Применение тригенерационной биогазовой установки, работающей на отходах мясопереработки (на примере ОАО «Калинковичский мясокомбинат»)

Материалы XIV Междунар. науч.-техн. конф. студентов, аспирантов и молодых ученых, Гомель, 24–25 апр. 2014 г

GABA-A Channel Subunit Expression in Human Glioma Correlates with Tumor Histology and Clinical Outcome

GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the CNS and is present in high concentrations in presynaptic terminals of neuronal cells. More recently, GABA has been ascribed a more widespread role in the control of cell proliferation during development where low concentrations of extrasynaptic GABA induce a tonic activation of GABA receptors. The GABA-A receptor consists of a ligand-gated chloride channel, formed by five subunits that are selected from 19 different subunit isoforms. The functional and pharmacological properties of the GABA-A channels are dictated by their subunit composition. Here we used qRT-PCR to compare mRNA levels of all 19 GABA-A channel subunits in samples of human glioma (n = 29) and peri-tumoral tissue (n = 5). All subunits except the ρ1 and ρ3 subunit were consistently detected. Lowest mRNA levels were found in glioblastoma compared to gliomas of lower malignancy, except for the θ subunit. The expression and cellular distribution of the α1, γ1, ρ2 and θ subunit proteins was investigated by immunohistochemistry on tissue microarrays containing 87 gliomas grade II. We found a strong co-expression of ρ2 and θ subunits in both astrocytomas (r = 0.86, p<0.0001) and oligodendroglial tumors (r = 0.66, p<0.0001). Kaplan-Meier analysis and Cox proportional hazards modeling to estimate the impact of GABA-A channel subunit expression on survival identified the ρ2 subunit (p = 0.043) but not the θ subunit (p = 0.64) as an independent predictor of improved survival in astrocytomas, together with established prognostic factors. Our data give support for the presence of distinct GABA-A channel subtypes in gliomas and provide the first link between specific composition of the A-channel and patient survival

Transcription factors PROX1 and p53 in cancer development

The majority of malignant brain tumors in adults are astrocytic gliomas. These are classified into four malignancy grades according to the World Health Organization (WHO) criteria, including grade I pilocytic astrocytoma, grade II astrocytoma, grade III anaplastic astrocytoma and grade IV glioblastoma. Understanding the underlying molecular defects of these tumors gives us the possibility to design new effective therapies. PROX1 is a transcription factor that has an important role in the developmental process of various organs during embryogenesis, including the central nervous system. In paper I we examined a group of 56 paraffin embedded astrocytic brain tumors for their expression of PROX1. We found that the number of PROX1+ cells was correlated to high tumor grade. We concluded that PROX1 maybe used as a diagnostic tool distinguishing between grade III, IV tumors and grade II astrocytomas. This finding led us to investigate PROX1 s prognostic and predictive value in grade II tumors, because these tumors have a highly unpredictable course of progression to a more malignant grade, which hinders the clinicians choice of treatment. In paper II we studied the expression levels of PROX1 in 128 grade II astrocytic brain tumors and found that it is a highly dependable, predictive factor for short term survival in patients with astrocytic gliomas but not for oligodendrogliomas. We propose that in the future PROX1 maybe used in the clinical routine as a biomarker to help in prediction of prognosis for astrocytic brain tumors. In order to understand why PROX1 is up regulated in these tumors, we determined its expression in different established glioma cell lines, to serve as a tool for the functional studies of the protein (paper III). A hallmark of glioblastoma is mutations in the p53 tumor suppressor gene. p53 is also frequently mutated in other tumors including those from lung, breast and skin. In paper IV we used a mouse skin model, p53QS-val135/QS-val135 (p53QS), to examine the importance of p53 transcriptional regulatory function in tumor suppression. This model contains a double mutationin the N-terminus of p53 abrogating the transactivation domain and a modification at amino acid 135 partially affecting DNA binding. Ras oncogene-induced senescence was lost in both p53QS and p53-/- keratinocytes. Likewise, p53QS, similar to p53-/-, cooperated with v-rasHa to enhance malignant conversion. The tumors arising in p53QS keratinocytes displayed strong nuclear p53 expression, thus the p53QS-val135 allele was maintained during tumor formation. While p53-/- keratinocytes displayed diminished response to TGF-beta, p53QS and p53wt keratinocytes responded equivalently, indicating that the requirement of p53 for maximizing TGF-beta-mediated growth regulation is independent of its transactivation domain and that TGF-beta-mediated growth regulation is not required for p53 mediated tumor suppression in the skin

NPM1 histone chaperone is upregulated in glioblastoma to promote cell survival and maintain nucleolar shape

