Hypoxia controls the expression of genes responsible for serine synthesis in U87MG cells on ERN1-dependent manner.

Objective. Serine synthesis as well as endoplasmic reticulum stress and hypoxia are important factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of ERN1 (endoplasmic reticulum to nucleus signaling) significantly suppressed the glioblastoma cell proliferation and modified the hypoxia regulation. The present study is aimed to investigate the impact of hypoxia on the expression of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine aminotransferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) in U87MG glioblastoma cells in relation to knockdown of ERN1 with the intent to reveal the role of ERN1 signaling pathway on the endoplasmic reticulum stress-dependent regulation of expression of these genes. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed to hypoxia introduced by dimethyloxalylglycine for 4 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH, PSAT1, PDPH, SHMT1, and ATF4 genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that hypoxia up-regulated the expression level of PHGDH, PSAT1, and ATF4 genes in control U87MG cells, but PSPH and SHMT1 genes expression was down-regulated. The expression of PHGDH, PSAT1, and ATF4 genes in glioblastoma cells with knockdown of ERN1 signaling protein was more sensitive to hypoxia, especially PSAT1 gene. At the same time, the expression of PSPH gene in ERN1 knockdown cells was resistant to hypoxia. The expression of SHMT1 gene, encoding the enzyme responsible for conversion of serine to glycine, showed similar negative sensitivity to hypoxia in both control and ERN1 knockdown glioblastoma cells. Conclusion. The results of the present study demonstrate that the expression of genes responsible for serine synthesis is sensitive to hypoxia in gene-specific manner and that ERN1 knockdown significantly modifies the impact of hypoxia on the expression of PHGDH, PSAT1, PSPH, and ATF4 genes in glioblastoma cells and reflects the ERN1-mediated reprograming of hypoxic regulation at gene expression level.

Expression of DNAJB9 and some other genes is more sensitive to SWCNTs in normal human astrocytes than glioblastoma cells.

Objective. Single-walled carbon nanotubes (SWCNTs) are considered to be one of the nanomaterials attractive for biomedical applications, particularly in the health sciences as imaging probes and drug carriers, especially in the field of cancer therapy. The increasing exploitation of nanotubes necessitates a comprehensive evaluation of the potential impact of these nanomaterials, which purposefully accumulate in the cell nucleus, on the human health and the function of the genome in the normal and tumor tissues. The aim of this study was to investigate the sensitivity of the expression of DNAJB9 and some other genes associated with the endoplasmic reticulum (ER) stress and cell proliferation to low doses of SWCNTs in normal human astrocytes (NHA/TS) and glioblastoma cells (U87MG) with and without an inhibition of ERN1 signaling pathway of the ER stress. Methods. Normal human astrocytes, line NHA/TS and U87 glioblastoma cells stable transfected by empty vector or dnERN1 (dominant-negative construct of ERN1) were exposed to low doses of SWCNTs (2 and 8 ng/ml) for 24 h. RNA was extracted from the cells and used for cDNA synthesis. The expression levels of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, CLU, and P4HA2 mRNAs were measured by a quantitative polymerase chain reaction and normalized to ACTB mRNA. Results. It was found that the low doses of SWCNTs up-regulated the expression of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, CLU, and P4HA2 genes in normal human astrocytes in dose-dependent (2 and 8 ng/ml) and gene-specific manner. These nanotubes also increased the expression of most studied genes in control (transfected by empty vector) U87 glioblastoma cells, but with much lesser extent than in NHA/TS. However, the expression of CLU gene in control U87 glioblastoma cells treated with SWCNTs was down-regulated in a dose-dependent manner. Furthermore, the expression of TOB1 and P4HA2 genes did not significantly change in these glioblastoma cells treated by lower dose of SWCNTs only. At the same time, inhibition of ERN1 signaling pathway of ER stress in U87 glioblastoma cells led mainly to a stronger resistance of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, and P4HA2 gene expression to both doses of SWCNTs. Conclusion. The data obtained demonstrate that the low doses of SWCNTs disturbed the genome functions by changing the levels of key regulatory gene expressions in gene-specific and dose-dependent manner, but their impact was much stronger in the normal human astrocytes in comparison with the tumor cells. It is possible that ER stress, which is constantly present in tumor cells and responsible for multiple resistances, also created a partial resistance to the SWCNTs action. Low doses of SWCNTs induced more pronounced changes in the expression of diverse genes in the normal human astrocytes compared to glioblastoma cells indicating for a possible both genotoxic and neurotoxic effects with a greater extent in the normal cells.

Glucose deprivation affects the expression of genes encoding cAMP-activated protein kinase and related proteins in U87 glioma cells in ERN1 dependent manner.

Objective. The aim of this investigation was to study the expression of genes encoding cAMP-activated protein kinase catalytic and regulatory A subunits (PRKACA and PRKAR1A) and related proteins such as cAMP-dependent protein kinase inhibitors A and G (PKIA and PKIG), catalytic subunit A of protein phosphatase 3 (PPP3CA), A-kinase anchoring protein 12 (AKAP12), and praja ring finger ubiquitin ligase 2 (PJA2) in U87 glioma cells in response to glucose deprivation in both control U87 glioma cells and cells with ERN1 (endoplasmic reticulum to nucleus signaling 1) knockdown, the major pathway of the endoplasmic reticulum stress signaling, for evaluation of possible significance of glucose deprivation in ERN1 dependent regulation of glioma growth.Methods. The expression level of PRKA related genes was studied in control (transfected by vector) and ERN1 knockdown U87 glioma cells under glucose deprivation by real-time quantitative polymerase chain reaction.Results. It was shown that the expression level of PRKACA and PKIA genes was down-regulated in control glioma cells treated by glucose deprivation, but PJA2 gene was up-regulated. At the same time, the expression of four other genes (PRKAR1A, PKIG, AKAP12, and PPP3CA) was resistant to this experimental condition. Furthermore, ERN1 knockdown of glioma cells significantly modified the effect glucose deprivation on the expression almost all studied genes. Thus, treatment of glioma cells with inhibited ERN1 enzymatic activity by glucose deprivation lead to a more significant down-regulation of the expression level of PKIA and to suppression PRKAR1A gene expressions. Moreover, the ERN1 knockdown introduced up-regulation of PKIG and AKAP12 gene expressions in glioma cells treated by glucose deprivation and eliminated the sensitivity of PJA2 gene to this experimental condition.Conclusions. Results of this investigation demonstrated that ERN1 knockdown significantly modified the sensitivity of most studied PRKA related gene expressions to glucose deprivation and that these changes are a result of complex interactions of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the suppression of glioma cell proliferation and their possibly chemoresistance.