Evaluation of expression profiles of microRNAs and two target genes, FOXO3a and RUNX2, effectively supports diagnostics and therapy predictions in breast cancer.

Breast cancer (BC) including its progression into bone metastasis is a complex process involving changes in gene expression and function of both, microRNAs (miRNAs) and their target genes. Deregulation of miRNAs has been described as a crucial factor responsible for the initiation and progression of BC, and specific miRNA expression profiles have been found to be associated with particular disease states, histological tumor types, and BRCA1/2 or HER status. BRCA1 tumor suppressor is involved in DNA damage response and repair and epigenetically controls miR-155 expression and its pre-cancerous potential. MiR-155 targets 3´UTR region of multiple components of the pro-oncogenic signaling cascades, including FOXO3a tumor suppressor and RUNX2 transcription factor regulating metastatic potential in BC. We employed qRT-PCR to determine expression level and examine possible regulatory role of selected miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-21, miR-27a and miR-155) and their impact on expression modulation of FOXO3a and RUNX2 in peripheral blood mononuclear cells (PBMCs) in healthy individuals, in women carrying BRCA1 mutations with no disease manifestation, in women carrying BRCA1 mutations after tumor resection and therapy and in women with BC of unknown BRCA1 status in acute stage before tumor resection. Our results showed significant increase of miR-17, miR-19a, miR-21, miR-27, miR-155 and RUNX2 expression in PBMCs in BRCA1 patients and patients in acute stage, while FOXO3a expression was significantly decreased in these patients. MiR-18a and miR-20a expression was not affected. We propose that expressional changes reported in this study could provide significant additive information for early BC diagnosis, disease development prediction and therapy outcome monitoring.

microRNA expression profiling as supportive diagnostic and therapy prediction tool in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cells carrying Philadelphia (Ph) chromosome and the oncogenic BCR-ABL1 fusion gene. microRNAs (miRNAs) belong to hematopoiesis transcription regulators and their deregulated expression associates with pathogenesis of CML. The current study assesses and validates expression profiles of selected oncogenic and tumor suppressing miRNAs that are associated with different imatinib mesylate (IM) response in CML patients carrying rare BCR-ABL variants. Microarray analysis has identified different expression of 70 miRNAs (46 up- and 24 down-regulated) when compared IM-resistant with IM-responsive patients carrying Ph chromosome. Significantly up-regulated expression of oncogenic miRNAs (miR-17, miR-18a, miR-20a, miR-21, miR-27a and miR-155) and significantly down-regulated expression of tumor supressing mRNAs (let-7d, miR-205, miR-320, miR-451 and miR-574) in IM-resistant compared to IM-responsive patients was confirmed and validated by qRT-PCR. This study confirms the involvement of the selected oncogenic and tumor suppressing miRNAs in CML pathogenesis and IM response and suggests that these miRNAs could be suitable biomarkers for differential diagnosis of CML patients carrying rare BCR-ABL transcripts, as well as for prediction of their IM response and therapy outcome.

miR-155 as a diagnostic and prognostic marker in hematological and solid malignancies.

MicroRNAs (miRNAs) are small RNAs that have emerged as potent regulators of the target genes messenger RNAs expression in the response of cell to both physiological and pathophysiological conditions. Reflecting pathological processes today, miRNAs are widely validated for their potential role in diagnostic, prognostic and novel therapeutic targeting for cancerous and other diseases. miR-155 is considered as a typical multifunctional miRNA including its role as oncomiR (cancer-associated miRNA). Expression of miR-155 is upregulated in cells with high proliferative activity and decreased apoptotic capability. It belongs to cluster of well-characterized tumor associated miRNAs detectable also in the peripheral blood. In this review we summarize the principles of miR-155 host gene expressional regulation, as well as its role in regulation of the target genes mRNAs. Altered expression of miR-155 has been described in multiple cancerous and other diseases, reflecting staging, progress and treatment outcomes. Therefore, miR-155 became a potential biomarker and candidate for clinical utilization as predictor of the presence of cancer, its staging and prognosis.

Simultaneous detection of multiple targets for ultrastructural immunocytochemistry.

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

5-azacitidine in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1 gene and cell differentiation capacity.

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34+ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte-macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

Healthy first-degree relatives of patients with type 1 diabetes exhibit significant differences in basal gene expression pattern of immunocompetent cells compared to controls: expression pattern as predeterminant of autoimmune diabetes.

