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.

[Changes of a2-macroglobulin activity and endothelin-1 concentration in tears of rabbits after transplantation of retinal pigment epithelium cells derived from the induced pluripotent stem cells]. / Izmenenie urovnia a2-makroglobulina i éndotelina-1 v sleznoi zhidkosti krolikov posle transplantatsii kletok retinal'nogo pigmentnogo épiteliia, differentsirovannykh iz indutsirovannykh pliuripotentnykh stvolovykh kletok.

Retinal diseases accompanied with the dysfunction or death of the retinal pigment epithelial (RPE) cells are widespread, hard to treat, and appear to be a leading case of visual loss and blindness among the persons older than 55 years. Transplantation of RPE cells derived from the induced pluripotent stem cells (IPSC-RPE) is a promising method of therapy for these diseases. To ensure the transplant survival instant follow-up is required. It can be based on biochemical analyses of tear fluid that can be easily non-invasively collected. For the post-transplantation process monitoring we have choosen such polyfunctional bioregulators as α2-macroglobulin (α2-MG) and endothelin-1 (ET-1). RPE atrophy in New Zealand Albino rabbits was modeled via the subretinal injection of bevacizumab. IPSC-RPE in suspension or as a monolayer on the scaffold were transplanted subretinally 1 month after the injection. α2-MG activity and ET-1 concentration in tears were estimated during the first month and after 2, 3 and 7 months after transplantation. On the 7-14 days after transplantation α2-MG activity increased in tears of the both operated and controlateral eye probably as a reaction on the corticosteroid therapy. In 50% rabbits there was one more increase after 2-3 months that could be due to the immune inflammation. Concentration of ET-1 in tears decreased dramatically on the 7-14 days and 7 months after transplantation, and it could have an influence upon the retinal vassal tone. The data obtained show that estimation of bioregulators in tears can help monitoring local metabolic processes after RPE transplantation that is necessary for the opportune, reasonable and focused medicamental correction of post-transplantation process.

[Experimental stem cell replacement transplantation in retinal pigment epithelium atrophy]. / Zamestitel'naya transplantatsiya stvolovymi kletkami pri atrofii retinal'nogo pigmentnogo epiteliya v eksperimente.

PURPOSE: To develop and evaluate the results of the modified surgical technique for transplantation of retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells (iPSC-RPE) in the form of a cell suspension into the subretinal space of rabbits with previously induced RPE atrophy. MATERIAL AND METHODS: The study was conducted on 10 New Zealand albino rabbits (20 eyes). One month after modeling RPE atrophy and retinal degeneration, rabbits were subjected to subretinal transplantation of iPSC-RPE cells in the form of a cell suspension. To prevent reflux of iPSC-RPE into the vitreal cavity, the injection site was sealed with 2-3 drops of autologous platelet-rich plasma (PRP). All rabbits underwent spectral optical coherence tomography (SOCT) and autofluorescence studies on the Heidelberg Spectralis system («Heidelberg Engineering¼, Germany). Enucleated animal eyes were studied with morphological and immunohistochemical methods. RESULTS: In this study we developed and evaluated a modified surgical technique of transplantation of iPSC-RPE in the form of a cell suspension into the subretinal space of rabbits with induced RPE atrophy. It was found that the use of PRP helps seal the defect and prevents cell suspension reflux into the vitreous cavity, effectively minimizing intra- and postoperative complications. Morphological in vivo study and examination of histological sections showed that implantable iPSC-RPEs were correctly integrated and adhered to the choroid in the surgery site. Immunohistochemical analysis involving fluorescence-marked antibodies confirmed the survival of iPSC-RPE integrated into the retina of model animals. CONCLUSION: This method improves the technology of iPSC-RPE transplantation on preclinical stages of the study, revealing new prospects in the treatment of degenerative diseases of the retina and the possibility of a personalized approach.

Derivation of induced pluripotent stem cells line (RCPCMi007-A-1) with inactivation of the beta-2-microglobulin gene by CRISPR/Cas9 genome editing.

