Prostate cancer (PCa) is one of the most common oncological diseases, which goes through two stages in its development. The first stage, localized prostate cancer, can proceed indefinitely in a dormant form that does not require active medical intervention, or suddenly turn into an aggressive metastatic form with lethal outcome. The pathogenesis of the transition of the dormant form of PCa to the metastatic form remains not fully understood. The signaling pathways of the tumor suppressor pRb and the proto-oncogene β-catenin are probably the most involved in the pathogenesis of PCa but the role of their interaction in the pathogenesis of prostate cancer has not been studied. The publication on the pathogenesis of tumors in other tissues suggests that pRb may lose some properties of a tumor suppressor at the initial stage of PCa development due to its interaction with β-catenin that enables tumor cells to gain competitive advantages for reproduction. In this work, we have shown that the RB and β-catenin (CTNNB1) genes are well expressed in tumor and normal prostate tissue. Unlike β-catenin, pRb is not detected by immunoblotting in tumor and normal prostate tissue, but is easily determined in this way in extracts of control T98G cells. Co-immunoprecipitation with antibodies to pRb from extracts of tumor and normal prostate tissue makes it possible to detect this protein and β-catenin by subsequent immunoblotting, which indicates the physical interaction of these proteins in prostate tissue. On the other hand, immunoprecipitation of β-catenin with antibodies to its C-terminal fragment does not detect this protein in prostate extracts by subsequent immunoblotting using the same antibody. In contrast to prostate tissue, β-catenin is readily detected by immunoprecipitation combined with immunoblotting in T98G control cell extracts. The obtained data suggest that pRb and β-catenin physically interact with each other in cells of different tissue specificity. In T98G cells, this interaction probably occurs through the C-terminal fragment of β-catenin, but in prostate cells it occurs in a different way, since the C-fragment of β-catenin is shielded from such interaction, possibly due to its physical association with pRb.

Purpose: The Phenotypes of COPD in Central and Eastern Europe (POPE) study assessed the prevalence and clinical characteristics of four clinical COPD phenotypes, but not mortality. This retrospective analysis of the POPE study (RETRO-POPE) investigated the relationship between all-cause mortality and patient characteristics using two grouping methods: clinical phenotyping (as in POPE) and Burgel clustering, to better identify high-risk patients.Patients and Methods: The two largest POPE study patient cohorts (Czech Republic and Serbia) were categorized into one of four clinical phenotypes (acute exacerbators [with/without chronic bronchitis], non-exacerbators, asthma-COPD overlap), and one of five Burgel clusters based on comorbidities, lung function, age, body mass index (BMI) and dyspnea (very severe comorbid, very severe respiratory, moderate-to-severe respiratory, moderate-to-severe comorbid/obese, and mild respiratory). Patients were followed-up for approximately 7 years for survival status.Results: Overall, 801 of 1,003 screened patients had sufficient data for analysis. Of these, 440 patients (54.9%) were alive and 361 (45.1%) had died at the end of follow-up. Analysis of survival by clinical phenotype showed no significant differences between the phenotypes (P=0.211). However, Burgel clustering demonstrated significant differences in survival between clusters (P<0.001), with patients in the "very severe comorbid" and "very severe respiratory" clusters most likely to die. Overall survival was not significantly different between Serbia and the Czech Republic after adjustment for age, BMI, comorbidities and forced expiratory volume in 1 second (hazard ratio [HR] 0.80, 95% confidence interval [CI] 0.65-0.99; P=0.036 [unadjusted]; HR 0.88, 95% CI 0.7-1.1; P=0.257 [adjusted]). The most common causes of death were respiratory-related (36.8%), followed by cardiovascular (25.2%) then neoplasm (15.2%).Conclusion: Patient clusters based on comorbidities, lung function, age, BMI and dyspnea were more likely to show differences in COPD mortality risk than phenotypes defined by exacerbation history and presence/absence of chronic bronchitis and/or asthmatic features.

