Early diagnosis of prostate cancer is a challenging issue due to the lack of specific markers. Therefore, a sensitive diagnostic marker that is expressed or upregulated exclusively in prostate cancer ...cells would facilitate diagnostic procedures and ensure a better outcome. We evaluated the expression of myosin 1C isoform A in 5 prostate cell lines, 41 prostate cancer cases, and 11 benign hyperplasias. We analyzed the expression of 12 surface molecules on prostate cancer cells by flow cytometry and analyzed whether high or low myosin 1C isoform A expression could be attributed to a distinct phenotype of prostate cancer cells. Median myosin 1C isoform A expression in prostate cancer samples and cancer cell lines was 2 orders of magnitude higher than in benign prostate hyperplasia. Based on isoform A expression, we could also distinguish clinical stage 2 from clinical stage 3. Among cell lines, PC-3 cells with the highest myosin 1C isoform A level had diminished numbers of CD10/CD13-positive cells and increased numbers of CD29 (integrin beta1), CD38, CD54 (ICAM1) positive cells. The surface phenotype of clinical samples was similar to prostate cancer cell lines with high isoform A expression and could be described as CD10-/CD13- with heterogeneous expression of other markers. Both for cell lines and cancer specimens we observed the strong correlation of high myosin 1C isoform A mRNA expression and elevated levels of CD29 and CD54, suggesting a more adhesive phenotype for cells with high isoform A expression. Compared to normal tissue, prostate cancer samples had also reduced numbers of CD24- and CD38-positive cells. Our data suggest that a high level of myosin 1C isoform A is a specific marker both for prostate cancer cells and prostate cancer cell lines. High expression of isoform A is associated with less activated (CD24/CD38 low) and more adhesive (CD29/CD54 high) surface phenotype compared to benign prostate tissue.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
High mobility group protein (HMGB1) is secreted by myeloid cells and cells of damaged tissues during inflammation, causing inflammatory reactions through various receptors, including TLR
and RAGE. ...TREM-1 is considered to be one of the potential HMGB1 receptors. In this work, we have shown that the HMGB1 protein is able to bind to the TREM-1 receptor at high affinity both in solution and on the cell surface. This binding causes lymphocytes to release cytokines IL-2, IL-1b, IL-6, TNF and Ifny into the medium, which leads to the appearance of cytotoxic lymphocytes in PBMC capable of lysing HLA-negative tumor cells. Expanding the spectra of proinflammatory receptor ligands and understanding the mechanisms of their action is essential for the creation of new immunotherapy pathways.
Understanding the exact mechanisms of the activation of proinflammatory immune response receptors is very important for the targeted regulation of their functioning. In this work, we were able to ...identify the sites of the molecules in the proinflammatory cytokine TNF (tumor necrosis factor) and its TNFR1 (tumor necrosis factor receptor 1), which are necessary for the two-stage cytotoxic signal transduction required for tumor cell killing. A 12-membered TNFR1 peptide was identified and synthesized, interacting with the ligands of this receptor protein's TNF and Tag7 and blocking their binding to the receptor. Two TNF cytokine peptides interacting with different sites of TNFR1 receptors were identified and synthesized. It has been demonstrated that the long 16-membered TNF peptide interferes with the binding of TNFR1 ligands to this receptor, and the short 6-membered peptide interacts with the receptor site necessary for the transmission of a cytotoxic signal into the cell after the ligands' interaction with the binding site. This study may help in the development of therapeutic approaches to regulate the activity of the cytokine TNF.
Chromatin architecture regulates gene expression and shapes cellular identity, particularly in neuronal cells. Specifically, polycomb group (PcG) proteins enable establishment and maintenance of ...neuronal cell type by reorganizing chromatin into repressive domains that limit the expression of fate-determining genes and sustain distinct gene expression patterns in neurons. Here, we map the 3D genome architecture in neuronal and non-neuronal cells isolated from the Wernicke's area of four human brains and comprehensively analyze neuron-specific aspects of chromatin organization. We find that genome segregation into active and inactive compartments is greatly reduced in neurons compared to other brain cells. Furthermore, neuronal Hi-C maps reveal strong long-range interactions, forming a specific network of PcG-mediated contacts in neurons that is nearly absent in other brain cells. These interacting loci contain developmental transcription factors with repressed expression in neurons and other mature brain cells. But only in neurons, they are rich in bivalent promoters occupied by H3K4me3 histone modification together with H3K27me3, which points to a possible functional role of PcG contacts in neurons. Importantly, other layers of chromatin organization also exhibit a distinct structure in neurons, characterized by an increase in short-range interactions and a decrease in long-range ones.
CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell ...regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines. Despite a substantial difference in the proliferative activities of cell populations with different levels of CD133 expression, transcriptomic and proteomic profiling revealed only minor distinctions between them. Nonetheless, a further in silico assessment of the differentially expressed transcripts and proteins revealed 16 proteins that could be involved in the regulation of CD133 expression; these were assigned ranks reflecting the apparent extent of their involvement. Among them, the TRIM28 transcription factor had the highest rank. The prominent role of TRIM28 in CD133 expression modulation was confirmed experimentally in the Caco2 cell line clones: the knockout, though not the knockdown, of the TRIM28 gene downregulated CD133. These results for the first time highlight an important role of the TRIM28 transcription factor in the regulation of CD133-associated cancer cell heterogeneity.
During evolution, viruses had to adapt to an increasingly complex environment of eukaryotic cells. Viral proteins that need to enter the cell nucleus or associate with nucleoli possess nuclear ...localization signals (NLSs) and nucleolar localization signals (NoLSs) for nuclear and nucleolar accumulation, respectively. As viral proteins are relatively small, acquisition of novel sequences seems to be a more complicated task for viruses than for eukaryotes. Here, we carried out a comprehensive analysis of the basic domain (BD) of HIV-1 Tat to show how viral proteins might evolve with NLSs and NoLSs without an increase in protein size. The HIV-1 Tat BD is involved in several functions, the most important being the transactivation of viral transcription. The BD also functions as an NLS, although it is substantially longer than a typical NLS. It seems that different regions in the BD could function as NLSs due to its enrichment with positively charged amino acids. Additionally, the high positive net charge inevitably causes the BD to function as an NoLS through a charge-specific mechanism. The integration of NLSs and NoLSs into functional domains enriched with positively charged amino acids might be a mechanism that allows the condensation of different functional sequences in small protein regions and, as a result, reduces protein size, influencing the origin and evolution of NLSs and NoLSs in viruses.
Here, we investigated the molecular mechanism of nuclear localization signal (NLS) and nucleolar localization signal (NoLS) integration into the basic domain of HIV-1 Tat (
RKKRRQRRR
) and found that these two supplementary functions (i.e., function of NLS and function of NoLS) are embedded in the basic domain amino acid sequence. The integration of NLSs and NoLSs into functional domains of viral proteins enriched with positively charged amino acids is a mechanism that allows the concentration of different functions within small protein regions. Integration of NLS and NoLS into functional protein domains might have influenced the viral evolution, as this could prevent an increase in the protein size.
The clinical course of the new coronavirus disease 2019 (COVID-19) has shown that patients with chronic lymphocytic leukemia (CLL) are characterized by a high mortality rate, poor response to ...standard treatment, and low virus-specific antibody response after recovery and/or vaccination. To date, there are no data on the safety and efficacy of the combined vector vaccine Sputnik V in patients with CLL. Here, we analyzed and compared the magnitudes of the antibody and T cell responses after vaccination with the Sputnik V vaccine among healthy donors and individuals with CLL with different statuses of preexposure to coronavirus. We found that vaccination of the COVID-19-recovered individuals resulted in the boosting of pre-existing immune responses in both healthy donors and CLL patients. However, the COVID-19-naïve CLL patients demonstrated a considerably lower antibody response than the healthy donors, although they developed a robust T cell response. Regardless of the previous infection, the individuals over 70 years old demonstrated a decreased response to vaccination, as did those receiving anti-CD20 therapy. In summary, we showed that Sputnik V, like other vaccines, did not induce a robust antibody response in individuals with CLL; however, it provided for the development of a significant anti-COVID-19 T cell response.
