Oncogene (2005) 24, 4301–4310 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Partial downregulation of MAD1 causes spindle checkpoint inactivation and aneuploidy, but does not confer resistance towards taxol Anne Kienitz1,2, Celia Vogel1,2, Ivonne Morales1, Rolf Mu¨ ller1 and Holger Bastians*,1 1Institute for Molecular Biology and Tumor Research (IMT), Philipps University Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany The mitotic spindle assembly checkpoint ensures proper promoting complex/cyclosome (APC/C) (Li et al., chromosome segregation during mitosis by inhibiting the 1997; Fang et al., 1998; Gorbsky et al., 1998). This onset of anaphase until all kinetochores are attached to large ubiquitin ligase complex is required for the the mitotic spindle and tension across the kinetochores is ubiquitination and destruction of securin and cyclin B, generated. Here, we report that the stable partial down- prerequisites for anaphase onset and mitotic exit, regulation of the spindle checkpoint gene MAD1, which is respectively (for a review see Peters, 2002). The spindle observed in human cancer, leads to a functional inactiva- checkpoint is also activated in response to various tion of the spindle checkpoint resulting in gross aneu- spindle poisons like nocodazole, a drug that depoly- ploidy. Interestingly, although Mad1is thought to act as merizes microtubules, and thus, prevents the attachment a kinetochore based activator of Mad2 during checkpoint of microtubules to the kinetochores. On the other hand, activation, we show that normal levels of Mad2, but not anticancer drugs, like taxanes (e.g. paclitaxel/taxol), of Mad1, are required for preventing premature sister inhibit the dynamic instability of the spindle and allow chromatid separation and for maintaining the timing of an microtubule attachment, but prevent the generation of undisturbed mitosis, suggesting a Mad1independent tension across kinetochores. Treatment of cells with function of Mad2 that operates independent of its these drugs inhibits chromosome alignment and results checkpoint function. Most significantly, a partial repres- in a mitotic arrest before anaphase, which is followed by sion of either MAD1 or MAD2 confers resistance to the induction of apoptosis (Jordan and Wilson, 2004). nocodazole, a drug that inhibits microtubule attachment. To date, the molecular pathways of the spindle In contrast, sensitivity to clinically relevant drugs like checkpoint are not well understood, but it is established taxol or monastrol that inhibit the generation of tension that several evolutionary conserved proteins including across kinetochores is not modulated by partial down- Cenp-E, BubR1, Bub1, Bub3, Mad1, Mad2 and Mps1 regulation of MAD1, suggesting a functional bifurcation are required for spindle checkpoint function (for a of spindle checkpoint dependent apoptotic pathways. review see Yu, 2002; Bharadwaj and Yu, 2004; Taylor Oncogene (2005) 24, 4301–4310. doi:10.1038/sj.onc.1208589 et al., 2004). These and most likely additional yet Published online 14 March 2005 unknown proteins are sequentially recruited to unat- tached or tension-lacking kinetochores leading to the Keywords: cell cycle; mitosis; mad2; apoptosis; genomic generation of a diffusible APC/C inhibitor (Shah and instability Cleveland, 2000; Musacchio and Hardwick, 2002; Vigneron et al., 2004). Mad2, BubR1, and also a mitotic checkpoint complex (MCC) consisting of Mad2, BubR1, Bub3 and Cdc20 can function as potent Introduction inhibitors for APC/C activity, but the mechanism of the MCC formation is still unclear (Fang et al., 1998; The faithful segregation of chromosomes during mitosis Sudakin et al., 2001; Tang et al., 2001; Fang, 2002). requires that all chromosomes are correctly attached to According to a current model, an unattached kineto- the mitotic spindle apparatus and properly aligned at chore serves as an activating platform for the diffusible the metaphase plate before anaphase onset is allowed. APC/C inhibitor. Consistently, Mad1 is required for During the early phases of mitosis when chromosomes recruitment of Mad2 to kinetochores and complex are not yet aligned, the mitotic spindle assembly formation of Mad1 and Mad2 at the kinetochore is checkpoint is activated (Campbell and Gorbsky, 1995; important for checkpoint activation, suggesting that Li and Nicklas, 1995; Rieder et al., 1995) and prevents Mad1 might function as an activator of Mad2 at the the onset of anaphase by inhibiting the anaphase kinetochore (Jin et al., 1998; Chen et al., 1999; Campbell et al., 2001; Sironi et al., 2001; Luo et al., 2002; Martin- Lluesma et al., 2002). *Correspondence: H Bastians; E-mail: [email protected] 2These authors contributed equally to this work Interestingly, Mad2 and BubR1 appear to be also Received 11 August 2004; revised 27 January 2005; accepted 4 February involved in regulating the normal timing of mitosis. 