Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA.
PLoS One. 2010 Apr 2;5(4):e9994. doi: 10.1371/journal.pone.0009994.
Truncating mutations in the tumor suppressor gene APC (Adenomatous Polyposis Coli) are thought to initiate the majority of colorectal cancers. The 15- and 20-amino acid repeat regions of APC bind beta-catenin and have been widely studied for their role in the negative regulation of canonical Wnt signaling. However, functions of APC in other important cellular processes, such as cell cycle control or aneuploidy, are only beginning to be studied. Our previous investigation implicated the 15-amino acid repeat region of APC (M2-APC) in the regulation of the G2/M cell cycle transition through interaction with topoisomerase IIalpha (topo IIalpha).
METHODOLOGY/PRINCIPAL FINDINGS: We now demonstrate that the 20-amino acid repeat region of APC (M3-APC) also interacts with topo IIalpha in colonic epithelial cells. Expression of M3-APC in cells with full-length endogenous APC causes cell accumulation in G2. However, cells with a mutated topo IIalpha isoform and lacking topo IIbeta did not arrest, suggesting that the cellular consequence of M2- or M3-APC expression depends on functional topoisomerase II. Both purified recombinant M2- and M3-APC significantly enhanced the activity of topo IIalpha. Of note, although M3-APC can bind beta-catenin, the G2 arrest did not correlate with beta-catenin expression or activity, similar to what was seen with M2-APC. More importantly, expression of either M2- or M3-APC also led to increased aneuploidy in cells with full-length endogenous APC but not in cells with truncated endogenous APC that includes the M2-APC region.
CONCLUSIONS/SIGNIFICANCE: Together, our data establish that the 20-amino acid repeat region of APC interacts with topo IIalpha to enhance its activity in vitro, and leads to G2 cell cycle accumulation and aneuploidy when expressed in cells containing full-length APC. These findings provide an additional explanation for the aneuploidy associated with many colon cancers that possess truncated APC.
肿瘤抑制基因 APC（结肠腺瘤性息肉病基因）的截断突变被认为是引发大多数结直肠癌的原因。APC 的 15 个和 20 个氨基酸重复区域与β-连环蛋白结合，并因其在经典 Wnt 信号通路的负调控中的作用而被广泛研究。然而，APC 在其他重要细胞过程（如细胞周期控制或非整倍体）中的功能才刚刚开始被研究。我们之前的研究表明 APC 的 15 个氨基酸重复区（M2-APC）通过与拓扑异构酶 IIα（topo IIalpha）相互作用参与 G2/M 细胞周期转换的调控。
方法/主要发现：我们现在证明 APC 的 20 个氨基酸重复区（M3-APC）也与结肠上皮细胞中的 topo IIalpha 相互作用。在具有全长内源性 APC 的细胞中表达 M3-APC 会导致细胞在 G2 期积累。然而，具有突变的 topo IIalpha 同工型且缺乏 topo IIbeta 的细胞不会停滞，这表明 M2-或 M3-APC 表达的细胞后果取决于功能性拓扑异构酶 II。纯化的重组 M2-和 M3-APC 均显著增强了 topo IIalpha 的活性。值得注意的是，尽管 M3-APC 可以与β-连环蛋白结合，但 G2 期停滞与β-连环蛋白的表达或活性无关，这与 M2-APC 相似。更重要的是，在含有全长内源性 APC 的细胞中表达 M2-或 M3-APC 也会导致细胞非整倍体增加，但在含有包括 M2-APC 区域的截断内源性 APC 的细胞中则不会。
结论/意义：总的来说，我们的数据表明 APC 的 20 个氨基酸重复区与 topo IIalpha 相互作用，在体外增强其活性，并在含有全长 APC 的细胞中表达时导致 G2 期细胞周期积累和非整倍体。这些发现为许多具有截断 APC 的结肠癌相关的非整倍体提供了另一种解释。
