MAD1) Increases the Incidence of Tumors in Mice

Research Article

Heterozygous Deletion of Mitotic Arrest–Deficient Protein 1 (MAD1) Increases the Incidence of Tumors in Mice

Yoichi Iwanaga,1 Ya-Hui Chi,1 Akiko Miyazato,1 Sergey Sheleg,1 Kerstin Haller,1 Jean-Marie Peloponese, Jr.,1 Yan Li, 1 Jerrold M.Ward, 2 Robert Benezra,3 and Kuan-Teh Jeang1

1Molecular Virology Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases and 2Infectious Disease Pathogenesis Section, Comparative Medicine Branch, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland; and 3Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York

Abstract Several knockout (KO) mouse models (Mad2, BubR1,orBub3) Mitotic arrest–deficient protein 1 (MAD1) is a component of have been constructed to investigate the physiologic functions of the mitotic spindle assembly checkpoint. We have created a SAC proteins. An underlying goal, among others, of these KO knockout mouse model to examine the physiologic conse- studies is to clarify whether a weakened SAC creates a proclivity for +/À in vivo quence of reduced MAD1 function. Mad1 mice were tumor development . Regrettably, the full biological import successfully generated, but repeated paired mating of of these KO studies has been unrealized because mice homozygous +/À +/À BubR1, Mad2 Bub3 Mad1 with Mad1 mice failed to produce a single null for ,or are embryonic lethal (12–14). À/À Mad1 animal, suggesting that the latter genotype is Nonetheless, analyses of heterozygous null animals have been instructive, albeit slightly confounding. Here, an increased inci- embryonic lethal. In aging studies conducted for >18 months, +/À +/À dence of constitutive tumors was revealed in Mad2 mice but not Mad1 mice compared with control wild-type (wt) litter- +/À +/À in either BubR1 or Bub3 mice (12–16). On the other hand, a mates showed a 2-fold higher incidence of constitutive +/À Mad1+/À P wt higher tumor incidence can be induced in BubR1 compared tumors. Moreover, 42% of ( < 0.03), but 0% of , wild-type wt mice developed neoplasia after treatment with vincristine, a with ( ) control mice when animals are treated with +/À microtubule depolymerization agent. Mad1 mouse embry- carcinogens (12). Whereas other interpretations are possible, these onic fibroblasts (MEF) were found to be more prone than wt data suggest potentially two qualitatively discrete paths within the +/À cells to become aneuploid; Mad1 ,butnotwt,MEFs SAC, in which attenuation of one, but not the other, suffices to trigger constitutive tumors. Alternatively, MAD2 and BUBR1 may produced fibrosarcomas when explanted into nude mice. +/À have different quantitative effects on the SAC because BubR1 Our results indicate an essential MAD1 function in mouse +/À Mad1 ApcMin compound mice exhibit greater genetic instability than development and correlate haploinsufficiency with BubR1+/À increased constitutive tumors. [Cancer Res 2007;67(1):160–6] mice and do developconstitutive tumors (17). To further clarify tumorigenic differences, if any, between a +/À +/À +/À Mad2 genotype and its BubR1 or Bub3 counterpart, we Introduction constructed a KO mouse model for the MAD2 partner protein, À/À À/À Cancer is a disease of damaged genes. Currently, the precise MAD1. Here, we report that Mad1 like Mad2 (13) confers +/À +/À mechanisms for cellular transformation remain incompletely embryonic lethality, and that Mad1 mice are similar to Mad2 elucidated. Abnormal chromosome numbers from losses or gains mice in developing increased constitutive tumors. in whole chromosomes (i.e., aneuploidy) are commonly seen in cancer cells. Eukaryotic cells have evolved a spindle assembly checkpoint (SAC) to monitor the fidelity of chromosomal Materials and Methods segregation in mitosis (1, 2) to guard against aneuploidy. In lower Construction of Mad1 KO mice. The Mad1 KO vector (Mad1 KO) was eukaryotes (i.e., yeasts), the SAC is composed of the mitotic arrest– constructed by cloning exon 10 of the mouse Mad1 gene into vector pGEM-7 deficient (MAD) proteins (MAD1, MAD2, and MAD3), the budding (Promega, Madison, WI). Neo was positioned downstream of exon 10. uninhibited by benzimidazole (BUB) proteins (BUB1, BUB2, and Introns 9 (1.4 kb) and 10 (6.1 kb) were placed before the 5¶ end of exon ¶ neo A HSV-TK BUB3), and the monopolar spindle 1 protein (3). In higher 10 and after the 3 end of , respectively (Fig. 1 ). The gene was ¶ loxP eukaryotes, SAC components also include BUBR1 (a vertebrate introduced at the 3 end for negative selection. In addition, three (locus of X-over of bacteriophage P1) sites were created between the 5¶ arm and variant of yeast MAD3 and BUB1), the ROD-ZW10-Zwilch complex, exon 10, exon 10 and neo, and neo and the 3¶ arm, respectively (Fig. 1A). and the microtubule motor centromere protein E (4). A link Mad1 KO mice were generated by the Mouse Genome Engineering Facility between SAC function and neoplasia is suggested, although not at the University of Nebraska. In brief, Mad1 KO was introduced into proven, by findings that expression of many checkpoint proteins is embryonic stem (ES) cells by electroporation and doubly selected using aberrant in cancers (5–11). G418 and ganciclovir. Surviving clones were confirmed by PCR. Heterozy- gous ES cells were injected into C57BL/6J blastocysts. Mosaic founder animals were screened for germ-line transmission of the KO genotype by