2005; published online 14 March 2005 Inhibition of Mad2 or BubR1, but none of the other MAD1 downregulation causes aneuploidy A Kienitz et al 4302 Mad and Bub proteins, causes an acceleration of mitosis A function of Mad1 together with Mad2 for the (Gorbsky et al., 1998; Canman et al., 2002; Meraldi spindle checkpoint has been demonstrated in cells et al., 2004). Moreover, the inhibition of their kineto- depleted of Mad1 by transient siRNA transfections chore localization does not shorten the time from (Luo et al., 2002; Martin-Lluesma et al., 2002; Meraldi prophase to anaphase indicating that Mad2 and BubR1 et al., 2004). However, severe depletion or homozygous might be part of a kinetochore independent mitotic disruption of spindle checkpoint genes results in cell timer mechanism (Meraldi et al., 2004). death indicating that spindle checkpoint genes are The majority of human cancer cells are aneuploid, essential for viability (Dobles et al., 2000; Kalitsis which can be caused by gaining or loosing whole et al., 2000; Kops et al., 2004; Michel et al., 2004). chromosomes during defective chromosome segregation Therefore, previous studies could not address the long- events (Duesberg et al., 2000; Sieber et al., 2003). This term physiological consequences of Mad1 inactivation. phenotype is called chromosomal instability (CIN) and Moreover, in human cancer, only partial downregula- although its molecular basis is still unknown, it is now tion of MAD1 or MAD2 is observed and it is important apparent that malfunction of the spindle checkpoint can to resolve whether a partial downregulation of MAD1 directly contribute to CIN (Lengauer et al., 1998; Michel or MAD2 is functionally redundant. Therefore, we et al., 2001; Masuda and Takahashi, 2002; Babu et al., investigated the phenotypes associated with partial 2003; Rajagopalan and Lengauer, 2004). Indeed, defects downregulation of MAD1 or MAD2. Our results in the spindle checkpoint are frequently found in various indicate a differential requirement of Mad1 and Mad2 types of human cancer including lung, breast, ovarian, for spindle checkpoint activation, mitotic timing and colon and hepatocellular tumors and these defects are induction of apoptosis in response to chemotherapeutic associated with CIN and aneuploidy (Cahill et al., 1998; drugs in human cancer cells. Takahashi et al., 1999; Masuda and Takahashi, 2002; Shichiri et al., 2002). While spindle checkpoint defects are frequent, spindle checkpoint genes appear to be Results rarely altered in human tumors (Cahill et al., 1998; Nomoto et al., 1999; Hernando et al., 2001). Instead, Generation of human colon carcinoma cells exhibiting downregulated expression of spindle checkpoint genes reduced levels of Mad1 might contribute to a deactivated spindle checkpoint in cancer cells. In fact, the genes of the MCC, MAD2, We wished to generate karyotypically stable human cells BUB3 and BUBR1, have been shown to be haplo- exhibiting partial downregulation of MAD1. For this, insufficient (Michel et al., 2001; Babu et al., 2003; Baker we stably expressed an siRNA targeting MAD1 in et al., 2004). Heterozygous deletion of MAD2 in human HCT116 cells, which are well characterized for their colon carcinoma cells and heterozygous MAD2, BUB3 intact cell cycle checkpoints and their relatively stable or BUBR1 disruptions in mice result in partially karyotype. In addition, HCT116 cells have been used downregulated checkpoint protein levels, an impaired previously for heterozygous gene deletion of MAD2 spindle checkpoint and aneuploidy (Michel et al., 2001; (HCT116-MAD2 þ /À) resulting in cells with approxi- Babu et al., 2003; Baker et al., 2004). Most significantly, mately 20% reduction of Mad2 protein level (Michel MAD2 þ /À, BUB3 þ /À and BUBR1 þ /À mice are prone to et al., 2001). This enabled us to investigate and to tumor development suggesting that aneuploidy can compare the long-term cellular consequences of MAD1 contribute directly to tumorigenesis (Michel et al., and MAD2 downregulation. Upon stable transfection 2001; Babu et al., 2003; Dai et al., 2004). To date, it is we selected two independent clones, termed knockdown unclear if a moderate downregulation of other spindle clone 2-2 (MAD1-kd-2-2) and knockdown clone 2-31 checkpoint genes directly contributes to aneuploidy and (MAD1-kd-2-31) for the experiments throughout this tumorigenesis; however, a reduced expression of MAD2 study. These clones showed about 35and 50%reduction and BUBR1, but also of BUB1 and MAD1, has been of endogenous Mad1 protein, respectively, when com- found in different