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MAD1) Increases the Incidence of Tumors in Mice

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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¶. Cancer Res 2007; 67: (1). January 1, 2007 160 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. MAD1 Mice and Tumors 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.
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