Supported by the literature, our initial hypothesis was that Estrogen Receptor alfa (ESR1) may function as a master regulator by influencing the expression of epithelial-to-mesenchymal transition (EMT)-related genes in cancer cells. To explore this further, we used the CRISPR/Cas9 gene editing system to create MCF-7 sublines with down-regulated ESR1 expression and analyzed its impact on EMT initiation. By applying two distinct types of gRNA for gene editing, we established six MCF-7 cell sublines with either nearly complete or partial down-regulation of the ESR1 isoforms. Unexpectedly, the data obtained revealed no discernible impact of ESR1 down-regulation on EMT manifestation as Western blot and Real-Time qPCR analysis of selected clones revealed no changes in EMT markers expression. We suggested that those of the ESR1 isoforms, the expression of which was not affected by gene editing, could be crucial for the initiation of EMT. The obtained cell models will be used further to evaluate the activity of ESR1 isoforms.

Keywords: CoA, S6 kinase 1, in situ proximity ligation assay, post-translation modification, oxidative stress, signal transduction, confocal microscopy, cell growth, antibodies

Ribosomal protein kinase S6 (S6K1) is one of the key components of the PI3K/mTOR/S6K1-dependent signaling cascade, which plays an important role in the regulation of translation, cell growth, survival, proliferation, and cell migration. Disturbances in the functioning of the PI3K/mTOR/S6K1 signaling cascade, in particular changes in the expression and activity of S6K1, accompany the development of a number of human pathologies, including oncological, neurodegenerative, and cardiovascular diseases; metabolic disorders; and pathologies associated with aging. Recently, in addition to the already known p70 and p85, the existence of a new isoform of the S6K1 kinase, p60, was confirmed. In addition, it was established that a change in the balance in the expression of various S6K1 isoforms towards an increase in p60-S6K1 leads to a multiple increase in the motility of model malignant cells, which is accompanied by significant changes in the expression of molecules involved in cell adhesion and intercellular interaction and may indicate that the cells acquire tumor characteristics important for invasion and metastasis. To find out whether the expression of the p60 isoform of S6K1 correlates with invasion and metastasis at the tumor level, the expression level of S6K1 isoforms was analyzed in three groups of human papillary thyroid carcinoma tumors, namely, without signs of invasion, with signs of invasion, and with lymph node metastases. Conditionally normal tissues adjacent to the tumors were also analyzed at the same time. The analysis was carried out by real-time PCR. A trend towards an increase in the expression level of all S6K1 isoforms, as well as the p60 isoform of S6K1 in tumors with manifestations of invasion and metastasis, was established. However, changes in the ratio of the total expression level of all S6K1 isoforms to p60-S6K 1 in the direction of an increase in the share of the latter in tumors with manifestations of metastasis turned out to be statistically reliable. The obtained results indicate an important role of the p60S6K1 isoform in the maintenance or initiation of invasion and metastasis of thyroid tumors.

Aim.To generate monoclonal antibodies to Coenzyme A (CoA) with antigenic epitope specificity different to previously developed anti-CoA mAb (1F10) by introducing alternative approach for antigen preparation animal immunization.Methods.Cross-linking of CoA to Keyhole Limpet Hemocyanin (KLH) and to Bovine serum albumin (BSA) using reversible chemical crosslinker SPDP.Hybridoma technique.Western blot analysis.Immunoprecipitation. ELISA.Results.Monoclonal antibody (A11) that specifically recognizes CoA in various immunoassays and has the same antigenic epitope specificity as existing anti-CoA mAb (1F10) has been generated.Conclusions.Application of different ways for CoA conjugation to the carrier proteins to create an antigen for immunization does not affect the antigenic epitope specificity of generated anti-CoA antibodies most probably due to the existence of only one immunogenic epitope in CoA molecule. K e y w o r d s:CoA,