breeding to C57BL/6J mice. F1 mice heterozygous for the KO allele were Note: Y. Iwanaga, Y-H. Chi, and A. Miyazato contributed equally to this work. mated to a Cre-expressing transgenic mouse [BALB/c-TgN(CMV-Cre)#Cgn; Requests for reprints: Kuan-Teh Jeang, NIH, Building 4, Room 306, 9000 Rockville The Jackson Laboratory, Bar Harbor, ME], resulting in deletion of exon 10 of Pike, Bethesda, MD 20892-0460. Phone: 301-496-6680; Fax: 301-480-3686; E-mail: Mad1 (Fig. 1A) in some of the offsprings. Loss of exon 10 was verified by [email protected]. ¶ ¶ I2007 American Association for Cancer Research. PCR using the following primers: 5 -cggacgaggtatttgcacgtgcagctctattttagg-3 doi:10.1158/0008-5472.CAN-06-3326 and 5¶-gcatgggtgagctcagtcacactgg-3¶.

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Figure 1. Characterization of Mad1 KO mice. A, schematic representation of the Mad1 gene-targeting strategy. LoxP-flanked exon 10 of Mad1 was removed by crossing mouse carrying Mad1 KO vector with a whole-body Cre transgenic mouse. Solid triangles, loxP sites. B, amino acid sequence of mouse MAD1. Sequence of exon 10 of mouse Mad1 is denoted in bold, italics, and is underlined. C, PCR analysis of Mad1 +/À mice. PCR of wt genomic DNA generated a 312-bp fragment, whereas the KO allele generated a 260-bp fragment.

