p53调控四倍体细胞命运的研究
摘要
p53异常和非整倍体通常共存于人类肿瘤中,而四倍体则被认为是正常的二倍体细胞转化到非整倍体细胞的中间体,且其也与p53的异常密切相关。本研究的目的就是研究p53是否以及如何影响四倍体向非整倍体的转化。我们利用长时间活细胞实时摄影研究了众多具有不同p53状态的人和小鼠细胞系的四倍体细胞的命运以及分裂行为,并用中心体和纺锤体的免疫荧光研究了四倍体细胞在有丝分裂中期时中心体的行为。我们的结果显示p53显性失活突变,点突变,或者敲除能导致N/TERT1,3T3以及小鼠胚胎成纤维细胞(MEF)发生多极分裂的比例上升了2~33,而四倍体细胞进入有丝分裂和死亡的比例则并没有受到影响。我们发现,p53缺失的的四倍体MEF细胞聚集多余中心体的能力削弱了,从而促进多极分裂的发生,但是失活多余中心体的能力则并没有改变。当使用C3转移酶或H1152特异性地抑制了RhoA或ROCK的活性后,p53缺失的四倍体MEF细胞聚集多余中心体的能力增强,而多极分裂的比例也从38%分别下降到20%和16%。在p53正常的3T3细胞中表达持续活化的RhoA后,其四倍体细胞进行多极分裂的比例从15%增加至35%。因此,我们认为p53的异常会通过RhoA/ROCK的过度活化损害四倍体细胞聚集多余中心体的能力,进而导致多极分裂的发生。本研究为肿瘤发生过程中非整倍体如何发生以及在人类肿瘤中常见的p53异常,多极分裂以及非整倍体的共存提供了一个新的解释。
p53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy. Live cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2~ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%. Thus, we conclude that p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway. This provides an alternative explanation for arising of aneuploidy during tumor initiation and the frequently observed coexistence of p53 abnormalities, multipolar mitosis, and aneuploidy in human tumors.
p53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy. Live cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2~ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%. Thus, we conclude that p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway. This provides an alternative explanation for arising of aneuploidy during tumor initiation and the frequently observed coexistence of p53 abnormalities, multipolar mitosis, and aneuploidy in human tumors.
引文
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