Background: COPD is a major cause of morbidity and mortality. Despite this, there are very little data regarding COPD mortality in Central and Eastern Europe (CEE). The Phenotypes of COPD in Central and Eastern Europe (POPE) study outlined four clinical COPD phenotypes in patients from the CEE region. Here, RETRO-POPE compared mortality in two independent POPE study cohorts. Methods: RETRO-POPE retrospectively assessed mortality in patients with COPD from the Czech Republic (CZ) and Serbia (SRB) 7 years after the POPE study. Patients were assigned to predefined phenotypes according to their clinical characteristics, or into five clusters based on comorbidities, lung function, age, body mass index and dyspnea (Burgel's clustering), and their survival status was assessed. Results: Overall, 1,003 POPE study patients were assessed for inclusion in the RETRO-POPE analysis, of whom 801 patients had sufficient data for analysis. At the time of data collection, 440 patients (54.9%) were alive and 361 had died (45.1%). When mortality was assessed by phenotype, no significant differences were seen. In the CZ, separation in mortality was seen between phenotypes (P=0.009), whereas in SRB, death seemed to be more independent of phenotype (P=0.479). Burgel's clustering of patients distinguished between mortality more than when stratified by phenotypes. Patients in SRB were found to have a greater long-term overall survival than patients in the CZ. Conclusion: RETRO-POPE found that clusters based on comorbidities, lung function, age, body mass index and dyspnea were more likely to show an association with mortality risk than phenotypes according to exacerbation history and presence or absence of chronic bronchitis and asthma features.

Prostate cancer (PC) currently occupies a leading position in Western countries in terms of morbidity and mortality among all tumor diseases. Prostate cancer appears in the form of adenocarcinoma (localized PC), which can remain indefinitely in a dormant form that does not threaten the life of the patient, or transform into an aggressive cancer that is insensitive to androgen-deprivation therapy—castration-resistant prostate cancer (CRPC) with metastasis and rapid lethal outcome. PC arises from the epithelium of the prostate gland, the formation and functioning of which occurs under the action of androgens. Androgens, mainly dihydrotestosterone, activate the androgen-receptor (AR) signaling pathway, which regulates the growth and division of the prostate epithelium under normal conditions and in localized PC. Androgen-deprivation therapy, for example, with androgen-receptor inhibitors (enzalutamide or abiraterone), inhibits the development of localized PC for 1.5–2 years, but then loses its effectiveness and inevitably leads to the transition of the disease to aggressive CRPC. Inactivation of the tumor suppressor RB contributes to the development of cancer of any tissue specificity due to mutations, gene loss, or posttranslational modification of its product. Assessment of the state of RB and its product shows that RB gene is altered in less than 1% of patients with localized PC, but its loss causes CRPC in 17–33% of patients. This brief literature review presents the role of the pRb signaling pathway in the pathogenesis of localized and castration-resistant PC.

Numerous evidences from prevention studies in humans, support the existence of an association between green tea polyphenols consumption and a reduced cancer risk. Prostate cancer is one of the most frequently diagnosed male neoplasia in the Western countries, which is in agreement with this gland being particularly vulnerable to oxidative stress processes, often associated with tumorigenesis. Tea polyphenols have been extensively studied in cell culture and animal models where they inhibited tumor onset and progression. Prostate cancer appears a suitable target for primary prevention care, since it grows slowly, before symptoms arise, thus offering a relatively long time period for therapeutic interventions. It is, in fact, usually diagnosed in men 50-year-old or older, when even a modest delay in progression of the disease could significantly improve the patients quality of life. Although epidemiological studies have not yet yielded conclusive results on the chemopreventive and anticancer effect of tea polyphenols, there is an increasing trend to employ these substances as conservative management for patients diagnosed with less advanced prostate cancer. Here, we intend to review the most recent observations relating tea polyphenols to human prostate cancer risk, in an attempt to outline better their potential employment for preventing prostate cancer.

