The cortical localization of dynamin did not require the presence of NME1, because we observed dynamin at the cortex of mitotic cells expressing the NME1 shRNA (Fig. cells. We thus provide evidence that the loss of Nm23-H1, an event suspected to promote metastasis, may additionally Sertindole function at an earlier stage of tumor development to drive the acquisition of chromosomal instability. Keywords:aneuploidy, RNAi screen, cell cycle, tetraploidy, p53 Chromosomal instability, or the inability to segregate chromosomes equally during cell division, plays a critical role in the development of the majority of solid tumors. One mechanism by which chromosomal instability evolves is through a transient tetraploidy state (1). In a companion article, we describe the execution of loss-of-function shRNA screens in untransformed diploid human cells to identify kinases whose loss leads to increased ploidy. We further show that sustained loss of one of the screening hits, NME1, leads to aneuploidy, chromosomal instability, loss of contact inhibition, and anchorage-independent growth. NME1 is the genomic locus that encodes the nucleoside diphosphate kinase (NDK) Nm23-H1; for the sake of consistency, we will refer to the gene, mRNA, and protein as NME1 in this article. NME1 is a member of the NDK family, whose biochemical Sertindole role is to use ATP to generate a different nucleoside triphosphate (2). Although initially thought of as a housekeeping gene that maintains cellular pools of nucleoside triphosphates, desire for NME1 was sparked by its identification as a potential metastasis suppressor by Steeg et al. (3). A comparison of expression levels across mouse melanoma cell lines revealed an inverse correlation between NME1 expression and metastasis (3). These expression data were supported by the observation that overexpression of NME1 in human carcinoma cells is sufficient to reduce tumor cell motility (46). Additional evidence points to a role for NME1 in the direct regulation of factors that may drive motility and invasion during metastasis (511). Given its connection with tumor progression, we were intrigued to identify NME1 as a regulator of chromosomal stability. To clarify the link between NME1, chromosomal instability, and tumor development, we have carried out an extensive analysis of the mechanism by which NME1 loss leads to chromosomal instability and contributes to tumor development. == Results == == NME1 Regulates Chromosomal Stability. == We first sought to link increased ploidy conclusively to a reduction in NME1 expression. First, we Sertindole established that multiple shRNAs targeting different regions of the NME1 mRNA were capable of generating increased ploidy in HBE135 cells. As shown inFig. 1A, four shRNAs targeting NME1 induced an increase in ploidy in HBE135 cells as measured by circulation cytometry 4 d after the constructs were introduced. Also shown inFig. 1Ais usually the DNA ARHA content histogram of an additional shRNA targeting NME1 (shRNA 5), which did not induce a significant increase in ploidy. Consistent with the phenotypes of the five NME1 shRNAs, Western blot analysis showed that only those four shRNAs that reduced NME1 protein levels caused an increase in ploidy (Fig. 1B). Because the ploidy phenotype induced by shRNA 1 was slightly stronger than that of the other shRNAs, all experiments discussed below made use of NME1 shRNA 1 unless otherwise indicated. == Fig. 1. == NME1 regulates chromosomal stability. (A) Multiple shRNAs against NME1 cause an increase in ploidy. A scrambled control shRNA or five different shRNAs against NME1 were transduced into HBE135 cells. Cells were fixed for circulation cytometric analysis at 4 d. (B) Protein knockdown for multiple NME1 shRNAs. Protein extracts were prepared in parallel when cells were fixed as inA. Normalized protein extracts were subjected to SDS/PAGE and Western blot analysis for the indicated proteins. (C) Overexpression of NME1 cDNA complements the NME1 knockdown phenotype. The indicated lentiviral shRNA and cDNA constructs were transduced into confluent HBE135 cells, and the cells were reseeded after 4 d and fixed for circulation cytometric analysis after an additional 4 d. Protein extracts were also prepared and subjected to Western blot analysis Sertindole to measure the level of endogenous (closed arrowhead) and Flag-tagged NME1 protein (open arrowhead). Ponceau S staining was used as a loading control. luc, luciferase; shNME1, short hairpin NME1. (D) Increase in tetraploid cells occurs 48 h following transduction of NME1 shRNA. Control or NME1 shRNAs were launched into HBE135 cells, and cells were prepared for circulation cytometric, mRNA, or Western blot analysis at the indicated time points. For quantification, data points were normalized to the mRNA or protein level at time 0. (E) Abortive mitosis occurs within 24 h of knockdown of NME1. HBE135 cells were infected at confluence with scrambled or NME1 shRNA.
