Predictive Potential of RNA Polymerase B (II) Subunit 1 (RPB1) Cytoplasmic Aggregation for Neoadjuvant Chemotherapy Failure.

We aimed to investigate the contribution of co-translational protein aggregation to the chemotherapy resistance of tumor cells. Increased co-translational protein aggregation reflects altered translation regulation that may have the potential to buffer transcription under genotoxic stress. As an indicator for such an event, we followed the cytoplasmic aggregation of RPB1, the aggregation-prone largest subunit of RNA polymerase II, in biopsy samples taken from patients with invasive carcinoma of no special type. RPB1 frequently aggregates co-translationally in the absence of proper HSP90 chaperone function or in ribosome mutant cells as revealed formerly in yeast. We found that cytoplasmic foci of RPB1 occur in larger sizes in tumors that showed no regression after therapy. Based on these results, we propose that monitoring the cytoplasmic aggregation of RPB1 may be suitable for determining-from biopsy samples taken before treatment-the effectiveness of neoadjuvant chemotherapy.

Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System.

DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., 18S rRNA, B2M, eEF1α1, GAPDH, GUSB, HPRT1, PPIA, and TBP) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using 18S rRNA, eEF1α1, GAPDH, GUSB, and HPRT1 genes for UVB-induced DNA damage-related studies. B2M, HPRT1, and TBP genes are recommended for NCS treatment, while 18S rRNA, B2M, and PPIA genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a "zero set" of potential reference gene candidates.

Application of Lacunarity for Quantification of Single Molecule Localization Microscopy Images.

The quantitative analysis of datasets achieved by single molecule localization microscopy is vital for studying the structure of subcellular organizations. Cluster analysis has emerged as a multi-faceted tool in the structural analysis of localization datasets. However, the results it produces greatly depend on the set parameters, and the process can be computationally intensive. Here we present a new approach for structural analysis using lacunarity. Unlike cluster analysis, lacunarity can be calculated quickly while providing definitive information about the structure of the localizations. Using simulated data, we demonstrate how lacunarity results can be interpreted. We use these interpretations to compare our lacunarity analysis with our previous cluster analysis-based results in the field of DNA repair, showing the new algorithm's efficiency.

The role of p53 in the DNA damage-related ubiquitylation of S2P RNAPII.

DNA double-strand breaks are one of the most deleterious lesions for the cells, therefore understanding the macromolecular interactions of the DNA repair-related mechanisms is essential. DNA damage triggers transcription silencing at the damage site, leading to the removal of the elongating RNA polymerase II (S2P RNAPII) from this locus, which provides accessibility for the repair factors to the lesion. We previously demonstrated that following transcription block, p53 plays a pivotal role in transcription elongation by interacting with S2P RNAPII. In the current study, we reveal that p53 is involved in the fine-tune regulation of S2P RNAPII ubiquitylation. Furthermore, we emphasize the potential role of p53 in delaying the premature ubiquitylation and the subsequent chromatin removal of S2P RNAPII as a response to transcription block.

The clinical significance of epigenetic and RNAPII variabilities occurring in clear cell renal cell carcinoma as a potential prognostic marker.

Patients diagnosed with clear cell renal cell carcinoma (ccRCC) have poor prognosis for recurrence and approximately 30-40% of them will later develop metastases. For this reason, the appropriate diagnosis and the more detailed molecular characterisation of the primary tumour, including its susceptibility to metastasis, are crucial to select the proper adjuvant therapy by which the most prosperous outcome can be achieved. Nowadays, clinicopathological variables are used for classification of the tumours. Apart from these, molecular biomarkers are also necessary to improve risk classification, which would be the most beneficial amongst modern adjuvant therapies. As a potential molecular biomarker, to follow the transcriptional kinetics in ccRCC patients (n=30), we analysed epigenetic changes (γH2A.X, H3K4me3, and H3K9me3) and the alterations in the level of RNA polymerase II (RNAPII) by immunohistochemical staining on dissected tissue sections. The variabilities between the tumorous and non-tumorous parts of the tissue were detected using quantitative image analysis by monitoring 30 cells from different positions of either the tumorous or the non-tumorous part of the tissue sections. Data obtained from the analyses were used to identify potential prognostic features and to associate them with the progression. These markers might have a value to predict patient outcomes based on their individual cellular background. These results also support that detection of any alteration in the level of H3K4me3, H3K9me3, and γH2A.X can account for valuable information for presuming the progression of ccRCC and the clinical benefits to select the most efficient personalised therapy.

