СРАВНИТЕЛЬНЫЙ АНАЛИЗ ЭКЗОСОМ КЛЕТОК ЭСТРОГЕН-РЕЗИСТЕНТНОГО РАКА МОЛОЧНОЙ ЖЕЛЕЗЫ

The exosomes involvement in the pathogenesis of tumors is based on their property to incorporate into the recipient cells resulting in the both genomic and epigenomic changes.  Earlier we have shown that exosomes from different types of estrogen-independent breast  cancer cells (MCF-7/T developed by long-term tamoxifen treatment, and MCF-7/M)  developed by metformin treatment were able to transfer resistance to the parent MCF-7  cells. To elucidate the common features of the both types of resistant exosomes, the  proteome and microRNA cargo of the control and both types of the resistant exosomes were  analyzed. Totally, more than 400 proteins were identified in the exosome samples. Of these  proteins, only two proteins, DMBT1 (Deleted in Malignant Brain Tumors 1) and THBS1  (Thrombospondin-1), were commonly expressed in the both resistant exosomes (less than  5% from total DEPs) demonstrating the unique protein composition of each type of the resistant exosomes. The comparative analysis of the miRNA differentially expressed in  the both MCF-7/T and MCF-7/M resistant exosomes revealed 180 up-regulated and 202  down-regulated miRNAs. Among them, 4 up-regulated and 8 down-regulated miRNAs were  associated with progression of hormonal resistance of breast tumors. The bioinformatical  analysis of 4 up-regulated exosomal miRNAs revealed 2 miRNAs, mir- 101and mir-181b, which up-regulated PI3K signaling  supporting the key role of PI3K/Akt in the development of the resistant phenotype of breast cancer cells.Участие экзосом в патогенезе злокачественных опухолей основано на их способности проникать внутрь  клеток-реципиентов, вызывая в последних каскад генетических и эпигенетических изменений. Ранее мы  показали, что экзосомы, продуцируемые различными вариантами эстроген-независимых сублиний клеток  рака молочной железы (MCF-7/T, полученной в результате длительного культивирования клеток в  присутствии антиэстрогена тамоксифена, и MCF-7/M, полученной в результате культивирования клеток с  метформином), способны индуцировать резистентность в родительских клетках MCF-7. В настоящей работе  для исследования характерных особенностей состава экзосом резистентных клеток был проведен  сравнительный анализ протеома и профиля микроРНК контрольных экзосом и экзосом, полученных от  резистентных сублиний. В целом в образцах экзосом было идентифицировано более 400 белков, из которых  только 2 белка, DMBT1 (Deleted in Malignant Brain Tumors 1) и THBS1 (Thrombospondin-1), были  гиперэкспрессированы в обоих типах резистентных экзосом (менее 5 % от общего количества белков,  дифференциально экспрессированных в экзосомах резистетных клеток), что свидетельствует об уникальном  составе экзосомальных белков для каждого типа резистентных клеток. Сравнительный анализ  состава микроРНК, дифференциально экспрессированных в обоих вариантах экзосом резистентных клеток,  выявил 180 гиперэкспрессированных микроРНК и 202 микроРНК с пониженной экспрессией. Среди них 4  гиперэкспрессированных и 8 гипоэкспрессированных микроРНК оказались ассоциированы с развитием  гормональной резистентности клеток рака молочной железы. Биоинформатический анализ 4  гиперэкспрессированных микроРНК выявил 2 микроРНК, mir-101и mir-181b, участвующих в стимуляции PI3K  сигналинга, свидетельствуя о важной роли последнего в развитии гормональной резистентности  клеток рака молочной железы.

