Cell stemness is maintained upon concurrent expression of RB and the mitochondrial ribosomal protein S18-2.

Stemness encompasses the capability of a cell for self-renewal and differentiation. The stem cell maintains a balance between proliferation, quiescence, and regeneration via interactions with the microenvironment. Previously, we showed that ectopic expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2) led to immortalization of primary fibroblasts, accompanied by induction of an embryonic stem cell (ESC) phenotype. Moreover, we demonstrated interaction between S18-2 and the retinoblastoma-associated protein (RB) and hypothesized that the simultaneous expression of RB and S18-2 is essential for maintaining cell stemness. Here, we experimentally investigated the role of S18-2 in cell stemness and differentiation. Concurrent expression of RB and S18-2 resulted in immortalization of Rb1-/- primary mouse embryonic fibroblasts and in aggressive tumor growth in severe combined immunodeficiency mice. These cells, which express both RB and S18-2 at high levels, exhibited the potential to differentiate into various lineages in vitro, including osteogenic, chondrogenic, and adipogenic lineages. Mechanistically, S18-2 formed a multimeric protein complex with prohibitin and the ring finger protein 2 (RNF2). This molecular complex increased the monoubiquitination of histone H2ALys119, a characteristic trait of ESCs, by enhanced E3-ligase activity of RNF2. Furthermore, we found enrichment of KLF4 at the S18-2 promoter region and that the S18-2 expression is positively correlated with KLF4 levels. Importantly, knockdown of S18-2 in zebrafish larvae led to embryonic lethality. Collectively, our findings suggest an important role for S18-2 in cell stemness and differentiation and potentially also in cancerogenesis.

Upregulation of PKN1 as a Prognosis Biomarker for Endometrial Cancer.

BACKGROUND: Several markers of survival among endometrial cancer (EC) patients have been proposed, namely, the oncoprotein stathmin, RAF kinase inhibitor (RKIP), Cyclin A, GATA-binding protein 3 (GATA3), and growth and differentiation factor-15 (GDF-15). Their elevated expression correlated significantly with a high stage, serous papillary/clear cell subtypes, and aneuploidy. In a previous study, we reported the elevated expression of the serine/threonine protein kinase N1 (PKN1) in cancerous cells. In the present paper, we studied PKN1 expression in EC tissues from a large cohort of patients, to determine whether PKN1 can serve as a marker for the aggressiveness and prognosis of EC, and/or as a marker of survival among EC patients. METHODS: Tissue samples from EC patients were examined retrospectively for tumor type, tumor size, FIGO stage and grade, depth of invasion in the myometrium, and presence of lymph node metastasis. The PKN1 protein expression in EC cells was assessed by immunohistochemistry. PKN1 mRNA levels were analyzed in publicly available databases, using bioinformatic tools. RESULTS: We found that expression of PKN1 at the mRNA and proteins levels tended to increase in high-grade EC samples (P = .0001 and P = .06, respectively). In addition, patients with metastatic disease had higher PKN1 mRNA levels (P = .02). Moreover, patients with high PKN1 expression could be characterized by poorer survival. CONCLUSIONS: We have shown a trend of the higher PKN1 expression levels in EC patients with poor prognosis. Therefore, PKN1 might be considered as a candidate prognostic marker for EC.

Expression of the Chemokine Receptor CCR1 in Burkitt Lymphoma Cell Lines Is Linked to the CD10-Negative Cell Phenotype and Co-Expression of the EBV Latent Genes EBNA2, LMP1, and LMP2.

Chemokines and their receptors regulate the migration of immune cells and the dissemination of cancer cells. CCR1, CCR2, CCR3, and CCR5 all belong to a single protein homology cluster and respond to the same inflammatory chemokines. We previously reported that CCR1 and CCR2B are induced upon Epstein-Barr virus (EBV) infection of B cells in vitro. EBV is present in almost all cases of endemic Burkitt lymphoma (BL); however, the contribution of EBV in the pathogenesis of the disease is not fully understood. Here, we analyzed the relation of the expression of CCR1, CCR2, CCR3, and CCR5, the EBV DNA load and expression of EBV latent genes in nine EBV-carrying and four EBV-negative BL cell lines. We revealed that CCR1 is expressed at high mRNA and protein levels in two CD10-negative BL cell lines with co-expression of the EBV latent genes EBNA2, LMP1, and LMP2. Low levels of CCR2 transcripts were found in three BL cell lines. CCR3 and CCR5 transcripts were hardly detectable. Our data suggest that in vivo, CCR1 may be involved in the dissemination of BL cells and in the selection of BL cells with restricted EBV gene expression programs.