Heat shock protein 60 (Hsp60) is considered as one of the possible autoantigens involved in the pathogenesis of a number of chronic diseases including periodontal diseases. The application of hyaluronic acid or hyaluronan (HA) in the treatment of periodontitis has been evaluated in several clinical trials, however, the effect of hyaluronic acid on heat shock protein 60 level in periodontal soft tissues has not been studied. The aim of this work was to evaluate the effect of HA injections on levels of Hsp60 in periodontal tissue of the rats. Samples of periodontal tissue of mandibular incisors of adult male Wistar rats at 10-12 months of age were investigated. Rats were distributed into the control group and the periodontitis group. Visual manifestations of hyperemia of the gums around the incisors were the criterion for selecting animals into the periodontitis group. There were two subgroups in the control group: intact rats (I); intact rats after HA “hyaDENT BG” 1.0 MDa (BioScience GmbH, Germany) treatment (I+“G-1.0”). There were four subgroups in the periodontitis group: rats with periodontitis (P); rats with periodontitis after HA “hyaDENT BG” 1.0 MDa (BioScience GmbH, Germany) treatment (P+“G-1.0”); rats with periodontitis after HA “SERTOBEC” 2.4 MDa (S.C. Rompharm Company S.R.L., Romania) treatment (P+“S-2.4”); rats with periodontitis after HA “SERTOBEC Tendon” 2.4 МDа (S.C. Rompharm Company S.R.L., Romania) treatment (P+“ST-2.4”). There were three animals in each subgroup. Rats were injected 0.05 ml HA in the area of alveolar processus of central incisors once a week, three times. Levels of Hsp60 in total lysates of periodontal tissue were tested by Western blotting method before and after the treatment with HA (one month after the last injection). There was no significant difference between levels of Hsp60 in total lysates of periodontal tissue of intact rats and rats with periodontitis before treatment (p>0.05). Rats with periodontitis showed decreased inflammation in the periodontal tissue after treatment with HA with different molecular weight. Intact rats and rats with periodontitis which were treated with HA “hyaDENT BG” 1.0 MDa showed reduced levels of Hsp60 in total lysates of periodontal tissue comparatively with levels of Hsp60 before treatment (by 15.4 and 10.7 times respectively, p<0.001). Rats with periodontitis which were treated with HA “SERTOBEC” 2.4 MDa or HA “SERTOBEC Tendon” 2.4 MDa also showed reduced levels of Hsp60 in total lysates of periodontal tissue com­paratively with levels of Hsp60 before treatment (by 21.3 and 16.4 times respectively, p<0.001). Rats with pe­riodontitis showed the decrease in inflammation in periodontal tissue after treatment with HA with different molecular weight. Injections of HA has contributed to reduce levels of Hsp60 in periodontal tissue of intact rats and rats with periodontitis.

The high mortality rate from ovarian cancer is due to the asymptomatic nature of the course of the disease, which leads to the diagnosis of ovarian cancer in later stages. The sodium-dependent phosphate transporter NaPi2b encoded by SLC34A2 gene is expressed in 80-90% of epithelial ovarian cancers and used as a target for therapeutic antibodies XMT-1536, and XMT-1592, which are derived from MX35 antibodies and used in clinical trials for the treatment of ovarian and lung cancers. In this work, we aimed to evaluate NaPi2b as a molecular marker for diagnostics and predicting the course and outcome of ovarian cancer disease. Quantitative analysis of SLC34A2 gene expression in ovarian tumor tissue was performed at the level of transcription and translation using real-time PCR, droplet digital PCR and Western blot analysis respectively. Statistical analysis was performed taking into account various clinicopathological characteristics of the ovarian cancer patients, including the stage of the disease, the tumor grade, the applying of neoadjuvant chemotherapy and the presence of ascites. In this work, we demonstrated that the expression of the human NaPi2b (hNaPi2b) transporter is downregulated in the tumors of patients receiving neoadjuvant therapy and during the development of disease. The data suggest that the level of expression of the SLC34A2 gene can serve as a potential marker for the monitoring and predicting responses to neoadjuvant and targeted therapy in patients with ovarian cancer.

Aim.To generate and characterize MCF-7 cell lines with altered expression of p85, p70 and p60 S6K1 isoforms: p85-/p70-/p60-MCF-7 and p85-/p70-/p60+MCF-7.Methods.CRISPR/ Cas9 gene editing, western blot analysis, immunofluorescence analysis, scratch assay.Results.Modified MCF-7 cells with knocked down expression of p85, p70, p60 or only p85 and p70 S6K1 isoforms were generated.Selective inhibition of only p85 and p70 isoforms in p85-/p70-/p60+MCF-7 cells was accompanied by actin cytoskeleton rearrangements, appearance of fibroblast-like cell morphology and significantly increased cell locomotor activity.Downregulation of all three S6K1 isoforms in p85-/p70-/p60 -MCF-7 cells inhibited cell migration with no changes in the cell morphology.Alterations in had a different impact on the ribosomal protein S6 phosphorylation and Akt signaling.Conclusion.Analysis of the modified MCF-7 cell lines revealed different impact of expression of S6K1 isoforms on MCF7 cell locomotor activity and the S6K1-and Akt-dependent signaling.Our data suggest that p60-S6K1 could be involved in regulation of the cell migration.The generated cells can be used for further analysis of functional activity of the S6K1 isoforms.