Focal adhesions (FAs) are mechanosensory structures that transform physical stimuli into chemical signals guiding cell migration. Comprehensive studies postulate correlation between the FA parameters ...and cell motility metrics for individual migrating cells. However, which properties of the FAs are critical for epithelial cell motility in a monolayer remains poorly elucidated. We used high-throughput microscopy to describe relationship between the FA parameters and cell migration in immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) with depleted or inhibited vinculin and focal adhesion kinase (FAK) FA proteins. To evaluate relationship between the FA morphology and cell migration, we used substrates with varying stiffness in the model of wound healing. Cells cultivated on fibronectin had the highest FA area values, migration rate, and upregulated expression of FAK and vinculin mRNAs, while the smallest FA area and slower migration rate to the wound were specific to cells cultivated on glass. Suppression of vinculin expression in both normal and tumor cells caused decrease of the FA size and fluorescence intensity but did not affect cell migration into the wound. In contrast, downregulation or inactivation of FAK did not affect the FA size but significantly slowed down the wound closure rate by both HaCaT and A549 cell lines. We also showed that the FAK knockdown results in the FA lifetime decrease for the cells cultivated both on glass and fibronectin. Our data indicate that the FA lifetime is the most important parameter defining migration of epithelial cells in a monolayer. The observed change in the cell migration rate in a monolayer caused by changes in expression/activation of FAK kinase makes FAK a promising target for anticancer therapy of lung carcinoma.
Microtubule (MT) inhibitors show anti-cancer activity in a wide range of tumors
and demonstrate high clinical efficacy. To date they are routinely included into many chemotherapeutic regimens. While ...the mechanisms of MT inhibitors' interactions with tubulin have been well-established, the relationship between their concentration and effect on neoplastic cells is not completely understood. The common notion is that tumor cells are most vulnerable during division and all MT inhibitors block them in mitosis and induce mitotic checkpoint-associated cell death. At the same time multiple evidence of more subtle effects of lower doses of MT inhibitors on cell physiology exist. The extent of efficacy of the low-dose MT inhibitor treatment and the mechanisms of resulting cell death currently present a critical issue in oncology. The prospect of MT inhibitor dose reduction is promising as protocols at higher concentration have multiple side effects. We assessed cell cycle changes and cell death induced by MT inhibitors (paclitaxel, nocodazole, and vinorelbine) on human lymphoid B-cell lines in a broad concentration range. All inhibitors had similar accumulation effects and demonstrated "trigger" concentrations that induce cell accumulation in G2/M phase. Concentrations slightly below the "trigger" promoted cell accumulation in sub-G1 phase. Multi-label analysis of live cells showed that the sub-G1 population is heterogeneous and may include cells that are still viable after 24 h of treatment. Effects observed were similar for cells expressing Tat-protein. Thus cell cycle progression and cell death are differentially affected by high and low MT inhibitor concentrations.
Prostate cancer (PC) diagnostics and treatment often present a challenging task due to cancer subtype heterogeneity and differential disease progression in patient subgroups. Hence, the critical ...issue is finding a reliable and sensitive diagnostic and prognostic PC marker, especially for cases of biopsies with low percentages of cancer cells. Isoform A of myosin 1C was shown to be expressed in PC cells and responsible for their invasive properties, however, its feasibility for diagnostic purposes remains to be elucidated.
To verify the role of myosin 1C isoform A mRNA expression as a putative prostate cancer marker we performed RT qPCR normalized by three reference genes (
) on PC3, RWPE-1, LNCaP and 22Rv1 cell lines. Myosin 1C isoform A detection specificity was confirmed by immunofluorescence staining, cancer and non-cancer prostate cell lines were immunophenotyped by flow cytometry.
Median normalized mRNA expression level of myosin 1C isoform A in PC cells (PC3 and 22Rv1) is two orders of magnitude higher compared to RWPE-1 cells, which functionally correspond to benign prostate cells. Myosin 1C isoform A expression allows PC cell detection even at a dilution ratio of 1:1000 cancer to non-cancer cells. At the protein level, the mean fluorescence intensity of myosin 1C isoform A staining in PC3 nuclei was only twice as high as in RWPE-1, while the immunophenotypes of both cell lines were similar (CD44+/CD90-/CD133-/CD57-/CD24+-).
We report a distinct difference in myosin 1C isoform A mRNA levels in malignant (PC3) and benign (RWPE-1) prostate cell lines and suggest a combination of three reference genes for accurate data normalization. For the first time we provide an immunophenotype comparison of RWPE-1 and PC3 cells and demonstrate that RT qPCR analysis of
using appropriate reference genes is sufficient for PC detection even in low-abundance cancer specimens.
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