Impact of the Uridine⁻Cytidine Kinase Like-1 Protein and IL28B rs12979860 and rs8099917 SNPs on the Development of Hepatocellular Carcinoma in Cirrhotic Chronic Hepatitis C Patients-A Pilot Study.

Background and objectives: The hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma (HCC) in the western world. The efficacy of surveillance programs for early detection of HCC is not satisfactory: many tumors are diagnosed at the late, incurable stages. Therefore, there is a need in reliable prognostic markers for the proper follow-up of HCV-positive patients. The aim of the present study was to assess the prognostic value of the uridine⁻cytidine kinase-like protein 1 (UCKL-1), a putative oncoprotein, together with genetically determined polymorphisms in the interleukin 28B (IL28B) gene (rs12979860, rs8099917) in the development of HCC in HCV-positive cirrhotic patients. Materials and Methods: We included 32 HCV cirrhotic patients, 21 (65.6%) of whom had HCC. The expression of UCKL-1 was assessed in liver tissue sections, using immunohistochemistry. For IL28B rs12979860 and rs8099917 genotype analysis, the corresponding genomic regions were amplified by polymerase chain reaction (PCR) with appropriate primers. Results: We have found that UCKL-1 expression was significantly increased in HCC (p = 0.003). The presence of rs8099917 TT single-nucleotide polymorphism (SNP) elevated the chances of HCC manifestation more than sevenfold (OR = 7.3, p = 0.0273). The presence of rs12979860 CC SNP also heightened HCC chances more than sevenfold (OR = 7.5, p = 0.0765). Moreover, in the HCC group, a combination of IL28B rs12979860 non-TT and rs8099917 TT genotypes was observed more often, compared with the non-HCC group. Other combinations of IL28B rs12979860 and rs8099917 SNIPs were associated with a reduced risk of HCC development, approximately at the same extent. Conclusions: The presence of IL28B rs8099917 TT and rs12979860 CC SNPs, but not the intensity of UCKL-1 expression, is strongly associated with increased chances of HCC development in HCV-positive cirrhotic patients.

Ancient permafrost staphylococci carry antibiotic resistance genes.

Background: Permafrost preserves a variety of viable ancient microorganisms. Some of them can be cultivated after being kept at subzero temperatures for thousands or even millions of years. Objective: To cultivate bacterial strains from permafrost. Design: We isolated and cultivated two bacterial strains from permafrost that was obtained at Mammoth Mountain in Siberia and attributed to the Middle Miocene. Bacterial genomic DNA was sequenced with 40-60× coverage and high-quality contigs were assembled. The first strain was assigned to Staphylococcus warneri species (designated MMP1) and the second one to Staphylococcus hominis species (designated MMP2), based on the classification of 16S ribosomal RNA genes and genomic sequences. Results: Genomic sequence analysis revealed the close relation of the isolated ancient bacteria to the modern bacteria of this species. Moreover, several genes associated with resistance to different groups of antibiotics were found in the S. hominis MMP2 genome. Conclusions: These findings supports a hypothesis that antibiotic resistance has an ancient origin. The enrichment of cultivated bacterial communities with ancient permafrost strains is essential for the analysis of bacterial evolution and antibiotic resistance.

The kinin-kallikrein system: physiological roles, pathophysiology and its relationship to cancer biomarkers.

The kinin-kallikrein system (KKS) is an endogenous multiprotein cascade, the activation of which leads to triggering of the intrinsic coagulation pathway and enzymatic hydrolysis of kininogens with the consequent release of bradykinin-related peptides. This system plays a crucial role in inflammation, vasodilation, smooth muscle contraction, cardioprotection, vascular permeability, blood pressure control, coagulation and pain. In this review, we will outline the physiology and pathophysiology of the KKS and also highlight the association of this system with carcinogenesis and cancer progression.

Comparative Analysis of the Embryonal Brain Tumors Based on Their Molecular Features.

