TP63-TRIM29 axis regulates enhancer methylation and chromosomal instability in prostate cancer.

BACKGROUND: Prostate adenocarcinoma (PRAD) is the second leading cause of cancer-related deaths in men. High variability in DNA methylation and a high rate of large genomic rearrangements are often observed in PRAD. RESULTS: To investigate the reasons for such high variance, we integrated DNA methylation, RNA-seq, and copy number alterations datasets from The Cancer Genome Atlas (TCGA), focusing on PRAD, and employed weighted gene co-expression network analysis (WGCNA). Our results show that only single cluster of co-expressed genes is associated with genomic and epigenomic instability. Within this cluster, TP63 and TRIM29 are key transcription regulators and are downregulated in PRAD. We discovered that TP63 regulates the level of enhancer methylation in prostate basal epithelial cells. TRIM29 forms a complex with TP63 and together regulates the expression of genes specific to the prostate basal epithelium. In addition, TRIM29 binds DNA repair proteins and prevents the formation of the TMPRSS2:ERG gene fusion typically observed in PRAD. CONCLUSION: Our study demonstrates that TRIM29 and TP63 are important regulators in maintaining the identity of the basal epithelium under physiological conditions. Furthermore, we uncover the role of TRIM29 in PRAD development.

Development of a 3D Tumor Spheroid Model from the Patient's Glioblastoma Cells and Its Study by Metabolic Fluorescence Lifetime Imaging.

Patient-specific in vitro tumor models are a promising platform for studying the mechanisms of oncogenesis and personalized selection of drugs. In case of glial brain tumors, development and use of such models is particularly relevant as the effectiveness of such tumor treatment remains extremely unsatisfactory. The aim of the study was to develop a model of a 3D tumor glioblastoma spheroid based on a patient's surgical material and to study its metabolic characteristics by means of fluorescence lifetime imaging microscopy of metabolic coenzymes. Materials and Methods: The study was conducted with tumor samples from patients diagnosed with glioblastoma (Grade IV). To create spheroids, primary cultures were isolated from tumor tissue samples; the said cultures were characterized morphologically and immunocytochemically, and then planted into round-bottom ultra low-adhesion plates. The number of cells for planting was chosen empirically. The characteristics of the growth of cell cultures were compared with spheroids from glioblastomas of patients with U373 MG stable line of human glioblastoma. Visualization of autofluorescence of metabolic coenzymes of nicotinamide adenine dinucleotide (phosphate) NAD(P)H and flavin adenine dinucleotide (FAD) in spheroids was performed by means of an LSM 880 laser scanning microscope (Carl Zeiss, Germany) with a FLIM module (Becker & Hickl GmbH, Germany). The autofluorescence decay parameters were studied under normoxic and hypoxic conditions (3.5% О2). Results: An original protocol for 3D glioblastoma spheroids cultivation was developed. Primary glial cultures from surgical material of patients were obtained and characterized. The isolated glioblastoma cells had a spindle-shaped morphology with numerous processes and a pronounced granularity of cytoplasm. All cultures expressed glial fibrillary acidic protein (GFAP). The optimal seeding dose of 2000 cells per well was specified; its application results in formation of spheroids with a dense structure and stable growth during 7 days. The FLIM method helped to establish that spheroid cells from the patient material had a generally similar metabolism to spheroids from the stable line, however, they demonstrated more pronounced metabolic heterogeneity. Cultivation of spheroids under hypoxic conditions revealed a transition to a more glycolytic type of metabolism, which is expressed in an increase in the contribution of the free form of NAD(P)H to fluorescence decay. Conclusion: The developed model of tumor spheroids from patients' glioblastomas in combination with the FLIM can serve as a tool to study characteristics of tumor metabolism and develop predictive tests to evaluate the effectiveness of antitumor therapy.

Examination of Collagen Structure and State by the Second Harmonic Generation Microscopy.

Collagen is the major component of the extracellular matrix in mammals and its characteristics provide important information about the state of connective tissue. There are only few methods of label-free visualization of collagen fibers; the most frequently used is the second harmonic generation (SHG) microscopy. SHG microscopy is a non-invasive technique for the assessment of the abundance and structure of fibrillar collagen with a high resolution and specificity. At constant measurement parameters (magnification, excitation power, resolution, digital gain of registration matrix), quantitative analysis of SHG images provides a reliable characterization of collagen state. Current approaches to the SHG signal quantification are numerous and typically should be adapted to a specific task. In this review, we systematize the variety of these approaches and present the examples of biomedical application of the SHG signal quantitative analysis, as well of combined application of SHG and autofluorescence imaging.

Global DNA dynamics of 8-oxoguanine repair by human OGG1 revealed by stopped-flow kinetics and molecular dynamics simulation.

The toxic action of different endogenous and exogenous agents leads to damage in genomic DNA. 8-Oxoguanine is one of the most often generated and highly mutagenic oxidative forms of damage in DNA. Normally, in human cells it is promptly removed by 8-oxoguanine-DNA-glycosylase hOGG1, the key DNA-repair enzyme. An association between the accumulation of oxidized guanine and an increased risk of harmful processes in organisms was already found. However, the detailed mechanism of damaged base recognition and removal is still unclear. To clarify the role of active site amino acids in the damaged base coordination and to reveal the elementary steps in the overall enzymatic process we investigated hOGG1 mutant forms with substituted amino acid residues in the enzyme base-binding pocket. Replacing the functional groups of the enzyme active site allowed us to change the rates of the individual steps of the enzymatic reaction. To gain further insight into the mechanism of hOGG1 catalysis a detailed pre-steady state kinetic study of this enzymatic process was carried out using the stopped-flow approach. The changes in the DNA structure after mixing with enzymes were followed by recording the FRET signal using Cy3/Cy5 labels in DNA substrates in the time range from milliseconds to hundreds of seconds. DNA duplexes containing non-damaged DNA, 8-oxoG, or an AP-site or its unreactive synthetic analogue were used as DNA-substrates. The kinetic parameters of DNA binding and damage processing were obtained for the mutant forms and for WT hOGG1. The analyses of fluorescence traces provided information about the DNA dynamics during damage recognition and removal. The kinetic study for the mutant forms revealed that all introduced substitutions reduced the efficiency of the hOGG1 activity; however, they played pivotal roles at certain elementary stages identified during the study. Taken together, our results gave the opportunity to restore the role of substituted amino acids and main "damaged base-amino acid" contacts, which provide an important link in the understanding the mechanism of the DNA repair process catalyzed by hOGG1.