Liver cancer is one of the most threatening diseases in Chinese population. Just like in other tissues, tumor initiation and development in liver involve multiple steps of genetic and epigenetic alterations with sever...Liver cancer is one of the most threatening diseases in Chinese population. Just like in other tissues, tumor initiation and development in liver involve multiple steps of genetic and epigenetic alterations with several unknown details. However, unlike in other tissues, a tissue specific inducible Cre recombinase system that allows temporal and spatial deletion of a target DNA fragment is still not available for in vivo functional gene annotation in hepatocytes. In our pursuit to establish such a mouse model, we designed a dual inducible Cre transgene system and tested it in cultured cells. By combining a CCAAT/enhancer binding protein β （C/EBP β） promoter derived Tet-off expression system and the estrogen receptor （ER） mediated functional control, we show a desirable profile of both hepatocyte-specificity and regulability of the Cre expression in a series of critical assessments in the cell culture system, which provides confidence in continuation of our ongoing pursuit in mouse.展开更多
Aim:The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen(HT)on their sensitivity to tubulin polymerization inhibitor docetaxel.Methods:The anal...Aim:The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen(HT)on their sensitivity to tubulin polymerization inhibitor docetaxel.Methods:The analysis of cell viability was performed by the MTT method.The expression of signaling proteins was analyzed by immunoblotting and flow cytometry.ERαactivity was evaluated by gene reporter assay.To establish hormone-resistant subline MCF7,breast cancer cells were treated with 4-hydroxytamoxifen for 12 months.Results:The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen,and the resistance index was 2.Increased Akt activity(2.2-fold)and decreased ERαexpression(1.5-fold)were revealed in MCF7/HT cells.The activity of the estrogen receptorαwas reduced(1.5-fold)in MCF7/HT.Evaluation of class Ⅲβ-tubulin expression(TUBB3),a marker associated with metastasis,revealed the following trends:higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells(P<0.05).The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells(MCF7/HT<MCF7<MDA-MB-231,approximately 1:2:4).High TUBB3 expression strongly correlated with docetaxel resistance:IC_(50)value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells,whereas resistant MCF7/HT cells were the most sensitive to the drug.The accumulation of cleaved PARP(a 1.6-fold increase)and Bcl-2 downregulation(1.8-fold)were more pronounced in docetaxel-treated resistant cells(P<0.05).The expression of cyclin D1 decreased(2.8-fold)only in resistant cells after 4 nM docetaxel treatment,while this marker was unchanged in parental MCF7 breast cancer cells.Conclusion:Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising,especially for cancers with low TUBB3 expression.展开更多
基金supported by Shanghai Science Foundation grants,National Science Foundation of China(No.30570850 and 10574134)National Research Program for Basic Research of China(No.2004CB518804)+1 种基金National Research Program for High Technology(No.2006AA02Z-320 and 2006AA 02Z197)European 6th Program(LSHBCT-2005-019067).
文摘Liver cancer is one of the most threatening diseases in Chinese population. Just like in other tissues, tumor initiation and development in liver involve multiple steps of genetic and epigenetic alterations with several unknown details. However, unlike in other tissues, a tissue specific inducible Cre recombinase system that allows temporal and spatial deletion of a target DNA fragment is still not available for in vivo functional gene annotation in hepatocytes. In our pursuit to establish such a mouse model, we designed a dual inducible Cre transgene system and tested it in cultured cells. By combining a CCAAT/enhancer binding protein β （C/EBP β） promoter derived Tet-off expression system and the estrogen receptor （ER） mediated functional control, we show a desirable profile of both hepatocyte-specificity and regulability of the Cre expression in a series of critical assessments in the cell culture system, which provides confidence in continuation of our ongoing pursuit in mouse.
基金supported by the Ministry of Science and Higher Education of the Russian Federation(agreement No.075-15-2020-789).
文摘Aim:The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen(HT)on their sensitivity to tubulin polymerization inhibitor docetaxel.Methods:The analysis of cell viability was performed by the MTT method.The expression of signaling proteins was analyzed by immunoblotting and flow cytometry.ERαactivity was evaluated by gene reporter assay.To establish hormone-resistant subline MCF7,breast cancer cells were treated with 4-hydroxytamoxifen for 12 months.Results:The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen,and the resistance index was 2.Increased Akt activity(2.2-fold)and decreased ERαexpression(1.5-fold)were revealed in MCF7/HT cells.The activity of the estrogen receptorαwas reduced(1.5-fold)in MCF7/HT.Evaluation of class Ⅲβ-tubulin expression(TUBB3),a marker associated with metastasis,revealed the following trends:higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells(P<0.05).The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells(MCF7/HT<MCF7<MDA-MB-231,approximately 1:2:4).High TUBB3 expression strongly correlated with docetaxel resistance:IC_(50)value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells,whereas resistant MCF7/HT cells were the most sensitive to the drug.The accumulation of cleaved PARP(a 1.6-fold increase)and Bcl-2 downregulation(1.8-fold)were more pronounced in docetaxel-treated resistant cells(P<0.05).The expression of cyclin D1 decreased(2.8-fold)only in resistant cells after 4 nM docetaxel treatment,while this marker was unchanged in parental MCF7 breast cancer cells.Conclusion:Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising,especially for cancers with low TUBB3 expression.