Tumorigenicity assay in nude mice. Mouse embryonic fibroblasts mouse from The Jackson Laboratory [strain BALB/c-TgN(CMV- 6 (MEF; 5 Â 10 ) were resuspended into 200 AL PBS containing 5% fetal Cre)#Cgn] that overexpress a CMV promoter-driven Cre expression bovine serum and injected s.c. into the posterior neck of 6-week-old vector (Fig. 1A). Cre is a site-specific DNA recombinase of athymic nude mice (Harlan, Inc., Indianapolis, IN). Tumor growth was bacteriophage P1 that recognizes a 34-bp site on the P1 genome monitored every 3 days. (i.e., loxP) and efficiently catalyzes reciprocal conservative DNA Immunofluorescence and confocal microscopy. MEFs were fixed in loxP 4% paraformaldehyde for 30 min and permeablized with 0.1% Triton X-100 recombination between pairs of sites (20). Cre-mediated loxP B in PBS for 5 min at room temperature. To prevent nonspecific binding, removal of -flanked exon 10 in F2 mice (Fig. 1 ) was confirmed cells were equilibrated in 1% bovine serum albumin in PBS for 30 min. by PCR. We checked by direct sequencing (data not shown) that Antibodies against a-tubulin (Sigma-Aldrich, St. Louis, MO) or anti– wt mouse DNA generated the expected 312-bp PCR fragment, phosphorylated histone H3 on Ser10 (Cell Signaling Technology, Beverly, whereas the deleted Mad1 genotype produced the expected 260-bp +/À MA) were added and incubated for 1 h at room temperature. Fluorescent- fragment (Fig. 1C). Western blotting of Mad1 mouse embryo conjugated secondary antibodies (Molecular Probes, Carlsbad, CA) were fibroblasts showed a one-third to one-half expression level of ¶ used for detection. Nuclei were stained with 4 ,6-diamidino-2-phenylindole MAD1 protein with no clear detection of truncated polypeptide (DAPI; Molecular Probes). Cells on the coverslips were mounted on glass (data not shown). This suggests that cell endogenous exon 10– slides with antifade reagent (Molecular Probes). Slides were monitored deleted MAD1 protein may be unstable. using a Leica (Wetzlar, Germany) TCS-NP/SP confocal microscope. Mad1+/À Mad1 Fluorescent in situ hybridization. Fluorescent in situ hybridization We paired mice to breed for homozygous null Mad1+/À Â Mad1+/À (FISH) was done using chromosome-specific Cy3-labeled probe for chro- mouse. To this end, 15 deliberate pairings mosome 2 (Cambio Ltd., Cambridge, United Kingdom) according to the were made, which produced a total of 319 pups (Table 1A). +/À manufacturer’s protocol. Briefly, cells were fixed in 3:1 (v/v) methanol/acetic Genotyping of the pups revealed that 209 were Mad1 and 110 +/+ À/À acid and dropped onto slides. Slides were denatured with 70% formamide at mice were wt (i.e., Mad1 ); no Mad1 mouse was born. The +/À 65jC for 1.5 min. After 16 h of hybridization, the slides were washed with Mad1 to wt ratio conformed to Mendelian inheritance and 50% formamide, 1Â SSC, and 4Â SSC/0.05% Tween 20 successively. Finally, suggested embryonic lethality as the explanation for the absence À/À slides were mounted onto coverslips with antifade reagent. of live Mad1 births. Subsequent matings have generated À/À another 1,000+ offsprings; no live Mad1 mouse has ever been detected. À Results Increased tumor incidence in Mad1+/ mice. It has been Construction of a Mad1 KO mouse. MAD1 is a protein-binding proposed that weakening of the SAC without a total loss in partner of MAD2. RNA interference– or dominant-negative mutant checkpoint function is relevant to the development of aneuploidy +/+ +/À protein–mediated knockdown of MAD1 in somatic cells has been (4). Compared with Mad1 , we reasoned that Mad1 represents shown to produce MAD2 mislocation from kinetochores and failed a weakened SAC. To ask if a MAD1-attenuated SAC influences SAC function (18). To investigate in vivo MAD1 function, we tumor development in mice, we monitored age- and sex- matched +/+ +/À embarked on generating a Mad1 KO mouse (see Materials and Mad1 and Mad1 cohorts (128 and 121 animals, respectively) À/À Methods). Mad1 has 17 coding exons; we chose to target exon 10 for >18 months. Although Mad1 is incompatible with live birth +/À from the coding sequence (Fig. 1B) because removal of this exon (Table 1A), Mad1 mice are born normally. Further, the life +/+ +/À abolishes the ability of MAD1 to bind MAD2 and creates loss of spans of Mad1 and Mad1 mice are similar as evidenced +/À SAC function (19). Independent founder mice with exon 10 of by 90% (114 of 128) of Mad1 and 93% (112 of 121) of wt mice Mad1 flanked by loxP sequences were created, and germ-line being alive at 18 months. This observation is similar to published +/À +/À +/À transmission from founder to F1 offsprings was achieved. To delete survival curves comparing wt with Mad2 , BubR1 ,orBub3 Mad1 exon 10 from , we mated F1 animals to a strain of transgenic mice (12, 16, 21). www.aacrjournals.org 161 Cancer Res 2007; 67: (1). January 1, 2007