The mismatch of HLA haplotypes between donor and recipient adversely affects the outcome of tissue transplantation. TheB2Mgene knockout (B2M-KO) disrupts the HLA I heterodimer formation; therefore,B2M-KO cells have reduced immunogenicity to allogeneic CD8+ T cells. Thus, theB2M-KO IPSCs and their derivatives can potentially solve a problem of the immunological compatibility in allogeneic transplantations. Using CRISPR/Cas9-mediated genome editing, we generated a human B2M-KO iPSC line (RCPCMi007-A-1). The RCPCMi007-A-1 iPSCs express pluripotency markers, have typical stem cell morphology, maintain normal karyotype, and the ability to differentiate into three germ layers.

[The Role of Mutant RNA in the Pathogenesis of Huntington's Disease and Other Polyglutamine Diseases].

Polyglutamine diseases are rare, inherited neurodegenerative pathologies that arise as a result of expansion of trinucleotide CAG repeats in the coding segment of certain genes. This expansion leads to the appearance of mRNA with abnormally long repetitive CAG triplets (mCAG-RNA) and proteins with polyglutamine (PolyQ) tracts in the cells, which is why these pathologies are commonly termed polyglutamine diseases, or PolyQ diseases. To date, nine PolyQ diseases have been described: Huntington's disease, dentatorubral pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), and six different types of spinocerebellar ataxia (SCA 1,2,3,6,7, and 17). PolyQ diseases lead to serious, constantly progressing dysfunctions of the nervous and/or muscular systems, and there currently exists no efficacious therapy for any of them. Recent studies have convincingly shown that mCAG-RNA can actively participate in the pathological process during the development of PolyQ diseases. Mutant RNA is involved in a wide range of molecular mechanisms, ultimately leading to disruption of the functions of transcription, splicing, translation, cytosol structure, RNA transport from the nucleus to the cytoplasm, and, finally, to neurodegeneration. This review discusses the involvement of mutant mCAG-RNA in neurodegenerative processes in PolyQ diseases.

[At Home among Strangers: Is It Possible to Create Hypoimmunogenic Pluripotent Stem Cell Lines?]

Human pluripotent stem cells, which include embryonic stem cells and induced pluripotent cells (iPSCs), are capable of unlimited division and differentiation into all cells of the body. These cells are considered as a potential source of various types of cells for transplantations. The use of autologous iPSCs is not potentially associated with immune rejection and does not require immunosuppression required for allogeneic grafts. However, the high cost of this technology and the duration of obtaining iPSCs and differentiated cells may limit the use of autologous iPSCs in clinical practice. In addition, full equivalence and immunological compatibility of autologous iPSCs and their derivatives have been repeatedly questioned. One approach to solving the problem of the immunological compatibility of allogeneic derivatives of iPSCs can be the establishment of cell lines with reduced immunogenicity. Differentiated derivatives of such iPSCs may be suitable for transplantation to any patient. This review discusses the strategies for evading immune surveillance in normal and tumor processes that can be used to establish stem cell lines with reduced immunogenicity.

Such Various Stem Cells.

Perhaps there is no more intriguing topic in modern biology than stem cells. The growing interest in stem cells is dictated by the ability of stem cells to both self-renew and differentiate, at least into several type cells. If we learn to influence these properties or reproduce them in vitro, it will be possible to effectively use stem cells or their differentiated derivatives in medicine. Fundamental knowledge of mechanisms of the self-maintenance and differentiation of stem cells is important for understanding a variety of processes - from embryogenesis to aging and oncogenic transformation. The purpose of this issue is to introduce readers to different areas in research on mammalian stem cells, including human stem cells. In the issue both review articles and research papers are presented, and the authors hope that they will be of interest for biochemists, cell biologists, and specialists in the field of biomedicine.

Development of Hematopoietic Stem Cells in the Early Mammalian Embryo.