Pediatric brain tumors currently show the highest incidence among solid childhood malignancies and, together with leukemia, are the leading cause of death from cancer in childhood. Embryonal brain tumors are the most common and frequent type of childhood brain cancer and are usually characterized by an extremely aggressive course of the disease with the worst outcomes in most cases. There is an urgent need for specific refined molecular diagnostics, which would help to develop personalized treatment. In the present review paper, the latest molecular characteristics of various classified forms of embryonal brain tumors were analyzed in detail. Overexpression of the MYC and MYCN genes is characteristic of many embryonal brain tumors, leading to enhanced cell proliferation and disturbances in the cell cycle. The functioning of the SWI2/SNF2 chromatin remodeling complex are distorted in such malignancies as well. Noteworthy, LIN28 and MYC discussed here are involved in the induction of pluripotency. We have to mention that molecular mechanisms underlying the development of embryonal brain tumors of the central nervous system (CNS) are still not well understood. Thus, it is important to uncover such mechanisms with the aim to provide a better prognosis of the course of disease and to create personalized therapy.

MTSS1 is downregulated in nasopharyngeal carcinoma (NPC) which disrupts adherens junctions leading to enhanced cell migration and invasion.

Loss of cell-cell adhesions is the indispensable first step for cancer cells to depart from the primary tumor mass to metastasize. Metastasis suppressor 1 (MTSS1) is frequently lost in metastatic tissues, correlating to advanced tumor stages and poor prognosis across a variety of cancers. Here we explore the anti-metastatic mechanisms of MTSS1, which have not been well understood. We found that MTSS1 is downregulated in NPC tissues. Lower levels of MTSS1 expression correlate to worse prognosis. We show that MTSS1 suppresses NPC cell migration and invasion in vitro through cytoskeletal remodeling at cell-cell borders and assembly of E-cadherin/ß-catenin/F-actin in adherens junctions. The I-BAR domain of MTSS1 was both necessary and sufficient to restore this formation of E-cadherin/ß-catenin/F-actin-mediated cell adherens junctions.

Analysis of protein-protein interactions in a complex environment: capture of an analyte-receptor complex with standard additions of the receptor (CARSAR) approach.

Traditional methods of analytical chemistry to detect an interaction between certain proteins in multicomponent mixtures (e.g. cell lysates, etc.) have limitations. This is due to difficulties in identification of a specific signal of an analyte (a molecule to be detected) against the background. In the present work, we propose the new analytical protocol for transducer-based sensors with a restricted sensitive area. It uses a combination of analyte-receptor complex precipitation with serial additions of the receptor (CARSAR). To test this new analytical strategy, we used a surface plasmon resonance technique to confirm an interaction between the Epstein-Barr virus-encoded nuclear antigen 6 and the mitochondrial ribosomal protein MRPS18-2.

MRPS18-2 protein immortalizes primary rat embryonic fibroblasts and endows them with stem cell-like properties.

We report that the overexpression of mitochondrial ribosomal protein MRPS18-2 (S18-2) can immortalize primary rat embryonic fibroblasts (REFs). The immortalized cells (18IM) lose contact inhibition, form foci, and are capable of anchorage-independent growth. Concurrently, mesodermal markers, such as vimentin, smooth muscle actin, and Fut4, disappear completely. 18IM cells express embryonic stem cell markers, such as SSEA-1, Sox2, and Oct3/4. In confluent cultures, a portion of cells also express ectoderm- and endoderm-specific pan-keratin, ectoderm-specific beta-III-tubulin, mesoderm-specific MHC class II, and become stainable for fat with Oil red O. None of these changes was detected in c-myc+Ha-ras (MR)-transformed cells. In immunodeficient mice, 18IM cells formed small transiently growing tumors that have down-regulated SSEA-1 and showed pan-keratin staining. We conclude that S18-2 can immortalize REFs and induces them to express stem cell traits.

A Comparative Study on the Viability of Normal and Cancerous Cells upon Irradiation with a Steady Beam of THz Rays.

Terahertz (THz) electromagnetic radiation is commonly used in astronomy, security screening, imaging, and biomedicine, among other applications. Such approach has raised the question of the influence of THz irradiation on biological objects, especially the human body. However, the results obtained to date are quite controversial. Therefore, we performed a comparative study on the viability of normal cells and cancer cells upon irradiation with a steady beam of THz rays. We used human peripheral blood mononuclear cells and cancer cell lines. Primary human mononuclear blood cells (monocytes, and B-, and T-cells) showed an increased death rate, determined by cell counting and fluorescence microscopy, upon 0.14 THz irradiation. The effect of THz radiation was different among malignant cells of B- and T-cell origin (Ramos and Jurkat cells) and epithelial cancer cells (MCF7 and LNCaP). This was demonstrated by cell counting and by the alamarBlue assay. In conclusion, THz radiation can result in the death of human primary and malignant cells. However, the mechanism of this phenomenon is largely unknown. Hence, more work should be done to shed some light on the mechanism of action of THz irradiation in living organisms to enhance technologic developments.