+/À At 18 months of age, both wt and Mad1 mice were sacrificed papillary lung adenocarcinomas (16). Interestingly, mice in our +/À and detailed necropsy and histopathology were done. Necropsies Mad1 cohort developed neoplasia in a wide range of tissues +/À of wt and Mad1 mice revealed a statistically significant (hepatocellular carcinoma, rhabdomyosarcoma, osteosarcoma, (P < 0.05, m2) difference in tumor incidence (Table 1B). Lymphoma hemangiosarcoma, and uterine sarcoma) in addition to the lung is a common background tumor in aging mice (22). The incidence (Table 1B). Spontaneous rhabdomyosarcoma and hemangiosar- +/À of lymphoma was statistically indifferent between wt and Mad1 coma are exceedingly rare in 18-month-old mice with BALB/c, mice and was excluded from our comparisons. Previously, C57BL/6, or B6:129 backgrounds (23); hence, the presence of these +/À +/À 18-month-old Mad2 mice were reported to have increased tumors in our Mad1 mice suggests specificity for Mad1 +/À haploinsufficiency. The Mad1 rhabdomyosarcoma and hemangiosarcoma showed typical morphologies of aggressively prolifer- À A, arrows Table 1. Embryonic lethality in Mad1+/ mice and tumor ating mitotic cells (Fig. 2 ). +/À Vincristine treatment increased tumor development in incidence in 18-month-old Mad1 mice À Mad1+/ mice. Vincristine is a microtubule-depolymerizing drug (A) Genotypes of pups from Mad1+/À Â Mad1+/À matings used frequently in cancer chemotherapy. Depolymerized micro- tubules attach poorly to kinetochores, and this inefficiency is Mating no. Mad1+/À wt Mad1À/À expected to elicit SAC-mediated mitotic arrest (24). Cells with weakened SAC may fail to arrest in mitosis despite microtubule 11680depolymerization; a failure to arrest in mitosis followed with 225140progression through cytokinesis could produce aneuploid progeny +/À 31050cells. To ask if the increased tumors in Mad1 mice could be 41340explained by a weakened SAC, we deliberately increased chromo- 531140somal missegregations by dosing animals with vincristine. We 61340hypothesized that such provocation would induce a higher 7400 +/À +/+ frequency of aneuploidy in Mad1 versus Mad1 cells, and 812100 induction of aneuploid cells might then engender more tumors in 91380+/À +/+ Mad1 versus Mad1 mice. 10 18 13 0 +/À 11 6 4 0 We took 6-month-old wt and Mad1 mice, in groups of 12, and 12 7 8 0 treated them i.p. with vincristine once every 7 days for three +/À 13 13 6 0 injections. As a control, we also treated 12 Mad1 mice with 14 20 6 0 etoposide, a topoisomerase II inhibitor, which does not affect 15 8 6 0 microtubule physiology (Table 1C). The mice were followed for Total 209 110 0 12 months after the conclusion of treatment and sacrificed to +/À examine for tumors (Table 1C). We found that none of the (B) Tumor incidence in 18-mo-old wt and Mad1 mice +/+;Vin 12 wt mice treated with vincristine (Mad1 ) nor any of the 12 +/À +/À;Eto Tumors* wt, n = 121 (%) Mad1+/À, n = 128 (%) Mad1 mice treated with etoposide (Mad1 ) developed +/À tumors. By contrast, 5 of 12 (42%) Mad1 mice treated with +/À;Vin Lung adenoma and 6 (5.0) 10 (7.8) vincristine (Mad1 ) did have tumors. These included three carcinoma cases of premalignant bronchioloalveolar adenoma (Fig. 2B), one Hemangiosarcoma 1 (0.8) 5 (3.9) case of hepatocellular carcinoma, and one case of Harderian gland Rhabdomyosarcoma 0 1 (0.8) adenoma. The significantly higher induced tumor incidence in +/À Hepatoma and hepatic 1 (0.8) 2 (1.6) vincristine-treated Mad1 than vincristine-treated wt mice carcinoma +/À or etoposide-treated Mad1 mice suggests that a weakened Osteosarcoma 0 1 (0.8) Mad1+/À Uterine sarcoma and 1 (0.8) 1 (0.8) SAC is less competent at arresting cells with deliberately carcinoma provoked chromosomal missegregations. Aneuploid progeny cells Melanoma 0 1 (0.8) that arise from uncensored missegregations could seed tumori- +/À Other tumors 2 (1.7) 3 (2.3) genesis in Mad1 mice. À Total tumors 11 (9) 24 (19) Genetic instability in dually haploinsufficient Mad1+/ À Mad2+/ MEFs. Above, we inferred that the increased tumor +/À presentation in 18-month-old Mad1 mice may be due to a +/À (C) Tumors in treated mice weakened SAC. To verify this inference, we isolated fresh Mad1 MEFs and compared them with wt MEFs for stringency of mitotic Genotype Treatment No. mice No. mice with arrest in response to treatment with nocodazole (Fig. 3A). Cells tumors (%) arrested in mitosis were identified by immunostaining for 10 A wt Vincristine 12 0 (0) phosphorylated Ser on histone H3 (Fig. 3 ). We counted 1,000 +/À wt Mad1 Vincristine 12 5 (42)c cells per group and found that 34% of nocodazole-treated and +/À +/À Mad1 Etoposide 12 0 (0) 10% of nocodazole-treated Mad1 MEFs were in mitosis. These +/À numbers support a weaker SAC-mediated arrest in Mad1 versus wt cells. +/À +/À *Excluding lymphoma. We next crossed Mad1 and Mad2 mice (16) to generate cP m2 +/À +/À < 0.03, . doubly heterozygous Mad1 Mad2 mice. We reasoned that cells doubly heterozygous null for Mad1 and Mad2 might have an