Роль трансглутаминазы 2 в регуляции баланса между аутофагией и апоптозом в опухолевых клетках

In normal tissue, cellular homeostasis is largely driven by two catabolic pathways: apoptosis and autophagy. Apoptosis, or programmed cell death, is regulated by pro-apoptotic factors, and promotes the removal of problematic cells. Autophagy, which in turn includes three forms: macro-, micro-, and chaperone-mediated autophagy, can promote both cell survival by selectively removing potentially apoptosis-inducing factors and raising the threshold of stress required for the induction of cell death. Recently, evidence has been accumulating suggesting the existence of common molecular pathways between autophagy and apoptosis, as well as the influence of the extracellular matrix on these processes. One of the important enzymes involved in the coordination and regulation of these processes is transglutaminase 2 (TG2). Different types of TG2 activities are involved in maintaining the dynamic balance between extracellular matrix and intracellular autophagy/apoptosis processes, while dysregulation of these processes may contribute to the pathogenesis of various human diseases, including oncogenesis. For example, TG2 can promote the degradation of pro-apoptotic proteins and the survival of renal cell carcinoma cells under nutrient-deficient conditions by modulating the autophagy process. In cells of various tissues deprived of TG2, aggregates of ubiquitinated proteins and damaged mitochondria are observed, which in turn induces proteotoxic stress and cell death. conversely, the transamidase activity of TG2 was observed to inhibit anti-apoptotic  signaling in a human leukemic monocytic lymphoma model. In the present review, a number of important functions of TG2 in oncogenesis are described, along with the dual role of TG2 in modulating such opposite processes as cell survival and cell death.В нормальной ткани клеточный гомеостаз в значительной степени обусловлен двумя катаболическими путями: апоптозом и аутофагией. Апоптоз, или запрограммированная клеточная гибель, регулируется проапоптотическими факторами и способствует уничтожению поврежденных клеток. Аутофагия, в свою очередь, включающая в себя 3 формы – макро-, микро- и шаперон-опосредованную аутофагию, – может как способствовать выживанию клеток путем избирательного  удаления факторов, потенциально вызывающих апоптоз, так и повышать порог стресса, необходимого для индукции клеточной гибели. В последнее время накапливаются данные, свидетельствующие о существовании общих молекулярных путей между аутофагией и апоптозом, а также о влиянии каспазного матрикса на данные процессы.  Одним из важных ферментов, участвующих в координации и регуляции этих процессов, является трансглутаминаза 2 (TG2). Различные типы активностей TG2 вовлечены в поддержание динамического баланса между внутриклеточным матриксом и внутриклеточными процессами аутофагии/апоптоза, в то время как их дерегуляция может способствовать развитию патогенеза различных заболеваний человека, включая онкогенез. Например, известно, что TG2 может благоприятствовать деградации проапоптотических белков и выживанию клеток почечно-клеточной карциномы в условиях недостатка питательных веществ, модулируя процесс аутофагии. В клетках различных тканей, лишенных TG2, наблюдается скопление агрегатов убиквитинированных белков и поврежденных митохондрий, что вызывает протеотоксический стресс и клеточную смерть. Наоборот, трансамидазная активность TG2 была замечена в ингибировании антиапоптотических сигналов на модели лейкемической моноцитарной лимфомы человека. В данном обзоре описываются важные функции TG2 в онкогенезе, а также подчеркивается двойственность роли этого фермента в модуляции таких противоположных процессов, как выживание клеток и их гибель

Modulation of EGFR activity by molecularly imprinted polymer nanoparticles targeting intracellular epitopes

In recent years, molecularly imprinted polymer nanoparticles (nanoMIPs) have proven to be an attractive alternative to antibodies in diagnostic and therapeutic applications. However, several key questions remain: how suitable are intracellular epitopes as targets for nanoMIP binding? And to what extent can protein function be modulated via targeting specific epitopes? To investigate this, three extracellular and three intracellular epitopes of epidermal growth factor receptor (EGFR) were used as templates for the synthesis of nanoMIPs which were then used to treat cancer cells with different expression levels of EGFR. It was observed that nanoMIPs imprinted with epitopes from the intracellular kinase domain and the extracellular ligand binding domain of EGFR caused cells to form large foci of EGFR sequestered away from the cell surface, caused a reduction in autophosphorylation, and demonstrated effects on cell viability. Collectively, this suggests that intracellular domain-targeting nanoMIPs can be a potential new tool for cancer therapy