The signal pathways associated with formation of the adipose cell phenotype converge on the regulation of expression of the tissue-specific PPARγ2 gene. Here we studied the interaction of Ezh2, Bmi1 and Utx proteins, which regulate the methylation levels of the H3K27, and p130 (a member of pRb family) with the promoter of PPARγ2, a regulator of adipogenic differentiation (AD), in the course of AD of murine mesenchymal stem cells (MSCs) using the chromatin immunoprecipitation (ChiP). In undifferentiated cells, promoters of the PPARγ2 and control RUNX2 gene, a regulator of bone differentiation, accumulated high levels of Bmi1, Ezh2 and low levels of Utx. The p130 was detected in undifferentiated cells at high levels on the PPARγ2, but not on the RUNX2 promoter. These protein-DNA interactions changed in the opposite way in the course of AD on the PPARγ2 promoter but did not change on the RUNX2 promoter. In cells with inactivated BMI1 under AD conditions there was observed a slight decrease in the H3K27me3 levels and an increase in the levels of Utx demethylase on the PPARγ2 promoter. Our results suggest that PPARγ2 expression in the terminal phase of AD in murine MSCs is activated by Utx, but is suppressed by Bmi1, Ezh2 and p130. These data support the hypothesis that in the course of differentiation there is a loss of the suppressive H3K27me3 mark in the bivalent domains of regulatory genes including PPARγ2 that contribute to formation of the tissue-specific phenotype.

The prostate gland (PG) is a small organ in the male reproductive system that is currently the focus of biomedical research due to its leading position in morbidity and mortality from the tissue-specific cancer prostate cancer (PC). The PG epithelium, which undergoes a cancerous transformation, is formed and functions under the control of androgens. At the beginning of the disease, epithelial cells produce an androgen receptor (AR) and are sensitive to androgen-deprivation therapy. However, such therapy inevitably leads to the transition of the disease to the castration-resistant prostate cancer (CRPC), which manifests itself in metastasis and rapid mortality. In CRPC, the cells of the prostate epithelium change their phenotype, that may be associated with AR mutation and loss the sensitivity to specific therapy. The mechanism of PG phenotypic transformation may be hidden in the interaction and formation of the stromal and epithelial cells, which are evident during the establishment of the primary cultures. The aim of this study was to investigate the generation of human PG stromal cells in primary stromal and organoid cultures. We found that, in contrast to the rapid appearance and formation of a homogeneous population of mesenchymal cells in primary stromal cultures of most tissues, human PG cell cultures are formed initially from epithelial cells. They appear in the second week of cultivation and produce cytokeratins (CKs). A homogeneous population of mesenchymal cells producing vimentin is formed only at the end of the fourth week of cultivation. It is accompanied by the disappearance of epithelial cells. At the same time, some epithelial cells simultaneously produce CKs and vimentin. In PG organoid cultures, there is often a concomitant growth of epithelial, but not mesenchymal, cells on culture plastic. During the cultivation of epithelial cells arising from the organoid cultures, they, like the cells of the primary epithelium, exhibit the ability to spontaneous transformation into mesenchymal cells and simultaneously produce CKs and vimentin. Our data suggest that in primary and organoid PG cultures, stromal cells can be formed from epithelium due to the epithelial-to-mesenchymal transition (EMT). The tendency of PG epithelium toward spontaneous EMT may contribute to the mechanism of high sensitivity of prostate tissue to malignant transformation and metastasis. Understanding this mechanism may contribute to the development of effective antitumor therapy of prostate cancer.

An effective regulation of quiescence plays a key role in the differentiation, plasticity, and prevention of stem cells from becoming malignant. The state of quiescence is being controlled by the pRb family proteins which show overlapping functions in cell cycle regulation; however, their roles in controlling the proliferation of mesenchymal stem cells (MSCs) remain to be understood. This study investigated the regulation of transient quiescence using growth curves, proliferation assay, the cytometric evaluation of cell cycle, Western blotting, and the electromobility gel shift assay (EMSA) on synchronized MSCs of the C3H10Т1/2 and control cells with different statuses of pRb proteins. It has been found that functional steady-state level of p130 but not pRb plays a critical role for entering, exiting, and maintenance of transient quiescence in multipotent mesenchymal stem cells.