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Figure 2. Histology of tumor examples from Mad1 +/À mice. A, section of rhabdomyosarcoma from the head of an 11.5-month-old Mad1+/À mouse (1–3) and a hemangiosarcoma from the leg of another 16-month-old Mad1 +/À mouse (4–6). Highly proliferating cells with mitotic feature (condensed chromosomes) are observed in both cases (2, 3, 5,and6; arrows). Sections were stained with H&E. B, 6-month-old wt and Mad1+/À mice were treated with vincristine and sacrificed 12 m later. Sections of normal lung from wt (left) and lung with bronchioloalveolar tumors from Mad1+/À (right) mice. Both sections were stained with H&E.

+/À +/À +/À even more attenuated SAC than either Mad1 or Mad2 cells gradation of spontaneous genetic instability with Mad1 +/À +/À +/À +/À +/À +/À (25). Mad2 and Mad1 Mad2 MEFs were also isolated and Mad2 > Mad2 z Mad1 > wt. Our comparisons of con- +/À evaluated for SAC arrest after nocodazole treatment. Nine percent stitutive in vivo tumor development in cohorts of aging Mad1 , +/À +/À +/À +/À +/À +/À of Mad2 and only 6% of Mad1 Mad2 MEFs were found in Mad2 , and Mad1 Mad2 mice are ongoing, and data will not M phase (Fig. 3A and B), verifying a further weakened SAC in the be available for >1 year. However, at this stage, we can assess the +/À doubly heterozygous cells. In our experiments, many Mad1 , relative efficiencies of the various MEFs as cell explants (f2 weeks +/À +/À +/À Mad2 , and Mad1 Mad2 cells that completed mitosis and after isolation from embryos) for tumorigenesis. Hence, we injected cytokinesis contained micronuclei; the finding of micronuclei is equal numbers of each cell type into athymic nude mice (Fig. 5A) consistent with increased missegregation of chromosomes in these and assessed tumor growth. Tumors appeared within 4 weeks +/À +/À cells (26). in 100% (eight of eight) of mice injected with Mad1 Mad2 +/À The above experiments measured SAC function after drug cells. By comparison, eight of eight mice injected with Mad2 provoked microtubule depolymerization. However, constitutive MEFs also developed tumors within 4 weeks; however, these +/À +/À tumors developed in drug-untreated Mad1 mice at a rate tumors averaged 40% smaller in size (243 F 142 mm3 for Mad2 +/À +/À higher than in wt mice (Table 1B). To assess and compare ambient MEFs compared with 399 F 141 mm3 for Mad1 Mad2 MEFs, SAC function, we carefully examined mitoses of drug-untreated measured at 6 weeks postexplant; Fig. 5B). On the other hand, only +/À +/À +/À +/À +/À wt, Mad1 , Mad2 , and Mad1 Mad2 MEFs. Interestingly, 37.5% (three of eight) of animals injected with Mad1 MEFs abnormal mitoses were frequent in the latter three cell types. developed tumors, and these tumors grew more slowly and did not Figure 3C shows several examples of chromosomal aberrations become visually apparent or palpable until weeks 6 to 10. None of (lagging chromosomes, DNA bridging between the separated the eight mice injected with wt MEFs developed tumor (Fig. 5A chromatids, and multipolar spindles) during various points of and B). Histologically, all tumors were fibrosarcomas with +/À +/À +/À +/À mitosis in Mad1 ,Mad2 ,andMad1 Mad2 MEFs. pleomorphic spindle cells (Fig. 5C), consistent with the expected +/À +/À Statistically, 11% of Mad1 , 14% of Mad2 , and 26% of cell type from the injected cells. The rapidity, size, and frequency +/À +/À Mad1 Mad2 MEFs had mitotic abnormalities (Fig. 3D). These of tumor development correlated with the relative degrees of +/À +/À +/À numbers indicated a rank order of Mad1 Mad2 > Mad2 z ambient genetic instability in MEFs subscribing to an ordering of +/À +/À +/À +/À +/À Mad1 in constitutive genetic instability. Mad1 Mad2 > Mad2 z Mad1 > wt. We independently checked chromosomal instability of wt, +/À +/À +/À +/À Mad1 , Mad2 , and Mad1 Mad2 MEFs by FISH using a mouse chromosome 2–specific probe (Fig. 4A). Here, we used Discussion losses and gains in chromosome 2 as measures of propensity for Complete loss of SAC proteins, MAD1, MAD2, BUBR1, or BUB3, aneuploidy. Consistent with results in Fig. 3, Mad1+Mad2 doubly is embryonic lethal in mice (present study; refs. 12, 14, 16), haploinsufficient MEFs were more prone to gain or lose suggesting essentiality of these factors for mouse development. chromosome 2 than their singly haploinsufficient counterparts Although the reasons for embryonic lethality are unknown, SAC +/À +/À (Fig. 4B). Thus, 14.5% of Mad1 Mad2 MEFs were aneuploid for normally functions to guard against missegregation of chromo- +/À +/À chromosome 2 compared with 6.0% of Mad1 , 7.2% of Mad2 , somes during somatic mitosis. Observations that cancer cells are and 1.4% of wt MEFs. frequently aberrant for expression of SAC proteins (4, 5) have raised À À Doubly haploinsufficient Mad1+/ Mad2+/ MEFs are more a suggestion that loss in or weakening of SAC may be causal for tumorigenic in nude mice. Collectively, the above data suggest a carcinogenesis. Thus far, in vivo verification of this hypothesis www.aacrjournals.org 163 Cancer Res 2007; 67: (1). January 1, 2007