How is Democracy Applied within the EU: Combining Elements of Traditional and Innovative Democratic Practice

The EU represents a new and complex political system which, according to numerous social scholars, suffers from the so-called democratic deficit. The basic argument behind this claim is that citizens lack control of the EU because, within its political system, national parliaments of member states possess only limited powers which have not been adequately compensated through steady empowerment of the European parliament (EP). Starting from this notion, the paper will explore the application of various concepts of democracy within the political system of the EU. First and foremost, it will analyse representative democracy in the EU, which stands as a foundation of all contemporary democratic systems. However, the paper will not stop at representative democracy, but it will also look at participatory, direct and deliberative democracy as applied within the political system of the EU. These concepts of democracy can only be viewed in relation and as an addition to representative democracy, but their application is very important for the EU due to limited possibilities for developing representative democracy at the supranational level. The paper will argue that, with regard to participatory and deliberative democracy, the EU can be viewed in many respects as a showcase for the national level, because it successfully developed various mechanisms related to implementation of these concepts. Particular attention will be paid to the Lisbon Treaty, which clarified many uncertainties that previously burdened the application of democracy within the EU. It will be argued that with the Lisbon Treaty the classic argument about the EU’s democratic deficit lost some of its appeal, because this treaty transformed the EP from secondary to equal participant in the EU’s legislative process

Interaction of the Transcription Start Site Core Region and Transcription Factor YY1 Determine Ascorbate Transporter SVCT2 Exon 1a Promoter Activity

Transcription of the ascorbate transporter, SVCT2, is driven by two distinct promoters in exon 1 of the transporter sequence. The exon 1a promoter lacks a classical transcription start site and little is known about regulation of promoter activity in the transcription start site core (TSSC) region. Here we present evidence that the TSSC binds the multifunctional initiator-binding protein YY1. Electrophoresis shift assays using YY1 antibody showed that YY1 is present as one of two major complexes that specifically bind to the TSSC. The other complex contains the transcription factor NF-Y. Mutations in the TSSC that decreased YY1 binding also impaired the exon 1a promoter activity despite the presence of an upstream activating NF-Y/USF complex, suggesting that YY1 is involved in the regulation of the exon 1a transcription. Furthermore, YY1 interaction with NF-Y and/or USF synergistically enhanced the exon 1a promoter activity in transient transfections and co-activator p300 enhanced their synergistic activation. We propose that the TSSC plays a vital role in the exon 1a transcription and that this function is partially carried out by the transcription factor YY1. Moreover, co-activator p300 might be able to synergistically enhance the TSSC function via a “bridge” mechanism with upstream sequences

TSPYL2 Is Important for G1 Checkpoint Maintenance upon DNA Damage

Nucleosome assembly proteins play important roles in chromatin remodeling, which determines gene expression, cell proliferation and terminal differentiation. Testis specific protein, Y-encoded-like 2 (TSPYL2) is a nucleosome assembly protein expressed in neuronal precursors and mature neurons. Previous studies have shown that TSPYL2 binds cyclin B and inhibits cell proliferation in cultured cells suggesting a role in cell cycle regulation. To investigate the physiological significance of TSPYL2 in the control of cell cycle, we generated mice with targeted disruption of Tspyl2. These mutant mice appear grossly normal, have normal life span and do not exhibit increased tumor incidence. To define the role of TSPYL2 in DNA repair, checkpoint arrest and apoptosis, primary embryonic fibroblasts and thymocytes from Tspyl2 deficient mice were isolated and examined under unperturbed and stressed conditions. We show that mutant fibroblasts are impaired in G1 arrest under the situation of DNA damage induced by gamma irradiation. This is mainly attributed to the defective activation of p21 transcription despite proper p53 protein accumulation, suggesting that TSPYL2 is additionally required for p21 induction. TSPYL2 serves a biological role in maintaining the G1 checkpoint under stress condition

p53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G1 phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state