Glioblastoma (grade IV glioma) is the most common and aggressive adult brain tumor. A better understanding of the biology of glioblastoma cells is crucial to identify molecular targets stimulating cell death. NPM1 (nucleophosmin) is a multifunctional chaperone that plays an important role in cancer development. Herein, NPM1 was analyzed by immunohistochemistry in human astrocytic gliomas. NPM1 was detected in all tumors but with a significantly higher staining intensity in grade IV than in low grade tumors. Depletion of NPM1 had only modest effects on the viability of U251MG, U1242MG, and U343MGa Cl2:6 glioma cells, despite alterations in nucleolar morphology. Glioma cell cultures depleted of NPM1 exposed to micromolar levels of actinomycin D were more prone to cell death (apoptosis) compared to cultures retaining NPM1. We had previously found that NPM1 binds to linker histone H1.5. Here we could show that silencing of H1.5 triggered glioma cell apoptosis as evidenced by a marked increase in both the numbers of cleaved caspase-3(+) cells and in the amounts of cleaved PARP. Enforced expression of NPM1 suppressed apoptosis in H1.5 depleted glioma cells. Although our studies would suggest little effectiveness of targeting NPM1 alone there could be potential using it as a combination treatment

PROX1 is a novel pathway-specific prognostic biomarker for high-grade astrocytomas; results from independent glioblastoma cohorts stratified by age and IDH mutation status

PROX1 is a transcription factor with an essential role in embryonic development and determination of cell fate. In addition, PROX1 has been ascribed suppressive as well as oncogenic roles in several human cancers, including brain tumors. In this study we explored the correlation between PROX1 expression and patient survival in high-grade astrocytomas. For this purpose, we analyzed protein expression in tissue microarrays of tumor samples stratified by patient age and IDH mutation status. We initially screened 86 unselected high-grade astrocytomas, followed by 174 IDH1-R132H1 immunonegative glioblastomas derived from patients aged 60 years and older enrolled in the Nordic phase III trial of elderly patients with newly diagnosed glioblastoma. Representing the younger population of glioblastomas, we studied 80 IDH-wildtype glioblastomas from patients aged 18-60 years. There was no correlation between PROX1 protein and survival for patients with primary glioblastomas included in these cohorts. In contrast, high expression of PROX1 protein predicted shorter survival in the group of patients with IDH-mutant anaplastic astrocytomas and secondary glioblastomas. The prognostic impact of PROX1 in IDH-mutant 1p19q non-codeleted high-grade astrocytomas, as well as the negative findings in primary glioblastomas, was corroborated by gene expression data extracted from the Cancer Genome Atlas. We conclude that PROX1 is a new prognostic biomarker for 1p19q non-codeleted high-grade astrocytomas that have progressed from pre-existing low-grade tumors and harbor IDH mutations

Illustration of the immunohistochemistry results in astrocytomas grade II (n = 42), and in oligodendrogliomas (n = 27) plus oligoastrocytomas grade II (n = 18).

<p>Positive immunoreactivity for each of the four different GABA-A channel subunits is visualized by a colored bar. Each number represents one tumor sample.</p

Photomicrographs of a gemistocytic astrocytoma grade II showing no immunoreactivity for: A) and A′) GABA-A channel α1 subunits, B) and B′) γ1 subunits, but positive immunoreactivity for: C) and C′) ρ2 subunits and D) and D′) θ subunits in a proportion of the tumor cells.

<p>(The scale bar shown in A represents respectively 200 µm in A, B, C, D; 30 µm in A′, B′, C′, D′).</p

Photomicrographs of an oligodendroglioma grade II showing positive immunoreactivity for: A) GABA-A channel α1 subunit, and B) γ1 subunit.

<p>The histological markers used for identification of specific cell types, CD34, IDH1, Ki67 and neuronal nuclear antigen (NeuN) illustrate respectively, C) haematopoetic cells in the tumor, D) accumulation of mutated IDH1 protein in tumor cells, E) a low percentage of proliferating tumor cells, F) an entrapped neuron in the tumor. (The scale bar shown in A represents 50 µm in A, B, F; 80 µm in C, D, E).</p

Results of immunostaining for GABA-A channel subunits on gliomas (n = 91).

*<p>0 = No immunoreactive tumor cells; + = <10% immunoreactive tumor cells; ++ = ≥10% immunoreactive tumor cells.</p

Summary of the qRT-PCR results showing mRNA levels of 17 different GABA-A subunits in gliomas grade II (n = 12), gliomas grade III (n = 10) and glioblastomas (n = 7).

<p>The normalized mRNA expression of each target gene relative to a reference gene TATA-binding protein (<i>TBP</i>) was calculated using the 2^−ΔCt method.</p