few studies investigating the antitumor properties of nerolidol have shown positive results in both cell culture and mouse models. In this study, we investigated the antitumor mechanism of cis-nerolidol in bladder carcinoma cell lines. The results of our experiments on two bladder carcinoma cell lines revealed that nerolidol inhibited cell proliferation and induced two distinct cell death pathways. We confirmed that cis-nerolidol induces DNA damage and ER stress. A mechanistic study identified a common cAMP, Casup.2+ , and MAPK axis involved in signal propagation and amplification, leading to ER stress. Inhibition of any part of this signaling cascade prevented both cell death pathways. The two cell death mechanisms can be distinguished by the involvement of caspases. The early occurring cell death pathway is characterized by membrane blebbing and cell swelling followed by membrane rupture, which can be prevented by the inhibition of caspase activation. In the late cell death pathway, which was found to be caspase-independent, cytoplasmic vacuolization and changes in cell shape were observed. cis-Nerolidol shows promising antitumor activity through an unorthodox mechanism of action that could help target resistant forms of malignancies, such as bladder cancer.
Several genes and their single nucleotide polymorphisms (SNPs) are associated with either spontaneous resolution of hepatitis C infection or better treatment-induced viral clearance. We tested a ...cohort of intravenous drug users (IVDU) diagnosed with chronic hepatitis C virus (HCV) for treatment response and its association with the SNPs in the interleukin-6 (rs1800795-IL6) and the interleukin-28B (rs12979860-IL28B) genes.
The study included 110 Croatian IVDU positive for anti-HCV antibody. Genotyping was performed by polymerase chain reaction (PCR) based approach. Patients were treated by standard pegylated-interferon/ribavirin and followed throughout a period of four years, during which sustained virological response (SVR) was determined. All data were analysed with statistical package SPSS 19.0 (IBM Corp, Armonk, NY, USA) and PLINK v1.07 software.
Patients showed a significantly better response to treatment according to the number of copies of the C allele carried at rs1800795-IL6 (P = 0.034). All but one of the patients with CC genotype achieved SVR (93%), whereas the response rate of patients with GG genotype was 64%. The association of rs1800795-IL6 with SVR status remained significant after further adjustment for patients' age, fibrosis staging, and viral genotype (OR 2.15, 95% CI 1.16-4.68, P = 0.019). Distributions of allele frequencies at the locus rs12979860-IL28B among the study cohort and the underlying general population were suggestive of a protective effect of CC genotype in acquiring chronic hepatitis C in the Croatian IVDU population.
The rs1800795-IL6 polymorphism is associated with positive response to treatment in IVDU patients positive for HCV infection. A protective role of rs12979860-IL28B CC genotype in acquiring chronic hepatitis C is suggested for Croatian IVDU population.
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