Usp28 is a deubiquitinating enzyme that removes ubiquitin from its conjugates with substrates and prevents their degradation in proteasomes. Modern publications show that Usp28 and the Fbw7 ubiquitin ligase create a functional pair of proteins that controls ubiquitin-mediated degradation of key regulators of cellular functions, including Myc, Jun, Nicd, and Hif1. In this pair of proteins, Usp28 counteracts the destructive activity of Fbw7 and plays the role of a factor promoting tumor growth. Since the Usp28 targets are the Myc and Jun proteins associated with the cell cycle, we suggested that Usp28 regulates cell division and its level may change during the cell cycle. The aim of this work was to assess changes in the level of Usp28 during the cell cycle in HCT116 human intestinal carcinoma cells. HCT116 cells were synchronized by 72-h cultivation in a growth medium with a low serum content. In asynchronously dividing cells, the level of Usp28 was low and its localization was detected as nuclear–cytoplasmic. The general level of Usp28 and the degree of its nuclear localization increased in cells from the state of asynchronous growth to the late phase G 1 followed by a decrease and redistribution of protein from the nucleus to the cytoplasm after the completion of phase G 1 . According to immunoblot data, the fluctuations in the levels of Usp28 and Cdc25A phosphatase in synchronized HCT116 cells in the cell cycle coincided. The immunofluorescence data directly corresponded to the results of immunoblotting. The results suggest that Usp28 can regulate the level and functional activity of the Cdc25A protein that controls the entry of cells into the DNA replication phase.

The retinoblastoma gene product (pRb) is a chromatin-associated protein that can either suppress or promote activity of key regulators of tissue-specific differentiation. We found that twelve weeks after transfection of the exogenous active (ΔB/X and Δр34) or inactive (ΔS/N) forms of RB into the 10T1/2 mesenchymal stem cells and clonal selection not a single cell line did contain exogenous RB, despite being G-418 resistant. However, the consequences of the transient production of exogenous RB had different effects on the cell fate. The ΔB/X and Δр34 cells transfected with active form of RB showed elevated levels of inducible adipocyte differentiation (AD). On the contrary, the ΔS/N cells transfected with inactive RB mutant were insensitive to induction of AD associated with abolishing of expression of the PPARγ2. Additionally, the PPARγ2 promoter in undifferentiated ΔS/N cells was hypermethylated, but all except -60 position CpG became mostly demethylated after cells exposure to AD. We conclude that while transient expression of inactive exogenous RB induces long term epigenetic alterations that prevent adipogenesis, production of active exogenous RBs results in an AD-promoting epigenetic state. These results indicate that pRb is involved in the establishment of hereditary epigenetic memory at least by creating a methylation pattern of PPARγ2.

In the context of rehabilitation therapy, the proliferation and migration of mesenchymal stem cells (MSCs) are functional properties of which the relationship remains unstudied. The aim of the present work consisted in parallel determination of proliferative and migration capacity of endometrial, adipose, and bonemarrow human MSCs (hMSCs) during long-term cultivation. The migration capacity of hMSCs was determined in comparison with those in epithelial cells of the established НСТ116, MCF7, and A-549 human lines. hMSCs obtained from different tissues demonstrate a comparable division rate on early passages, which progressively slows down by the tenth passage, which corresponds to a decrease in the expression of Ki67 proliferation marker and D1 cyclin. The mobility of MSCs during the determination of the cell migration through 8-μm filters and a “scratch regeneration” method does not change by the tenth passage. A high level of N-cadherin, vimentin production, and absence of E-cadherin, which level out in epithelial cells of the HCT116, MCF7, and A-549 lines is inverted as compared with those in MSCs, corresponding to the active mobility of MSCs of different tissue origin. Our results demonstrate that a decrease of the proliferative activity of MSCs of different tissue specificity during passaging is not associated with changes in their ability to migrate. It is likely that the mechanisms of division and cell-mobility regulation are genetically and functionally not related to each other and change autonomously during continuous cultivation.

Development of local or generalized forms of prostate cancer (PC) depends on metastasis formation which biological nature is based on the epithelial mesenchymal transition (EMT). ЕМТ presents a highly conservative reversible program maintained by specific transcription factors suppressing E-cadherin expression and supporting production of mesenchymal polarity factors. The goal of this work was to study functionally distinct markers in malignant prostate tissue of patients with prostate cancer using histological evaluation, reverse-transcription polymerase chain reaction and immunoblotting. Our results showed that most evident alterations in the prostate tissue of patients with prostate cancer were related to the basal-cell layer. Expression of Е-cadherin and СК5 markers decreased, while AMACR expression increased. These results support the idea that the primary targets of tumor transformation are basal cells. During cancer progression, these cells acquire a luminal phenotype. Functional analysis of these changes may be performed in the future using isolated prostate cancer stem cells.

