Transcriptomic analysis of HEK293A cells with a CRISPR/Cas9-mediated TDP1 knockout.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a human DNA repair protein. It is a member of the phospholipase D family based on structural similarity. TDP1 is a key enzyme of the repair of stalled topoisomerase 1 (TOP1)-DNA complexes. Previously, with the CRISPR/Cas9 method, we obtained HEK293A cells with a homozygous knockout of the TDP1 gene and used the TDP1 knockout cells as a cellular model for studying mechanisms of action of an anticancer therapy. In the present work, we hypothesized that the TDP1 knockout would alter the expression of DNA repair-related genes. By transcriptomic analysis, we investigated for the first time the effect of the TDP1 gene knockout on genes' expression changes in the human HEK293A cell line. We obtained original data implying a role of TDP1 in other processes besides the repair of the DNA-TOP1 complex. Differentially expressed gene analysis revealed that TDP1 may participate in cell adhesion and communication, spermatogenesis, mitochondrial function, neurodegeneration, a cytokine response, and the MAPK signaling pathway.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

Circulating Ouabain Modulates Expression of Claudins in Rat Intestine and Cerebral Blood Vessels.

The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 µg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression.

Cholera toxin perturbs the paracellular barrier in the small intestinal epithelium of rats by affecting claudin-2 and tricellulin.

Cholera toxin is commonly known to induce chloride secretion of the intestine. In recent years, effects on epithelial barrier function have been reported, indicating synergistic co-regulation of transporters and tight junction proteins. Our current study focused on the analysis of cholera toxin effects on transepithelial resistance and on tight junction proteins, the latter known as structural correlates of barrier function. Ligated segments of the rat jejunum were injected with buffered solution containing cholera toxin (1 µg/ml) and incubated for 4 h. Subsequently, selfsame tissue specimens were mounted in Ussing chambers, and cholera toxin (1 µg/ml) was added on the apical side. Transepithelial resistance and permeability of sodium fluorescein (376 Da) were analyzed. Subsequently, tissues were removed, expression and localization of claudins were analyzed, and morphological studies were performed employing transmission electron microscopy and confocal laser scanning microscopy. Cholera toxin induced a marked decrease in transepithelial resistance in the rat jejunal epithelium and an increase in paracellular permeability for sodium fluorescein. Immunoblotting of tight junction proteins revealed an increase in claudin-2 signals, which was verified by confocal laser scanning immunofluorescence microscopy, and a decrease in tricellulin, whereas other tight junction proteins remained unchanged. Transmission electron microscopy showed a reduction in the number of microvilli after incubation with cholera toxin. Moreover, cholera toxin led to a widening of the intercellular space between enterocytes. In accordance with the commonly known prosecretory effect of cholera toxin, our study revealed a complementary effect on small intestinal barrier function and integrity, which might constitute a pathomechanism with high relevance for prevention and therapeutic approaches.

Increased paracellular permeability of tumor-adjacent areas in 1,2-dimethylhydrazine-induced colon carcinogenesis in rats.

OBJECTIVE: The morphology and functions of the proximal and distal large intestine are not the same. The incidence of colorectal cancer in these regions is also different, as tumors more often appear in the descending colon than in the ascending colon. Inflammatory bowel disease and colorectal cancer can increase transepithelial permeability, which is a sign of reduced intestinal barrier function. However, there is not enough evidence to establish a connection between the difference in colorectal cancer incidence in the proximal and distal colon and intestinal permeability or the effects of carcinogenesis on the barrier properties in various areas of the colon. The aim of the study was to assess the permeability of different segments of the large intestine according to a developed mapping methodology in healthy rats and rats with 1,2-dimethylhydrazine (DMH)-induced colon adenocarcinoma. METHODS: The short circuit current, the transepithelial electrical resistance and the paracellular permeability to fluorescein of large intestine wall of male Wistar rats were examined in the Ussing chambers. The optical density of the solution from the serosa side to assess the concentration of the diffused fluorescein from mucosa to serosa was analyzed by spectrophotometry. The morphometric and histological studies were performed by optical microscopy. RESULTS: Rats with DMH-induced colon adenocarcinomas showed elevated transepithelial electrical resistance in the areas of neoplasm development. In contrast, there was no change in the electrophysiological properties of tumor adjacent areas, however, the paracellular permeability of these areas to fluorescein was increased compared to the control rats and was characterized by sharply reduced barrier function. CONCLUSIONS: The barrier properties of the colon vary depending on tumor location. The tumors were less permeable than the intact intestinal wall and probably have a negative influence on tumor-adjacent tissues by disrupting their barrier function.

p38 MAP kinase enhances EGF-induced apoptosis in A431 carcinoma cells by promoting tyrosine phosphorylation of STAT1.

While epidermal growth factor (EGF) is a well known mitogen, high doses of EGF result in a paradoxical apoptotic response in the cells that overexpress EGF receptor such as A431 epidermoid carcinoma cells. EGF-induced apoptosis in A431 cells is dependent upon activation of transcription factor STAT1. In this study, we demonstrate that p38 MAP kinase is another important mediator of EGF-dependent pro-apoptotic response in A431 cells. By utilizing p38 MAP kinase inhibitors, SB203580 and BIRB0796, we significantly reduced the integral growth-inhibiting as well as pro-apoptotic effects of EGF. Moreover, we observed that inhibition of p38 MAP kinase markedly decreased phosphorylation of tyrosine 701 in STAT1, while neither EGF-induced accumulation nor serine phosphorylation of STAT1 was decreased. We propose that p38 MAP kinase mediates STAT1 tyrosine phosphorylation, thereby enforcing EGF-induced apoptosis.