Hematopoietic stem cells (HSCs) were the first stem cells discovered in humans. A. A. Maximov proposed an idea of blood stem cells that was confirmed later by McCulloch and Till experimentally. HSCs were the first type of stem cells to be used in clinics and ever since are being continually used. Indeed, a single HSC transplanted intravenously is capable of giving rise to all types of blood cells. In recent decades, human and animal HSC origin, development, hierarchy, and gene signature have been extensively investigated. Due to the constant need for donor blood and HSCs suitable for therapeutic transplants, the experimental possibility of obtaining HSCs in vitro by directed differentiation of pluripotent stem cells (PSCs) has been considered in recent years. However, despite all efforts, it is not yet possible to reproduce in vitro the ontogenesis of HSCs and obtain cells capable of long-term maintenance of hematopoiesis. The study of hematopoiesis in embryonic development facilitates the establishment and improvement of protocols for deriving blood cells from PCSs and allows a better understanding of the pathogenesis of various types of proliferative blood diseases, anemia, and immunodeficiency. This review focuses on the development of hematopoiesis in mammalian ontogenesis.

Possibilities for Using Pluripotent Stem Cells for Restoring Damaged Eye Retinal Pigment Epithelium.

The retinal pigment epithelium is a monolayer of pigmented, hexagonal cells connected by tight junctions. These cells compose part of the outer blood-retina barrier, protect the eye from excessive light, have important secretory functions, and support the function of photoreceptors, ensuring the coordination of a variety of regulatory mechanisms. It is the degeneration of the pigment epithelium that is the root cause of many retinal degenerative diseases. The search for reliable cell sources for the transplantation of retinal pigment epithelium is of extreme urgency. Pluripotent stem cells (embryonic stem or induced pluripotent) can be differentiated with high efficiency into the pigment epithelium of the retina, which opens up possibilities for cellular therapy in macular degeneration and can slow down the development of pathology and, perhaps, restore a patient's vision. Pioneering clinical trials on transplantation of retinal pigment epithelial cells differentiated from pluripotent stem cells in the United States and Japan confirmed the need for developing and optimizing such approaches to cell therapy. For effective use, pigment epithelial cells differentiated from pluripotent stem cells should have a set of functional properties characteristic of such cells in vivo. This review summarizes the current state of preclinical and clinical studies in the field of retinal pigment epithelial transplantation therapy. We also discuss different differentiation protocols based on data in the literature and our own data, and the problems holding back the widespread therapeutic application of retinal pigment epithelium differentiated from pluripotent stem cells.

Differentiation of Human Pluripotent Stem Cells into Mesodermal and Ectodermal Derivatives Is Independent of the Type of Isogenic Reprogrammed Somatic Cells.

Induced pluripotent stem cells (iPSCs) have the capacity to unlimitedly proliferate and differentiate into all types of somatic cells. This capacity makes them a valuable source of cells for research and clinical use. However, the type of cells to be reprogrammed, the selection of clones, and the various genetic manipulations during reprogramming may have an impact both on the properties of iPSCs and their differentiated derivatives. To assess this influence, we used isogenic lines of iPSCs obtained by reprogramming of three types of somatic cells differentiated from human embryonic stem cells. We showed that technical manipulations in vitro, such as cell sorting and selection of clones, did not lead to the bottleneck effect, and that isogenic iPSCs derived from different types of somatic cells did not differ in their ability to differentiate into the hematopoietic and neural directions. Thus, the type of somatic cells used for the generation of fully reprogrammed iPSCs is not important for the practical and scientific application of iPSCs.

No DNA damage response and negligible genome-wide transcriptional changes in human embryonic stem cells exposed to terahertz radiation.

Terahertz (THz) radiation was proposed recently for use in various applications, including medical imaging and security scanners. However, there are concerns regarding the possible biological effects of non-ionising electromagnetic radiation in the THz range on cells. Human embryonic stem cells (hESCs) are extremely sensitive to environmental stimuli, and we therefore utilised this cell model to investigate the non-thermal effects of THz irradiation. We studied DNA damage and transcriptome responses in hESCs exposed to narrow-band THz radiation (2.3 THz) under strict temperature control. The transcription of approximately 1% of genes was subtly increased following THz irradiation. Functional annotation enrichment analysis of differentially expressed genes revealed 15 functional classes, which were mostly related to mitochondria. Terahertz irradiation did not induce the formation of γH2AX foci or structural chromosomal aberrations in hESCs. We did not observe any effect on the mitotic index or morphology of the hESCs following THz exposure.