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Research based on mice heterozygous null for Mad2, BubR1,orBub3 have correlating the degree of genetic instability with quantitative tumor +/À yielded inconsistent results. Mad2 mice show higher than wt development remains lacking. In an attempt to explore this +/À +/À constitutive tumor development (16), whereas BubR1 and correlation, we investigated the ex vivo genetic stability of Mad1 , +/À +/À +/À +/À Bub3 mice do not (12, 14, 21). Nevertheless, one should be Mad2 , and Mad1 Mad2 MEFs. Our analyses within the cautious in making cross-comparisons because it remains un- narrowly restricted MAD pathway, using several independent known how MAD2, BUBR1, and BUB3 may differ in additional non- measures of genetic instability (Figs. 3–5), indicate that the degree +/À +/À +/À +/À SAC functions and whether noncheckpoint differences might of CIN is that Mad1 Mad2 > Mad2 z Mad1 > wt. influence in vivo tumor development. For example, BubR1 mice Interestingly, when the same MEFs were explanted into nude mice, have reduced fertility and a premature aging phenotype (15), which the in vivo rapidity, frequency, and size of tumors also ranked +/À +/À +/À +/À makes difficult age-matched comparisons of tumor development. Mad1 Mad2 > Mad2 z Mad1 > wt (Fig. 5), consistent +/À Here, our Mad1 results are consistent with previous results with the ex vivo quantification of chromosomal instability. Pending +/À from Mad2 animals (16) and add credence to the notion that replication of results using larger numbers of mice, our findings are weakening of the SAC contributes to increased constitutive consistent with the hypothesis that the degree of weakness in SAC tumorigenesis. Empirically, although chromosome instability correlates with the number of aneuploid cells, which emerge and (CIN) is a feature of most human cancers, conclusive evidence then developinto tumors. We caution that this correlation in mice