Expression features of genetic landscape which predispose an individual to the type 1 diabetes are poorly understood. We addressed this question by comparing gene expression profile of freshly isolated peripheral blood mononuclear cells isolated from either patients with type 1 diabetes (T1D), or their first-degree relatives or healthy controls. Our aim was to establish whether a distinct type of 'prodiabetogenic' gene expression pattern in the group of relatives of patients with T1D could be identified. Whole-genome expression profile of nine patients with T1D, their ten first-degree relatives and ten healthy controls was analysed using the human high-density expression microarray chip. Functional aspects of candidate genes were assessed using the MetaCore software. The highest number of differentially expressed genes (547) was found between the autoantibody-negative healthy relatives and the healthy controls. Some of them represent genes critically involved in the regulation of innate immune responses such as TLR signalling and CCR3 signalling in eosinophiles, humoral immune reactions such as BCR pathway, costimulation and cytokine responses mediated by CD137, CD40 and CD28 signalling and IL-1 proinflammatory pathway. Our data demonstrate that expression profile of healthy relatives of patients with T1D is clearly distinct from the pattern found in the healthy controls. That especially concerns differential activation status of genes and signalling pathways involved in proinflammatory processes and those of innate immunity and humoral reactivity. Thus, we posit that the study of the healthy relative's gene expression pattern is instrumental for the identification of novel markers associated with the development of diabetes.

The syn3 strain HSZP of herpes simplex virus type 1 (HSV-1) is not pathogenic for mice and shows limited neural spread.

Strain HSZP of the herpes simplex virus type 1 (HSV-1) forms large giant cells in vitro. This property was found associated with a mutation that alters the codon CGC (in the strain KOS or 17 sequence) to CAC (in the HSZP sequence), changing the amino acid 857 from arginine to histidine in the cytoplasmic domain of the glycoprotein B (gB) polypeptide chain. Giant cell formation by ANGpath was attributed to a mutation that alters the codon GCC (in KOS and strain 17 sequences) to GTC (in ANGpath sequence) changing the amino acid 854 in the same (syn3) region of the gB molecule. In contrast to the ANGpath virus, which is pathogenic (1 LD50 < 1 x 10(4) PFU) for adult DBA/2 mice after peripheral inoculation, strain HSZP was never found to be lethal for adult mice. Whereas ANGpath-infected mice which survived acute infection frequently (79%) developed latency in the regional sensory ganglion (as proved by virus reactivation during explantation), latent HSZP reactivated in ganglion culture at a considerably reduced rate (21%). Only 10-day-old DBA/2 mice were sensitive to HSZP infection. In these, HSZP spread from the site of peripheral administration mainly by hematogenous route. The neural spread of HSZP in suckling DBA/2 mice was manifested by the involvement of vegetative neurons in the wall of the small intestine and in the retroperitoneal vegetative ganglia. We conclude that HSZP, a polykaryocyte-forming strain with a mutation in the syn3 region II, shows limited neuroinvasity for mice after peripheral administration.

Specific binding of herpes simplex virus type 1 (HSV-1) strain HSZP to host cell surface can provoke interference with early shutoff of host protein synthesis induced by HSV-1 strain KOS.

The HSZP strain of herpes simplex virus type 1 (HSV-1) is defective with respect to the early shutoff of host protein synthesis but is effective at interfering with the early shutoff function of the HSV-1 KOS strain, even when heat-inactivated or neutralized by antibody. The interference was not due to exclusion of strain KOS by HSZP at the level of adsorption or penetration. The component responsible for the interference is an integral part of HSZP virions. Strain HSZP inactivated with zinc ions failed to interfere with the early shutoff function of the superinfecting strain KOS. The same effect was also found with strain HSZP purified from cells treated with 2-deoxy-D-glucose. This finding supports the idea that a specific interaction between HSZP virions and the cell surface can be responsible for the interference phenomenon.

Herpes simplex virus type 1 (HSV-1) HSZP interferes also after antibody neutralization with early shutoff of host protein synthesis induced by HSV-1 KOS.

The HSZP strain of herpes simplex virus type 1 (HSV-1) is defective with respect to the early shutoff of host protein synthesis. However, in superinfection experiments using Vero cells, the HSZP strain was effective, even after neutralization by antibody, at interfering with the early shutoff function of the HSV-1 KOS strain. Evidence was given that the observed interference was not due to exclusion of the KOS by HSZP at the level of adsorption or penetration. The neutralized KOS strain failed to induce early shutoff of host protein synthesis.

Immunoprecipitation of herpes simplex virus polypeptides with human sera is related to their ELISA titre.