STK4 protein expression pattern follows different trends in endometrioid and serous endometrial adenocarcinoma upon tumor progression.

In a previous study, we showed that serine/threonine-protein kinase 4 (STK4) is involved in the control on proliferation and migration of endometrial cancer (EC) cells in vitro. In the present paper, we studied STK4 expression in EC tissues from a large cohort of patients to determine whether STK4 can serve as a marker for the aggressiveness and prognosis of EC. Tissue samples from patients with EC were examined for tumor type, grade, and stage. The STK4 protein expression in EC cells was assessed by immunohistochemistry and related to clinicopathological data of patients, such as progression and patient survival rate. The STK4 mRNA levels and its relation to the survival rate were analyzed also in publicly available databases. The STK4 gene expression was low at both, the mRNA and protein levels in EC, especially in serous tumors. Comparison of STK4 expression with the patient survival rate shows that the higher expression is associated with worse prognosis in serous EC, while no such dependence was found in endometrioid EC. Hence, the determination of the SKT4 expression pattern could be used as a putative prognostic marker for serous EC.

Evaluation of dyskerin expression and the Cajal body protein WRAP53ß as potential prognostic markers for patients with primary vaginal carcinoma.

Primary vaginal cancer (PVC) is a rare gynaecological malignancy, which, at present, lacks appropriate biomarkers for prognosis. The proteins dyskerin and WD repeat containing antisense to TP53 (WRAP53ß), both of which exert their functions in the telomerase holoenzyme complex, have been shown to be upregulated in different cancer types. These proteins have also been proposed as prognostic markers in some types of cancer. The aim of the present study was to examine the expression patterns of dyskerin and WRAP53ß in patients with PVC. Moreover, as part of a search for effective biomarkers to evaluate prognosis in PVC, the expression of these two proteins and their potential association with clinical variables and survival were also evaluated. The expression of dyskerin and WRAP53ß was assessed in PVC tumour samples from 68 patients using immunohistochemistry. The majority of tumour samples showed low and moderate expression levels of dyskerin. Upregulation of dyskerin in tumour samples was significantly associated with a shorter survival time and a poorer cancer-specific survival rate. WRAP53ß was also expressed in most of the cells but was not significantly associated with clinical variables or survival. This study demonstrates that upregulation of dyskerin is significantly associated with poor prognosis. Thus, dyskerin may serve as a promising prognostic marker and a potential putative therapeutic target in PVC.

Epstein-Barr virus-encoded EBNA-5 forms trimolecular protein complexes with MDM2 and p53 and inhibits the transactivating function of p53.

We report that MDM2, a negative regulator of p53, can bind to EBNA-5. Using GST pull-down assay, immunoprecipitation, surface plasmon resonance and immunostaining of lymphoblastoid cells, we found that trimolecular complexes are formed between EBNA-5, MDM2 and p53, where MDM2 serves as a bridge. The EBNA-5 binding to MDM2 counteracted destabilizing effect of the latter on the p53. In ubiquitination and degradation assays in vitro, EBNA-5 inhibited p53 polyubiquitination (but not monoubiquitination) in a concentration-dependent manner. This resembles the effect of p14ARF on p53. Moreover, EBNA-5 was found to inhibit the degradation of p53 in vitro. High levels of p53 expression were maintained in LCLs. The binding of EBNA-5 to MDM2 also could impair the functional activity of p53. The p53-dependent genes P21 and VDR were not induced in EBV-infected, in contrast to mitogen-activated cells. This may explain the tolerance of established LCLs to high levels of p53 without undergoing apoptosis.

Epstein-Barr virus encoded EBNA-3 binds to vitamin D receptor and blocks activation of its target genes.

Epstein-Barr virus (EBV) is a human gamma herpes virus that infects B cells and induces their transformation into immortalized lymphoblasts that can grow as cell lines (LCLs) in vitro. EBNA-3 is a member of the EBNA-3-protein family that can regulate transcription of cellular and viral genes. The identification of EBNA-3 cellular partners and a study of its influence on cellular pathways are important for understanding the transforming action of the virus. In this work, we have identified the vitamin D receptor (VDR) protein as a binding partner of EBNA-3. We found that EBNA3 blocks the activation of VDR-dependent genes and protects LCLs against vitamin-D3-induced growth arrest and/or apoptosis. The presented data shed some light on the anti-apoptotic EBV program and the role of the EBNA-3-VDR interaction in the viral strategy.