SerpinB10, a Serine Protease Inhibitor, Is Implicated in UV-Induced Cellular Response.

UV-induced DNA damage response and repair are extensively studied processes, as any malfunction in these pathways contributes to the activation of tumorigenesis. Although several proteins involved in these cellular mechanisms have been described, the entire repair cascade has remained unexplored. To identify new players in UV-induced repair, we performed a microarray screen, in which we found SerpinB10 (SPB10, Bomapin) as one of the most dramatically upregulated genes following UV irradiation. Here, we demonstrated that an increased mRNA level of SPB10 is a general cellular response following UV irradiation regardless of the cell type. We showed that although SPB10 is implicated in the UV-induced cellular response, it has no indispensable function in cell survival upon UV irradiation. Nonetheless, we revealed that SPB10 might be involved in delaying the duration of DNA repair in interphase and also in S-phase cells. Additionally, we also highlighted the interaction between SPB10 and H3. Based on our results, it seems that SPB10 protein is implicated in UV-induced stress as a "quality control protein", presumably by slowing down the repair process.

Emerging Roles of Post-Translational Modifications in Nucleotide Excision Repair.

Nucleotide excision repair (NER) is a versatile DNA repair pathway which can be activated in response to a broad spectrum of UV-induced DNA damage, such as bulky adducts, including cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). Based on the genomic position of the lesion, two sub-pathways can be defined: (I) global genomic NER (GG-NER), involved in the ablation of damage throughout the whole genome regardless of the transcription activity of the damaged DNA locus, and (II) transcription-coupled NER (TC-NER), activated at DNA regions where RNAPII-mediated transcription takes place. These processes are tightly regulated by coordinated mechanisms, including post-translational modifications (PTMs). The fine-tuning modulation of the balance between the proteins, responsible for PTMs, is essential to maintain genome integrity and to prevent tumorigenesis. In this review, apart from the other substantial PTMs (SUMOylation, PARylation) related to NER, we principally focus on reversible ubiquitylation, which involves E3 ubiquitin ligase and deubiquitylase (DUB) enzymes responsible for the spatiotemporally precise regulation of NER.

Ubiquitylation-Mediated Fine-Tuning of DNA Double-Strand Break Repair.

The proper function of DNA repair is indispensable for eukaryotic cells since accumulation of DNA damages leads to genome instability and is a major cause of oncogenesis. Ubiquitylation and deubiquitylation play a pivotal role in the precise regulation of DNA repair pathways by coordinating the recruitment and removal of repair proteins at the damaged site. Here, we summarize the most important post-translational modifications (PTMs) involved in DNA double-strand break repair. Although we highlight the most relevant PTMs, we focus principally on ubiquitylation-related processes since these are the most robust regulatory pathways among those of DNA repair.

Quantification of DNA damage induced repair focus formation via super-resolution dSTORM localization microscopy.

In eukaryotic cells, each process, in which DNA is involved, should take place in the context of a chromatin structure. DNA double-strand breaks (DSBs) are one of the most deleterious lesions often leading to chromosomal rearrangement. In response to environmental stresses, cells have developed repair mechanisms to eliminate the DSBs. Upon DSB induction, several factors play roles in chromatin relaxation by catalysing the appropriate histone posttranslational modification (PTM) steps, therefore promoting the access of the repair factors to the DSBs. Among these PTMs, the phosphorylation of the histone variant H2AX at its Ser139 residue (also known as γH2AX) could be observed at the break sites. The structure of a DNA double-strand break induced repair focus has to be organized during the repair as it contributes to the accessibility of specific repair proteins to the damaged site. Our aim was to develop a quantitative approach to analyse the morphology of single repair foci by super-resolution dSTORM microscopy to gain insight into chromatin organization in DNA repair. We have established a specific dSTORM measurement process by developing a new analytical algorithm for gaining quantitative information about chromatin morphology and repair foci topology at an individual γH2AX enriched repair focus. Using this method we quantified single repair foci to show the distribution of γH2AX. The image of individual γH2AX referred to as the Single target Molecule response scatter Plot (SMPlot) was obtained by using high lateral resolution dSTORM images. Determination of the average localization numbers in an SMPlot was one of the key steps of quantitative dSTORM. A repair focus is made up of nanofoci. Such a substructure of repair foci can only be resolved and detected with super-resolution microscopy. Determination of the number of γH2AXs in the nanofoci was another key step of quantitative dSTORM. Additionally, based on our new analysis method, we were able to show the number of nucleosomes in each nanofocus that could allow us to define the possible chromatin structure and the nucleosome density around the break sites. This method is one of the first demonstrations of a single-cell based quantitative measurement of a discrete repair focus, which could provide new opportunities to categorize the spatial organization of nanofoci by parametric determination of topological similarity.