Figure 3. Attenuated SAC in Mad1 +/ÀMad2 +/À doubly heterozygous null MEFs. A, MEFs were mock-treated or treated with nocodazole for 18 h. Numbers of mitotic cells were verified by immunostaining with anti–histone H3 phosphorylated Ser10 (red). Top right, mitotic indices for each cell type are denoted as percentage. DNA was stained with DAPI (blue). B, tabulation of mitotic indices of wt, Mad1 +/À, Mad2 +/À, and Mad1 +/ÀMad2 +/À MEFs arrested with 200 nmol/L nocodazole for 18 h. Cells positive for histone H3 phosphorylated on Ser10 were scored as mitotic cells. Columns, mitotic index (%) from three independent experiments. Bars, SD. C, examples of aberrant mitosis of Mad1 +/À, Mad2 +/À, and Mad1 +/ÀMad2 +/À MEFs in prometaphase, metaphase, anaphase, and telophase. Anti–a-tubulin (green) staining and DNA staining with DAPI (red) were done. D, statistical analyses of aberrant wt, Mad1 +/À, Mad2 +/À, and Mad1 +/ÀMad2 +/À MEFs in interphase (I) and anaphase/telophase (M). Interphase cells (1,000) and anaphase/telophase cells (100) were counted.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. MAD1 Mice and Tumors may not extrapolate directly to humans because there are differences between the natural stringencies of mouse and human SAC (27, 28), and there are well-described mechanistic divergences between mice and men, which render the former far more +/À +/À cancer prone (29). Nonetheless, the Mad1 , Mad2 ,and +/À +/À Mad1 Mad2 mice represent comparative tools restricted to a specific checkpoint pathway useful for investigating how SAC weakness translates into tumorigenesis. We note that others have also proposed a relationship between chromosomal instability, aneuploidy, and tumorigenesis (30, 31). +/À Finally, constitutive tumors developed in Mad1 mice in +/À multiple organs. Curiously, Mad1 mice treated with vincristine showed a heightened propensity to develop lung tumors, similar to +/À the tumor tissue type observed in Mad2 mice (16). To date, up to 40% of human lung cancer cells have been found to carry mitotic checkpoint defects, including in MAD1 (32–34). It is intriguing that

Figure 5. Tumorigenicity of Mad1 +/ÀMad2 +/À doubly haploinsufficient MEFs. A, Mad1 +/À, Mad2 +/À,orMad1 +/ÀMad2 +/À MEFs were injected s.c. into the posterior neck of 6-week-old athymic nude mice. Tumors appeared within 4 weeks after injection with Mad2 +/À or Mad1 +/ÀMad2 +/À MEFs. Tumors grew slower in mice injected with Mad1 +/À MEFs, whereas no tumor was observed in mice injected with wt MEFs. B, tumor volumes were measured in athymic nude mice 6 weeks after being individually injected with wt, Mad1 +/À, Mad2 +/À, or Mad1 +/ÀMad2 +/À MEFs. The average tumor volumes are denoted by horizontal lines. C, sections (H&E; magnification, Â400) of fibrosarcomas from athymic nude mice injected with Mad1 +/À, Mad2 +/À,orMad1 +/ÀMad2 +/À MEFs. Pleomorphic structures of the tumor cells (poorly differentiated, locally invasive fibrosarcoma) were observed.

from our current results (Fig. 2B; Table 1B), mouse lung tissue seems to be relatively more sensitive to deregulation in the MAD components of the SAC. Future studies are needed to shed light on whether there are MAD-SAC functions specific to lung tissue development and pulmonary cell division.

Acknowledgments Received 9/8/2006; revised 11/10/2006; accepted 11/15/2006. Grant support: National Institute of Allergy and Infectious Diseases (NIAID)/NIH intramural funds and a NIAID contract (SoBran, Inc., Dayton, OH). The costs of publication of this article were defrayed in part by the payment of page Figure 4. Aneuploidy occurs with increased frequency in doubly heterozygous charges. This article must therefore be hereby marked advertisement in accordance À À À null Mad1 +/ Mad2 +/ MEFs. A, representative examples of wt, Mad1 +/ , with 18 U.S.C. Section 1734 solely to indicate this fact. À À À Mad2 +/ , and Mad1 +/ Mad2 +/ MEFs analyzed by FISH using whole We thank Drs. Mathew Starost and Michael Eckhaus (Division of Veterinary chromosome 2–specific probe (red). All chromosomes were stained with Resources, NIH, Bethesda, MD) and Dr. Torgny Fredrickson (Contract Management À À DAPI (blue). B, tabulation of chromosome 2 in wt, Mad1 +/ , Mad2 +/ , and Branch, NIAID, NIH) for necropsy and histopathology analyses, Cindy Erexson and À À Mad1 +/ Mad2 +/ MEFs. Six hundred metaphase spreads from each genotype Larry Faucette for excellent technical assistance, and Dr. Dong-yan Jin and members of were profiled. the Jeang laboratory for critical readings of the manuscript. www.aacrjournals.org 165 Cancer Res 2007; 67: (1). January 1, 2007

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