EBV-encoded EBNA-6 binds and targets MRS18-2 to the nucleus, resulting in the disruption of pRb-E2F1 complexes.

Epstein-Barr virus (EBV), like other DNA tumor viruses, induces an S-phase in the natural host cell, the human B lymphocyte. This is linked with blast transformation. It is believed that the EBV-encoded nuclear antigen 6 (EBNA-6) is involved in the regulation of cell cycle entry. However, the possible mechanism of this regulation is not approached. In our current study, we found that EBNA-6 binds to a MRPS18-2 protein, and targets it to the nucleus. We found that MRPS18-2 binds to both hypo- and hyperphosphorylated forms of Rb protein specifically. This binding targets the small pocket of pRb, which is a site of interaction with E2F1. The MRPS18-2 competes with the binding of E2F1 to pRb, thereby raising the level of free E2F1. Our experimental data suggest that EBNA-6 may play a major role in the entry of EBV infected B cells into the S phase by binding to and raising the level of nuclear MRPS18-2, protein. This would inhibit pRb binding to E2F1 competitively and lift the block preventing S-phase entry.

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes.

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.

Persistent Roseoloviruses Infection in Adult Patients with Epilepsy.

BACKGROUND: Human herpesviruses (HHV)-6A, HHV-6B and HHV-7 are considered to be involved in the pathogenesis of epilepsy, a common neurological disorder. The objective of this study was to determine the association of roseoloviruses infection with epilepsy. METHODS: 53 epilepsy patients and 104 ordinary blood donors were analyzed to determine presence of virus-specific antibodies by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), genomic sequences, viral load and gene expression by polymerase chain reactions (PCRs) and restriction analysis, HHV-6 protein expression by IFA and level of cytokines by ELISA. RESULTS: Roseoloviruses genomic sequences in DNA samples from whole blood were found in 86.8% of patients versus 54.8% of controls and active infection was revealed only in patients with epilepsy (19.6% of roseolovirus-positive patients). Significantly higher viral load and more frequent gene expression was detected in patients compared to the controls. HHV-6-encoded protein expression was demonstrated in 53.3% of patients with previously detected HHV-6 DNA. Changes in level of cytokines were determined in patients with elevated viral load compared to the patients without elevated viral loads and to the controls. CONCLUSIONS: Results on frequent active HHV-6 and HHV-7 infection in epilepsy patient' peripheral blood indicate on possible involvement of these viruses in the disease development.

PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein.

The low molecular weight compound, PRIMA-1MET restores the transcriptional transactivation function of certain p53 mutants in tumor cells. We have previously shown that PRIMA-1MET induces nucleolar translocation of p53, PML, CBP and Hsp70. The Epstein-Barr virus encoded, latency associated antigen EBNA-5 (also known as EBNA-LP) is required for the efficient transformation of human B lymphocytes by EBV. EBNA-5 associates with p53-hMDM2-p14ARF complexes. EBNA-5 is a nuclear protein that translocates to the nucleolus upon heat shock or inhibition of proteasomes along with p53, hMDM2, Hsp70, PML and proteasome subunits. Here we show that PRIMA-1MET induces the nucleolar translocation of EBNA-5 in EBV transformed B lymphoblasts and in transfected tumor cells. The PRIMA-1MET induced translocation of EBNA-5 is not dependent on the presence of mutant p53. It also occurs in p53 null cells or in cells that express wild type p53. Both the native and the EGFP or DSRed conjugated EBNA-5 respond to PRIMA-1MET treatment in the same way. Image analysis of DSRed-EBNA-5 expressing cells, using confocal fluorescence time-lapse microscopy showed that the nucleolar translocation requires several hours to complete. FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) measurements on live cells showed that the nucleolar translocation was accompanied by the formation of EBNA-5 aggregates. The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET. Our results suggest that mutant p53 is not the sole target of PRIMA-1MET. We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET.

Study on the spatial architecture of p53, MDM2, and p14ARF containing complexes.

We have developed a surface plasmon resonance (SPR)-based immunocapture approach to study multimeric protein-protein complexes. A composition and spatial architecture of protein complexes that contained GST-tagged p53, p14ARF, and MDM2 was examined by the developed approach. Obtained results verified that the p53 protein possesses two binding sites for MDM2. Ternary complexes containing p14ARF, MDM2, and p53 proteins could only be formed when MDM2 protein functions as a bridging molecule. That was confirmed by immunoprecipitation and immunostaining.