the phosphatidylinositol-3-kinase (PI3k)/mechanistic target of rapamycin complex 1 (mtorc1) signaling pathway controls plenty of cellular functions regulating phosphorylation one of its mediators ribosomal protein S6 kinase 1 (S6k1).alternative translation of the common S6k1 transcript can generate three protein kinase isoforms, including p85-S6k1, p70-S6k1 and p60-S6k1.the catalytic activity of S6k1 is modulated by mitogens and growth factors via phosphorylation at three critical sites such as the activation loop (T-loop site), turn motif (TM site), and hydrophobic motif (HM site).Both members of the PI3K/mTORC1 pathway, PDk1 and mtorc1, directly phosphorylate the t-loop site and Hm site, respectively.Indeed, most studies aimed at elucidating S6k1 regulation have focused on p70-and p85-S6k1.meanwhile, however, the activity of p60-S6K1 and its regulation have not been elucidated so far.To test whether p60-S6K1 was an active kinase isoform that was regulated similar to p70/p85-S6k1, we employed previously generated p85 -/p70 -/ p60 + HEK-293 cells.First, an in vitro kinase assay confirmed the ability of p60-S6K1 to phosphorylate ribosomal protein S6 (rpS6), a well-known S6K1 substrate.Next, analysis of p60-S6K1 phosphorylation under different cell growth conditions showed that p60-S6K1 does not have detectable levels of phosphorylation at PDk1-and mtorc1-regulated sites, yet this isoform undergoes phosphorylation at the tm site.Finally, we found that activity of p60-S6K1 was not sensitive to mitogenic stimulation and cell treatment by potent inhibitor of the PI3K1/mTORC1-dependent signaling pathway rapamycin suggesting the existence of a PI3K/ mtorc1-independent mechanism of p60-S6k1 regulation in the data of the current study suggest that the p60-S6k1 isoform possesses intrinsic kinase activity that is independent of PI3k/mtorc1 signaling regulation in Hek-293 cells.What is more, modulation of p60-S6k1 activity via the PI3k/mtorc1 signaling pathway seems to be cell-type specific, since the p60-S6K1 isoform undergoes PDK1-and mTORC1-mediated phosphorylation in breast cancer cell line MCF-7.K e y w o r d s: p60-S6 kinase 1 (p60-S6K1), PI3K/mTORC1 signaling pathway, kinase activity, regulation by protein phosphorylation.

Aim.To identify a splicing mRNA of S6K1 kinase specific for the p60-S6K1 isoform alone.Methods.RT-PCR, DNA sequencing, Western blotting.Results.RT-PCR analysis of total RNA extracted from MCF-7 cells and subsequent DNA sequencing revealed a novel S6K1 splice variant that possesses additional exon 1a and encodes the p60 isoform of S6K1 only.Moreover, RT-PCR and western blotting were applied to estimate the expression levels of the p60-S6K1 mRNA and protein.A heterogeneous expression of the p60-S6K1 transcript was observed; it did not correlate with the total S6K1 mRNA expression and with the protein content of p60-S6K1 in the studied cell lines.Conclusions.We provide the evidence for the existence of a novel S6K1 splice variant coding for the p60-S6K1 isoform.The cell can employ two distinct pathways of the p60-S6K1 expression based on alternative translation of mRNA common for the main S6K1 isoforms mRNA and/or translation of the novel p60-S6K1 specific mRNA.Since the levels of the p60-S6K1 transcript expression do not correlate with the expression of total S6K1 mRNA, it is plausible that the p60-S6K1 isoform plays a role distinct from that of other isoforms in cellular physiology, as well as in the development of S6K1related pathologies.

