the spindle midzone is critical for completing furrow ingres- sion (Rappaport, 1961; Glotzer, 2001; Saint and Somers, 2003; Inoue et al., 2004; D’Avino et al., 2005). The ability of APC mutants to dominantly interfere with mitotic spindle assem- bly led us to ask whether a single APC allele might induce fail- ures in cytokinesis that contribute to genomic instability and cancer progression. Results APC1–1,450 inhibits cytokinesis To address the possibility that APC mutants compromise cyto- kinesis, we examined the long-term effect of expressing the dominant-negative APC1–1,450 (amino acids 1–1,450) mutant on chromosomal ploidy. This allele is representative of APC muta- tions observed in human colorectal cancer (Lamlum et al., 1999; Sieber et al., 2000; Fearnhead et al., 2001). We created HEK-293 cell lines stably expressing APC1–1,450 under an ecdysone-inducible promoter and control cells lacking the hormone receptor re- quired for expression (labeled as the control in figures; Green and Kaplan, 2003). Immunoblotting for the tagged APC allele showed relatively constant expression similar to endogenous levels over the course of the experiment (Green and Kaplan, 2003; Green et al., 2005; and unpublished data). In control cells, we observed a constant and very low incidence of binucleate cells (1–2%) and few multinucleated (more than two nuclei) cells over 10 d (Fig. 1, A and B, control). In contrast, cells ex- pressing APC1–1,450 exhibited a steady increase in the numbers of both binucleate and multinucleate cells during the course of the experiment (Fig. 1, A [binucleate and multinucleate] and B), with each category reaching 10% of the total cell population. Multiple stable cell lines all exhibited the same trend: after >3 d of APC1–1,450 expression, we observed a mean of 10.26% bi- nucleate (SD = 6.34%) and 8.34% multinucleate (SD = 5.27%) compared with control cells with 0.63% bincucleate (SD = 0.37%) and 0.33% multinucleate (SD = 0.1%). These results are Figure 1. Expression of APC1–1,450 results in the accumulation of binucleate and multinucleate cells. (A) Cells were ﬁxed and stained with DAPI to visualize normal, binucleate, and multinucleate cells. (B) The percentage of control cells or cells expressing APC1–1,450 with more than one nuclei were determined after ﬁxing and stain- ing cells to visualize chromosomes 0, 2, 4, 6, 8, and 10 d after induction. The percentage of cells that were binucleate (orange) or multinucleate (purple) were calculated for a minimum of 300 cells. The data presented are representative of three separate experi- ments performed at different time intervals. The range of multi- nucleated cells observed for all experiments is presented in Table I. DIC, differential interference contrast. Bars, 10 μm. 1110 JCB • VOLUME 178 • NUMBER 7 • 2007