Within the quick treatment experiment, the viabil ity of EGFP p31 overexpressing HeLa cells that had been treated with nocodazole or taxol was dramatically ele vated, however the identical observation was not created with monastrol, These benefits indicated that p31 overexpression induced the resistance to micro tubule poisons by inactivating the Mad2 dependent SAC directly. p53 independent adaptation by p31 overexpression Mad2 and p53 double knockout mice and embryonic fibroblast cells were viable and exhibited chromosomal instability, even though Mad2 single knockout mice were lethal, These studies indicated that p53 protein guarded the chromo somal loss and or achieve with SAC machinery. To address the p53 dependency in aneuploidy from p31 overex pression, p31 was overexpressed in HCT116 cells, which are colorectal carcinoma cells, and also the p53 dependent checkpoint was functional.
Simply because p53 pro tein is much less or not expressed in HeLa cells compared with typical cells, p31 overexpressing HeLa cells that have been treated with anti mitotic drugs had been capable to more than ride SAC like Mad2 and p53 double knockout mice along with the cells, Interestingly, p31 more than expressing HCT116 cells in the presence of nocodazole had been also capable to lead aneuploidy like HeLa cells with equivalent inhibitor SB 431542 kinetics, H1299 cells, that are non little cell lung cancer cells do not express p53 protein, have been made use of to overexpress p31 within the presence of anti mitotic drugs. Working with these cells, the overexpression of p31 didn’t override the nocodazole induced SAC, but it could override taxol induced SAC within a comparable manner like HeLa and HCT116 cells. These final results recommended that cells overexpressing p31 within the presence of spindle poisons exit mitosis within a p53 independent adaptation pathway.
Expression of p31 in cancer cell lines and resistance against taxol The overexpression of p31 contributed to aneu ploidy and resistance to anti mitotic drugs. To address p31 function with respect to drug sensitivity, we ob served the expression level of p31 in numerous cancer cell lines, Cycling cells had been TG100115 subjected to western blotting analysis, and monitored the p31, Mad2, and APC2 protein levels. The protein levels of Mad2 and p31 in the indicated cell lines showed fantastic variations. Quantitative evaluation on the Mad2 and p31 protein expression was performed working with the in tensity on the APC2 loading control as a regular. Each protein level was normalized for the expression level in HeLa cells, The quantitative p31 Mad2 expression ratios had been shown in Figure 6a. In U2OS, PC3, and HepG2 cells, the p31 Mad2 expression level ratio was greater than in other cell lines. Yet, in HEK293 and HT 29 cells, the p31 Mad2 expression level ratio was reduced, though the p31 signal was not detected in HT 29 cells. In A549, HCT116, DLD 1, MCF7, and SK N SH cells, the p31 Mad2 expression level ratio was equal to in HeLa cells.