Ribosomal protein S6 kinase 1 (S6K1) is a well-known downstream effector of mTORC1 (mechanistic target of rapamycin complex 1) participating primarily in the regulation of cell growth and metabolism.Deregulation of mTOR/S6K1 signaling can promote numerous human pathologies, including cancer, neurodegeneration, cardiovascular disease, and metabolic disorders.As existing data suggest, the S6K1 gene encodes several protein isoforms, including p85-S6K1, p70-S6K1, and p60-S6K1.The two of these isoforms, p85-S6K1 and p70-S6K1, were extensively studied to date.The origin and functional significance of the p60-S6K1 isoform remains a mystery, however, it was suggested that the isoform could be a product of alternative S6K1 mRNA translation.Herein we report the generation of HEK-293 cells exclusively expressing p60-S6K1 as a result of CRISPR/Cas9-mediated inactivation of p85/p70-S6K1 translation.Moreover, the generated modified cells displayed the elevated level of p60-S6K1 expression compared to that in wild-type HEK-293 cells.Our data confirm an assumption that p60-S6K1 is alternatively translated, most probably, from the common for both p70-and p85-S6K1 mRNA transcript and reveal a link between p60-S6K1 expression and such cellular processes as cell proliferation and motility.In addition, our findings indicate that the p60-S6K1 isoform of S6K1 may undergo a mode of regulation distinct from p70-and p85-S6K1 due to the absence of mTOR-regulated p60-S6K1 phosphorylation at T389 that is important for S6K1 activation.K e y w o r d s: CRISPR/Cas9 technology, p60-S6 kinase 1 (p60-S6K1) isoform, Akt/mTOR/S6K1 signaling pathway.

Aim.To generate HEK-293 cells with disrupted expression of S6K1 isoforms: p85, p70 and p60.Methods.CRISPR/Cas9 gene editing, Western blotting, immunofluorescent analysis, RT-PCR analysis, MTT assay, scratch assay.Results.Several clones of HEK-293 cells with a complete loss of p85/p70/p60-S6K1 protein expression were generated.The effects of p85/ p70/p60-S6K1 knockout on Akt/mTORC1/S6K1 signaling and cell proliferation and migration were assessed.Conclusions.The generated cell lines can be used to study a role played by S6K1 in cell physiology and to gain more detailed insight into cellular functions of the S6K1 isoforms.The HEK-293 cells exhibit downregulation of Akt phosphorylation on Ser473 and subsequent attenuation of cell growth rate, as well as inhibition of cell motility.K e y w o r d s: mTOR/S6K1 signaling,

Breast cancer (BC) is common and aggressive malignancy. Resistance to drugs often develops and is not fully understood. One of the possible reasons of this is increased activity of the DNA repair system in cancer cells. Since molecular disruption of RAD50, which implicated in DNA double-break repair, sensitizes breast tumor cells to cisplatin-based chemotherapy in cell culture, one may be hypothesized that level of RAD50 gene expression in breast tumors could be considered as a potential predictive and prognostic marker of BC. The aim of this investigation was to examine the impact of RAD50 gene features (level of expression and Copy Number Alteration (CNA)) on survival of BC patients taking into account their clinical characteristics: stage of disease and molecular subtype of tumors. Data on the clinical behavior, RAD50 gene expression (RNA-Seq (V2)) and CNA (deletion, gain, amplification) of the BC patients were obtained from three independent studies (TCGA, Provisional (n = 1105); TCGA, Cell 2015 (n = 1105); TCGA, Nature 2012(n = 825) using the cBioPortal (www.cbioportal.org/). Cases with absence of necessary data were excluded, leading to data sets from 2359 to 1220 samples. Testing intergroup differences (ANOVA, Tukey's HSD, Wilcoxon tests and Spearman's correlation) and analysis of BC patients' survival (Kaplan-Meier and Log-rank test) were performed using RStudio. A pairwise comparison groups of BC patients showed that level of RAD50 gene expression fluctuates significantly in breast tumors of various molecular subtypes (except pair of Luminal A and B), between patients on 1st and 2nd stages of disease and patients with different CNA. The patients with high level of RAD50 gene expression had significantly reduced overall survival in comparison with the patients that had low level of RAD50 gene expression only among patients with CNA. Correlation analysis of CNA and level of RAD50 gene expression has shown that high level of RAD50 gene expression bonded with gain and amplification of RAD50 gene. Our finding allows us to consider the gain and amplification of RAD50 gene as traits which are associated with poor survival of BC patients and RAD50 as potential prognostic marker of BC.