Out of 485 human sera tested by neutralization and enzyme immunoassay 13 were negative in both tests, while a positive correlation was found in 457 samples (94.3%). The rest of sera (3.2%) showed discordant results. Selected sera were examined, in addition, by Western blot and immunoprecipitation assays with the aim to analyse the precipitation profile of discordant sera and correlate the ELISA titre with the precipitation profiles. High titre sera precipitated the main glycoproteins (gC, gB, gE, gD), the capsid polypeptides (VP 5, VP 19, VP 21, VP 22) and several other structural and nonstructural proteins. The ability of sera to precipitate viral structural proteins was related to their ELISA titre rather than to their neutralizing activity.

The synthesis of the major DNA-binding protein (ICP8) in cells infected with the strain HSZP or KOS of herpes simplex virus type 1.

Synthesis of the major DNA-binding protein (ICP8) was investigated in primary rabbit kidney (RK) and Vero cells infected with the syncytial (syn) strain HSZP or with the non-syn strain KOS of herpes simplex virus type 1 (HSV-1). Results showed the following: 1. In contrast to strain KOS, the rate of viral polypeptide synthesis was accelerated in Vero cells infected with strain HSZP. The ICP8 could be detected in the nuclei of cells by one hour post-infection (hr p. i.) where it became associated with the viral DNA (DNase sensitive form). Later on (7 hr p.i.), the synthesis of viral polypeptides decreased and no further translocation of ICP8 from the cytoplasm into the nucleus was observed. 2. Strain HSZP was approx. three times more resistant to the action of phosphonoacetic acid (PAA) than strain KOS. In order to block the synthesis of HSZP gamma-2 polypeptides, a concentration of 600 micrograms PAA/ml had to be used. Under this condition, the HSZP ICP8 was translocated into the cell nucleus at later interval only (7 hr p.i.), and it was still possible to release this polypeptide from the nucleus by DNase treatment. The failure of the HSZP ICP8 to associate with the nuclear matrix (DNase resistant form) of infected cells in the absence of viral DNA replication may reflect its predominant affinity for the viral DNA which, in turn, may be responsible for the observed accelerated synthesis of the HSZP polypeptides in infected Vero cells. 3. In primary RK cells infected with strain HSZP the ICP8 did not translocate into the cell nucleus. Therefore, no gamma-2 polypeptides were synthesized.

Mechanism of altered cytoskeleton organization in influenza virus infection.

We followed the autophosphorylation of cytoskeleton (CS) isolated from control chick embryo cell membranes (CS-C) and from these membranes after influenza virus adsorption (CS-V) under conditions allowing to determine the activity of a single type proteinkinase. The Ca2+ dependent calmodulin (CaM) kinase used different substrates from CS-V than did the c'AMP dependent proteinkinase. The catalytic subunit (c-subunit) of the c'AMP dependent proteinkinase added from outside phosphorylated the same polypeptides than the endogenous c'AMP dependent proteinkinase, the further being more active than the latter. The purified influenza virus incorporated 32P in the presence of the c-subunit only. Incubation of influenza virus with the c-subunit caused morphological changes visible by electron microscopy. The pleomorphy of the particles as well as their electron transmissibility were enhanced in result of structural alterations and rarefaction of surface spikes of the haemagglutinin and neuraminidase. The contractibility of CS isolated from normal CEC and of the CS from CEC by 15 min postinfection (p.i.) was determined according to the actomyosin ATPase activity. The ATPase activity of the cytoskeleton in the presence of the Ca2+/CaM and that in the presence of c'AMP were used as controls. The virus as well as the Ca2+/CaM increased the ATPase activity. EGTA had no effect but did not interfere with virus stimulation, while c'AMP blocked the virus-induced enhancement of the ATPase activity.

Herpes simplex virus type 1 (HSV-1) HSZP interferes also heat inactivated with early shutoff of host protein synthesis induced by HSV-1 KOS.

The ability of two strains of herpes simplex virus type 1 (HSZP and KOS) to shut off the host protein synthesis in the presence of Actinomycin D was investigated. The HSZP strain proved to be defective with respect to the so-called early shutoff function. In superinfection experiments, the HSZP was effective at interfering with the early shutoff function of the KOS strain provided that the HSZP infection preceded KOS superinfection. Heat inactivation of the HSZP did not lead to the loss of its interfering ability. Evidence was given that this interference was neither due to the hindrance of the KOS by HSZP at adsorption nor due its exclusion during penetration.

Marked difference in electrophoretic migration rates between two influenza A viruses.

Comparative moving boundary electrophoresis revealed that influenza virus A/PR/8/34 (H0N1) has a 2.5 times higher electrophoretic migration rate at pH 7 than influenza virus A/Singpore/1/57 (H2N2). This difference was the same whether the compared viruses were purified first by either ammonium sulphate precipitation or adsorption onto and elution from red blood cells and then by density gradient centrifugation. The same electrophoretic methods was used for testing the homogeneity of influenza virus preparations purified by either method.