SerpinB2 is involved in cellular response upon UV irradiation.

Ultraviolet light induced pyrimidine dimer is a helix distortion DNA damage type, which recruits repair complexes. However, proteins of these complexes that take part in both DNA damage recognition and repair have been well-described, the regulation of the downstream steps of nucleotide excision repair (NER) have not been clearly clarified yet. In a high-throughput screen, we identified SerpinB2 (SPB2) as one of the most dramatically upregulated gene in keratinocytes following UV irradiation. We found that both the mRNA and the protein levels of SPB2 were increased upon UV irradiation in various cell lines. Additionally, UV damage induced translocation of SPB2 from the cytoplasm to the nucleus as well as the damage induced foci formation of it. Here we show that SPB2 co-localizes with XPB involved in the NER pathway at UV-induced repair foci. Finally, we demonstrated that UV irradiation promoted the association of SPB2 with ubiquitylated proteins. In basal cell carcinoma tumour cells, we identified changes in the subcellular localization of SPB2. Based on our results, we conclude that SPB2 protein has a novel role in UV-induced NER pathway, since it regulates the removal of the repair complex from the damaged site leading to cancerous malformation.

Coordinated activation of a cluster of MMP genes in response to UVB radiation.

Ultraviolet (UV) B radiation is a dangerous environmental stressor, which can lead to photoaging, inflammation, immune suppression and tumour formation. A recent report has shown the transcriptional activation of several skin-specific genes including matrix metalloproteases (MMPs) in response to UV irradiation. Here, we use a novel human keratinocyte model, HKerE6SFM, to demonstrate that UVB activates the transcription of most members of the 11q22.3 MMP gene cluster including MMP13, MMP12, MMP3, MMP1 and MMP10. Curiously, the expression of the well-characterized UVB-inducible MMP9, which is located outside of the cluster, remains unchanged. In accordance with the increased expression of the MMP gene cluster upon UVB irradiation, RNA polymerase II showed increased occupancy at their promoters following UVB irradiation. The results also demonstrate increased acetylated histone H3K9 levels at the promoters of the MMP13, MMP12, MMP3, MMP1 and MMP10 genes. These findings suggest a coordinated transcriptional activation of genes in the MMP cluster at 11q22.3 and that acetylation of histone H3 at lysine 9 has an important role in the UVB-dependent enhancement of transcription of MMP genes in this region.

Human p53 interacts with the elongating RNAPII complex and is required for the release of actinomycin D induced transcription blockage.

The p53 tumour suppressor regulates the transcription initiation of selected genes by binding to specific DNA sequences at their promoters. Here we report a novel role of p53 in transcription elongation in human cells. Our data demonstrate that upon transcription elongation blockage, p53 is associated with genes that have not been reported as its direct targets. p53 could be co-immunoprecipitated with active forms of DNA-directed RNA polymerase II subunit 1 (RPB1), highlighting its association with the elongating RNA polymerase II. During a normal transcription cycle, p53 and RPB1 are localised at distinct regions of selected non-canonical p53 target genes and this pattern of localisation was changed upon blockage of transcription elongation. Additionally, transcription elongation blockage induced the proteasomal degradation of RPB1. Our results reveal a novel role of p53 in human cells during transcription elongation blockage that may facilitate the removal of RNA polymerase II from DNA.

Detection of invasive fungal pathogens by real-time PCR and high-resolution melting analysis.

BACKGROUND/AIM: Causative agents most frequently encountered in systemic infections are bacteria, although fungi that cause invasive infections have also emerged, mostly in immune-compromised patients. The early detection and adequate treatment of bloodstream infections are critical for successful treatment. The aim of this study was to develop a rapid and efficient method for the detection and differentiation of the most common fungal pathogens. MATERIALS AND METHODS: Real-time Polymerase chain reaction (PCR) and consecutive high-resolution melting analysis was used for the detection and differentiation of fungal pathogens. RESULTS: The developed analysis procedure proved appropriate for discrimination of the ten most relevant Candida species, four Aspergillus species, and Cryptococcus neoformans. The sensitivity of the PCR reaction was 5, which is suitable for the detection of these fungi in blood. CONCLUSION: This technique is not adaptable as a general identification method, but it is highly useful when certain fungal species are to be expected in clinical samples.