Aim.To study the subcellular localization of phospho-mTOR (Ser2481) in cultured cells of human cancer cell lines.Methods.Immunofluorescent analysis of phospho-mTOR (Ser2481) subcellular localization in cultured MCF-7 (human breast adenocarcinoma) and HepG2 (human liver carcinoma) cells.Results.For the first time phospho-mTOR (Ser2481) was detected as bright spots situated in the equatorial region of chromosomes and co-localized with condensed chromosomes during metaphase.Conclusions.In this study, we describe for the first time the co-localization of phospho-mTOR (Ser2481) and chromosomes of the metaphase plate in the MCF-7 and HepG2 cells.Our data support the hypothesis that phospho-mTOR (Ser2481) could act as a CPP (chromosomal passenger protein)-like kinase, which takes part in the regulation of mitosis progression. K e y w o r d s: mTOR, phospho-mTOR (

Aim. Generation of monoclonal antibodies specifi c to Coenzyme A. Methods.Hybridoma technique.KLH carrier protein conjugated with CoA was used for immunization.Screening of positive clones was performed with BSA conjugated to CoA.Results.Monoclonal antibody that specifi cally recognizes CoA and CoA derivatives, but not its precursors ATP and cysteine has been generated.Conclusion.In this study, we describe for the fi rst time the production and characterization of monoclonal antibodies against CoA.The monoclonal antibody 1F10 was shown to recognize specifi cally CoA in Western blotting, ELISA and immunoprecipitation.These properties make this antiboby a particularly valuable reagent for elucidating CoA function in health and disease.K e y w

This review summarizes experimental data related to the studies of PI3K/mTOR/S6K signaling conducted at the department of cell signaling.Analysis of novel S6Ks protein-protein interactions provided valuable information for understanding molecular mechanisms of regulation of S6Ks functional activity and subcellular localization mediated by PKC, CK2 and ROC1 ubiquitin ligase.We discuss the identification and functional analysis of novel isoform of ribosomal protein S6 kinase -S6K2 and of mTOR kinase -mTORb, as well as their oncogenic properties.Identification of CoA synthase responsible for last two steps in CoA biosynthesis and characterization of its interactions with S6K1 and other signaling molecules uncovere a potential link between mTOR/S6K signaling pathway and energy metabolism through regulation of CoA biosynthesis.The data concerning new molecular mechanisms of CoA synthase regulation are presented.

Our previous studies have shown that S6K1 and S6K2 protein kinases form the complexes with newly identified adaptor protein TDRD7, which is involved in regulation of cytoskeleton dynamics, mRNA transport, protein translation, piRNAs processing and transposons silensing.Aim.Determination the subcellular localization of S6K1-TDRD7 and S6K2-TDRD7 protein complexes.Methods.Immunofluorescense microscopy was used to study co-localization of S6K1/S6K2 and TDRD7 in HEK293, HEPG2 cell lines as well as in rat primary hippocampal neurons using primary polyclonal anti-S6K1 antibodies, monoclonal anti-S6K2 and anti-TDRD7 antibodies.Results.It was found that S6K1 is co-localized with TDRD7 in perinuclear region of HEK293 cells.S6K1 and S6K2 were also co-localized with TDRD7 in perinuclear region of HEPG2 cells and in soma of primary rat hippocampal neurons.Conclusions.In this report we provide an additional experimental evidences of possible S6K1-TDRD7 and S6K2-TDRD7 complexes formation in cells of different tissue origins that may reflect their potential physiological importance.However, elucidation of the exact composition of these complexes and their role in cell physiology requires additional studies.

The aim of this study was to produce monoclonal antibodies directed to the N-terminal regulatory region of S6K1, which shows very low homology to S6K2. Methods. Hybridoma technology, ELISA, Western blot, Immunoprecipitation. Results. Three hybridoma clones (A1, A2 and A3) producing mAbs specific to ribosomal protein S6 kinase 1 have been generated. Specificity of mAbs has been confirmed by Western blot and immunoprecipitation technique. Conclusions. The obtained antibodies are suitable for elucidating signal transduction pathways involving S6K1.