Mitosis-targeted anti-cancer therapies gained much attention in recent years. However, lack of tumor selectivity poses limitations to the current anti-mitotic drugs to be used as broad-spectrum anti-cancer agents. In ...Mitosis-targeted anti-cancer therapies gained much attention in recent years. However, lack of tumor selectivity poses limitations to the current anti-mitotic drugs to be used as broad-spectrum anti-cancer agents. In this study, we show that combination treatment of colcemid, an inhibitor of microtubule polymerization with geldanamycin, an inhibitor of cancer chaperone, Hsp90 irreversibly targets mitosis through mitotic kinase bubR1 stabilization. When the individual and combination drugs treatments were tested against tumor cells (IMR-32 and HeLa) and non-tumor cells (SRA01), the combination treatment showed significant increase in cytotoxicity only in tumor cells followed by G2/M cell cycle block. The IMR-32 cells showed enhanced cytotoxicity in response to combination treatments, compared to HeLa cells. Further studies revealed that the G2/M arrest in IMR-32 correlates with both increased bubR1 nuclear localization and metaphase arrest. The siRNA knockdown of bubR1 has decreased tumor cell response to geldanamycin suggesting Hsp90-dependent regulation of bubR1. The combination treatment also showed inactivation of non-canonical β-catenin signaling suggesting inhibition of cancer growth. In addition, the combination treatment has significantly affected the distribution and functions of bubR1 downstream mitotic kinases such as aurks and plk1 indicating the combinatorial attack of combination treatment. In conclusion, we demonstrate that colcemid and GA combination treatment compromises the division potential of tumor cells interfering with the mitosis through bubR1 kinase.展开更多
目的研究胃癌组织中有丝分裂关卡基因BUB1和BUBR1的表达。方法采用半定量RT-PCR技术检测39例胃癌组织及正常胃组织中BUB1及BUBR1基因的表达。结果胃癌组织中BUB1mRNA/β-actin mRNA和BUBR1mRNA/β-actin mRNA拷贝数比值均明显高于正常...目的研究胃癌组织中有丝分裂关卡基因BUB1和BUBR1的表达。方法采用半定量RT-PCR技术检测39例胃癌组织及正常胃组织中BUB1及BUBR1基因的表达。结果胃癌组织中BUB1mRNA/β-actin mRNA和BUBR1mRNA/β-actin mRNA拷贝数比值均明显高于正常胃组织(1.84±0.43 vs. 0.86±0.44和2.00±1.36 vs. 0.96±0.60)(P<0.05)。结论 BUB1基因和BUBR1基因在胃癌中高表达可能与癌细胞的异常增殖有关。展开更多
Protein phosphatase 2A （PP2A） accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a cat- alyti...Protein phosphatase 2A （PP2A） accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a cat- alytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kine- tochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interac- tion plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attach- ment. Here we present the crystal structure of a PP2A B56-BubRI complex, which demonstrates that a con- served BubRl LxxlxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxlxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.展开更多
文摘Mitosis-targeted anti-cancer therapies gained much attention in recent years. However, lack of tumor selectivity poses limitations to the current anti-mitotic drugs to be used as broad-spectrum anti-cancer agents. In this study, we show that combination treatment of colcemid, an inhibitor of microtubule polymerization with geldanamycin, an inhibitor of cancer chaperone, Hsp90 irreversibly targets mitosis through mitotic kinase bubR1 stabilization. When the individual and combination drugs treatments were tested against tumor cells (IMR-32 and HeLa) and non-tumor cells (SRA01), the combination treatment showed significant increase in cytotoxicity only in tumor cells followed by G2/M cell cycle block. The IMR-32 cells showed enhanced cytotoxicity in response to combination treatments, compared to HeLa cells. Further studies revealed that the G2/M arrest in IMR-32 correlates with both increased bubR1 nuclear localization and metaphase arrest. The siRNA knockdown of bubR1 has decreased tumor cell response to geldanamycin suggesting Hsp90-dependent regulation of bubR1. The combination treatment also showed inactivation of non-canonical β-catenin signaling suggesting inhibition of cancer growth. In addition, the combination treatment has significantly affected the distribution and functions of bubR1 downstream mitotic kinases such as aurks and plk1 indicating the combinatorial attack of combination treatment. In conclusion, we demonstrate that colcemid and GA combination treatment compromises the division potential of tumor cells interfering with the mitosis through bubR1 kinase.
文摘目的研究胃癌组织中有丝分裂关卡基因BUB1和BUBR1的表达。方法采用半定量RT-PCR技术检测39例胃癌组织及正常胃组织中BUB1及BUBR1基因的表达。结果胃癌组织中BUB1mRNA/β-actin mRNA和BUBR1mRNA/β-actin mRNA拷贝数比值均明显高于正常胃组织(1.84±0.43 vs. 0.86±0.44和2.00±1.36 vs. 0.96±0.60)(P<0.05)。结论 BUB1基因和BUBR1基因在胃癌中高表达可能与癌细胞的异常增殖有关。
文摘Protein phosphatase 2A （PP2A） accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a cat- alytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kine- tochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interac- tion plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attach- ment. Here we present the crystal structure of a PP2A B56-BubRI complex, which demonstrates that a con- served BubRl LxxlxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxlxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.
