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We examined the role of Tie2 in regulating wound healing after tooth extraction. Wistar rats underwent maxillary incisor tooth extraction, and immunodetection techniques were used to determine Tie2 expression in the healing wound. The wound was initially filled with blood coagulum containing densely aggregated erythrocytes, leukocytes, fibrin, and endothelial progenitor cells, indicating that blood vessel formation started in the socket. Tie2 was detected on monocytic cell membranes. On day 3, fibroblastic cells proliferated in the coagulum, small vessels appeared by day 5, and new bone formed in the vessel-rich area. Robust woven bone trabeculae were present around vessels by day 7, and woven bone and osteoclast-like giant cells were present on day 10. Woven bone surrounded sinusoidal capillary-like vessels. Full-length (140-160 kDa) Tie2 was not detected at any time, although Tie2 fragments were present in the healing wound. N-terminus- and C-terminus-specific Tie2 antibodies detected 40-kDa and 60-kDa fragments or 70-kDa and 50-kDa fragments, respectively. The levels of these fragments decreased during the first 3 days and started to increase by day 5-10. The Tie2 extracellular domain initially inhibited angiogenesis, and its degradation relieved inhibition of new vessel formation. The onset of vessel formation in the wound may be induced by scattered endothelial progenitor cells. [ABSTRACT FROM AUTHOR]

In recent years, it has been shown that Z-DNA formation in DNA plays functionally significant roles in nucleic acid metabolism, such as gene expression, chromosome recombination, and epigenetic regulation. The reason for the identification of these effects is mainly due to the advancement of Z-DNA detection methods in target genome regions in living cells.The heme oxygenase-1 (HO-1) gene encodes an enzyme that degrades an essential prosthetic heme, and environmental stimuli, including oxidative stress, lead to robust induction of the HO-1 gene. Many DNA elements and transcription factors are involved in the induction of the HO-1 gene, and Z-DNA formation in the thymine-guanine (TG) repetitive sequence in the human HO-1 gene promoter region is required for maximum gene induction.Here, we describe a detailed protocol for Z-DNA detection in the human HO-1 gene promoter region based on chromatin immunoprecipitation with quantitative PCR. We also provide some control experiments to consider in routine lab procedures., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignant disease that is resistant to various chemotherapeutic agents and commonly relapses. Efficient elimination of metastasized PDA is critical for a positive post-surgical treatment outcome. The present study analyzed the effect of the B-cell lymphoma-2 (Bcl-2)/B-cell lymphoma extra-large (Bcl-x L ) inhibitor, ABT-737, on paclitaxel-induced PDA cell death. A total of 8 PDA cell lines were subjected to immunoblotting to compare the expression of Bcl-2/Bcl-x L and other factors associated with taxane resistance, including myeloid cell leukemia 1 and βIII-tubulin (TUBB3). The viability of PDA cells was analyzed following treatment with paclitaxel alone or a combination treatment with ABT-737 and paclitaxel. Treatment with the ABT-737/paclitaxel combination induced PDA cell death at a lower concentration of paclitaxel compared with paclitaxel alone. In addition, the viable cell population at the saturation point of paclitaxel was also decreased by co-treatment with ABT-737. ABT-737 lowered the half maximal inhibitory concentration (IC 50 ) by >2-fold in PDA cells with high Bcl-2/Bcl-x L expression, but not in PDA cells with low Bcl-2/Bcl-x L expression and high TUBB3 expression. Knockdown of Bcl-x L lowered the IC 50 of paclitaxel, but knockdown of TUBB3 did not. ABT-737 sensitized PDA to paclitaxel-induced cell death, and Bcl-x L expression was a key determinant of its sensitivity. ABT-737 is potential candidate for combination chemotherapy of PDA with high Bcl-x L expression levels.

The expression of βIII-tubulin (TUBB3) is generally restricted to neurons, but its mRNA is often expressed at low levels in non-neuronal cells. Interestingly, however, a number of non-neural tumors occasionally express high levels of TUBB3 protein, leading to a significant resistance to taxane derivatives. However, the molecular mechanisms controlling TUBB3 expression and its turnover during normal cell growth are largely unknown. Here, we present evidence that TUBB3 expression occurs in a cell cycle-dependent manner, and that its protein levels are controlled by the ubiquitin-proteasome system. Both mRNA and protein of TUBB3 accumulated around the G2/M stage of the cell cycle, and reduction of TUBB3 expression by siRNA resulted in partial inhibition of cell growth. Furthermore, the cell cycle-dependent expression of TUBB3 was mediated by the RE-1-silencing transcription factor REST through its binding to the RE-1 element that is present in the first intron of the TUBB3 gene. These results demonstrate a novel role of TUBB3 in cell cycle progression in non-neuronal cells, and further suggest that dysregulation of the REST-TUBB3 system could be a primary cause of the TUBB3 overexpression.