Molecular barriers to processes of genetic reprogramming and cell transformation.

Genetic reprogramming by ectopic expression of transcription factor genes induces the pluripotent state in somatic cells. This technology provides an opportunity to establish pluripotent stem cells for each person, as well as to get better understanding of epigenetic mechanisms controlling cell state. Interestingly, some of the molecular processes that accompany somatic cell reprogramming in vitro are also characteristic for tumor manifestation. Thus, similar "molecular barriers" that control the stability of epigenetic state exist for both processes of pluripotency induction and malignant transformation. The reprogramming of tumor cells is interesting in two aspects: first, it will determine the contribution of epigenetic changes in carcinogenesis; second, it gives an approach to evaluate tumor stem cells that are supposed to form the entire cell mass of the tumor. This review discusses the key stages of genetic reprogramming, the similarity and difference between the reprogramming process and malignant transformation.

Cloning and characterization of TNKL, a member of tankyrase gene family.

By serological screening of a breast tumor cDNA library we have identified a novel human gene, tnkl, encoding an ankyrin-related protein with a high degree of similarity to tankyrase, the poly(ADP-ribose)polymerase associated with human telomeres (Smith et al, Science 282: 1484). The tnkl gene maps to chromosome 10, while the tnks gene encoding tankyrase is located on chromosome 8. The predicted 1166-aa protein product of the tnkl gene is 78% identical to human tankyrase and 62% to a putative D. melanogaster protein. Since the proteins have essentially identical domain structures, the corresponding genes form a distinct gene family. The possible link between TNKL and cancer justifies its further functional analysis.

Human cortactin as putative cancer antigen.

Humoral immune response against overexpressed oncogenic or tumor supressor proteins has been demonstrated for many types of cancer. In this study we report on the detection of the autologous antibody response to putative oncogene, human cortactin using serological analysis of breast carcinoma expression library. Cortactin maps to chromosome 11q13, the region amplified in about 15% of primary breast carcinomas and 30% of head and neck squamous cell carcinomas. Cortactin overexpression due to such amplification might affect adhesive properties of human cancer cells and is associated with poor disease prognosis. Accordingly, we detected overexpression of cortactin transcript in autologous tumor and amplification/overexpression of cortactin in tumors of breast cancer patients serologically positive for this marker. We demonstrate that 15% of breast cancer patients elicit humoral immune response against human cortactin.

Excision of chromosomal DNA loops by treatment of permeabilised cells with Bal 31 nuclease.

The specificity of long-range fragmentation of human nucleolar genes by Bal 31 nuclease was studied using fractionation of cleavage products by pulsed field gel electrophoresis, followed by Southern hybridization. It was found that limited treatment of permeabilised cells with this nuclease results in accumulation of DNA scissions in matrix attachment areas. Consequently, chromosomal DNA loops and their oligomers are released from the genome.

Large-scale fragmentation of mammalian DNA in the course of apoptosis proceeds via excision of chromosomal DNA loops and their oligomers.

It has been shown recently that apoptotic degradation of genomic DNA in mammalian cells starts by excision of large DNA fragments ranging in size from 50 kilobases to more than 300 kilobases. Although it was suggested that the above fragments could represent chromosomal DNA loops, the supposition was not supported by direct experimental evidence. In present work, we have studied the specificity of nucleolar and euchromatic gene long-range fragmentation in mouse and human cells triggered to undergo apoptosis either by tumor necrosis factor or by serum deprivation. Separation of the excised large DNA fragments by pulsed field gel electrophoresis followed by Southern analysis has demonstrated that in all cases studied the above fragmentation proceeds in a specific way. Furthermore, the patterns of DNA long-range fragmentation in the cells undergoing apoptosis were indistinguishable from the patterns of DNA cleavage into chromosomal loops by the high salt-insoluble topoisomerase II of the nuclear matrix. These results suggest the conclusion that apoptotic degradation of chromosomal DNA starts by excision of DNA loops and their oligomers.