AIM:In patients with prostate cancer to trace the pathway of the malignant cells of the basal layer of the prostate epithelium during their differentiation into luminal cells and/or migration in the mesenchyme.MATERIALS AND METHODS:We used histological and immunohistochemical staining of the markers of the basal layer of the prostate: cytokeratin 5 (CK5), E-cadherin and AMACR, and Western blot to assess the production of the same markers in epithelial and stromal compartments of malignant and normal prostate tissue in patients with prostate cancer.RESULTS:Our findings revealed that prostate cancer is associated with losing of the basal epithelial layer in the prostate tumor tissue, which is accompanied by a complete loss of CK5 secretion, increased levels of E-cadherin and AMACR in luminal epithelium and the emergence of cells producing E-cadherin and AMACR in the stromal compartment of the prostate.DISCUSSION:These findings suggest that in prostate cancer the transformation the basal layer of the epithelial cells is associated with their differentiation into luminal cells and migration into the surrounding mesenchyme due to epithelial-mesenchymal transition.CONCLUSION:Prostate cancer pathogenesis of associated with changes in epithelial cell pathways and the levels of the markers expression. Their assessment can be used for studying the disease mechanisms and seeking new diagnosis and treatment options.

Development of local or general forms of prostate cancer (PC) depends on formation of metastasis the biologicalnature of which is based on epithelial mesenchymal transition (EMT). ÅÌÒ presents highly conservativereversible program that is maintained by specific transcription factors which suppress E-cadherin expressionand support production of mesenchymal polarity factors. The goal of this work was to study the functionallydistinct markers in malignant prostate tissue of patients with prostate cancer using the histological evaluation,reverse polymerase chain reaction and immunobloting. Our results showed that mostly evident alterations inprostate tissue of patients with prostate cancer were associated with the cells of basal layer. Expression levels ofthe markers of this layer: Å-cadherin and ÑK5, were decreased, while that of AMACR — increased. These resultssupport an idea that the basal cells are primarily targeted during transformation and acquired the luminalphenotype in the course of the following differentiation. The functional analysis of these results may be performedin future using selected prostate cancer stem cells.

Bmil is a key component of Polycomb (PcG), which in mammals controls the basic functions of mammalian somatic stem cells (SSC) such as self-renewal and differentiation. Bmi1 supports SSC via transcriptional suppression of genes associated with cell cycle and differentiation. The most studied target genes of Bmi1 are the genes of Ink4 locus, CdkI p16(Ink4a) and p1(Arf), suppression of which due to activating mutations of the BMI1 results in formation of cancer stem cells (CSC) and carcinomas in various tissues. In contrast, inactivation of BMI1 results in cell cycle arrest and cell senescence. Although clinical phenomena of hypo- and hyperactivation of BMI1 are well known, its targets and mechanisms of regulation of tissue specific SSC are still obscure. The goal of this study was to evaluate the regulatory role of BMI1 in adipocyte differentiation (AD) of mouse mesenchymal stem cells (MSC). Induction of AD in mouse MSC of the C3H10T1/2 cell line was associated with an increase in the expression levels of BMI1, the genes of pRb family (RB, p130) and demethylase UTX, but not methyltransferase EZH2, whose products regulate the methylation levels of H3K27. It was observed earlier that H3K27me3 may play the role of the epigenetic switch by promoting AD of human MSC via activating expression of the PPARγ2, the master gene of AD (Hemming et al., 2014). Here we show that inactivation of BMI1 using specific siRNA slows and decreases the levels of AD, but does not abolish it. This is associated with a complete inhibition of the expression of adipogenic marker genes--PPARγ2, ADIPOQ and a decrease in the expression of RB, p130, but not UTX. The results obtained give evidence that the epigenetic mechanism regulating AD differentiation in mouse and human MSC is different.

Using stable constitutive expression of retinoblastoma gene product (pRb) in polypotent mesenchymal 10T1/2 cells we obtained stable cell lines hyperexpressing functionally active or inactive mutant pRb. The cells producing active exogenous pRb demonstrated high sensitivity to adipocyte differentiation inductors, whereas production of inactive form of the exogenous protein suppressed adipocyte differentiation. The obtained lines can serve as the experimental model for studying the role of pRb in determination of adipocyte differentiation.