膀胱癌細胞株からの中心体分離 : p53 mutationと中心体過剰複製について

Abstract

膀胱癌細胞株を用いて中心体の分離を試み, 中心体複製周期をコントロールするnucleophosmin/B23(NPM)の中心体の局在を検討した.ヒト膀胱癌細胞株(RT-4, HT-1197, HT-1376)を用いた.RT-4は中心体の複製周期が保たれている細胞株であり, HT-1197, HT-1376は中心体過剰複製を認める細胞株であった.HT-1197, HT-1376においては, 3個以上の中心体も観察できた.RT-4とHT-1197において, γチューブリンとNPMの局在が一致した

Centrosome hyperamplification occurs frequently in human cancers, and is the major contributing factor for chromosomal instability and aneuploidy. We examined centrosome hyperamplification in bladder cancer cell lines. Samples were incubated with antibodies to the centrosome protein gamma-tubulin. The cell line (RT-4), which has a wild-type p53 status, showed a well-regulated centrosome replication cycle. On the other hand, centrosome hyperamplification was observed in HT-1197 and HT-13r cancer cells by discontinuous sucrose gradient fractionation. We used sucrose gradient fractions enriched for centrosomes by the immunoblot analysis for the presence of gamma-tubulin, a major component of centrosomes. The fractions were then immunoblotted with anti-nucleophosmin/B23 (NPM) antibody. NPM is a primary target of CDK2-cyclin E in the initiation of centrosome duplication. The profile of NPM closely paralleled that of gamma-tubulin, suggesting the association of NPM with the centrosome. Identification of the mechanism underlying the replication of the centrosome should lead to the understanding of the mechanism of chromosomal instability in bladder cancer, and enable us to develop cancer therapeutics targeted to centrosome replication.