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Expression and Mutational Analyses of the Human MAD2L1 Gene in Breast Cancer Cells

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Expression and Mutational Analyses of the Human MAD2L1 Gene in Breast Cancer Cells GENES,CHROMOSOMES&CANCER29:356–362(2000) BRIEFCOMMUNICATION ExpressionandMutationalAnalysesoftheHuman MAD2L1GeneinBreastCancerCells MelanieJ.Percy,1 KenuteA.Myrie,2 ChristopherK.Neeley,1 JamesN.Azim,1 StephenP.Ethier,3 and ElizabethM.Petty1,2* 1DepartmentofInternalMedicine,UniversityofMichiganMedicalCenter,AnnArbor,Michigan 2DepartmentofHumanGenetics,UniversityofMichiganMedicalCenter,AnnArbor,Michigan 3DepartmentofRadiationOncology,UniversityofMichiganComprehensiveCancerCenter,AnnArbor,Michigan Breastcancerisaheterogeneousdisorderinwhichmosttumorsdisplaysomedegreeofaneuploidy,especiallythoseatlater stagesofthedisease.Aneuploidyandassociatedchromosomeinstabilitymaybeimportantintheprogressionofmammary tumorigenesis.Aneuploidyispreventedduringnormalcelldivisioninpartthroughregulationofamitoticspindlecheckpoint wheremitoticarrestpreventssegregationofmisalignedchromosomesintodaughtercellsatanaphase.Mitoticarrestgenes, includingtheMADfamily,whichwasoriginallycharacterizedinyeast,helpregulatenormalfunctionofthemitoticspindle checkpoint.DecreasedexpressionofthehumangeneMAD2L1waspreviouslyreportedinabreastcancercelllineexhibiting chromosomeinstabilityandaneuploidy.ToexplorefurtherthepotentialroleofMAD2L1inbreastcancer,weanalyzed MAD2L1geneexpressionin13minimallytogrosslyaneuploidhumanbreastcancercelllinesandfoundsignificantdifferences ofexpressioninthreelines.SequenceanalysisofMAD2L1cDNAintheseaswellasnineadditionalaneuploidbreastcancer andfiveimmortalizednormalhumanmammaryepithelialcelllinesrevealedoneheterozygousframeshift(572delA)mutation inacancercelllinethatdemonstratedahighleveloftranscriptexpression.Inaddition,two3ЈUTRsequencevariantswere notedinbreastcancercelllines.The572delAmutationcreatesatruncatedMAD2proteinproduct.Furtherfunctionalstudies inprimarybreasttumorsarethereforewarrantedtodeterminethepotentialroleMAD2L1mayplayinbreastcancer. ©2000Wiley-Liss,Inc. Breastcancercontinuestobeasignificantcause laterstagesofthedisease(Shackneyetal.,1995). ofmorbidityandmortalityintheWesternworld.It Thesignificanceofobservableaneuploidyinbreast iswidelyhypothesizedthatbreastcancerarises cancerisnotwellestablished,butcorrelateswith fromtheclonalaccumulationofmutationsingenes bothdiseaseaggressiveness(Tsudaetal.,1998)and regulatingnormalcellulargrowth,akintotheele- chromosomalinstability(CIN)(Duesbergetal.,1998; gantmultistepparadigmfirstrecognizedincolorec- Lengaueretal.,1998).Moleculardefectsresultingin talcancer(Fearon,1997).Themultisteppath- CINandaneuploidy,therefore,maycontributetothe way(s)inbreastcancerremainspoorlyunderstood, progressionofmammarytumorigenesis. however,especiallyforthecommonheterogeneous Normally,amitoticspindlecheckpointmonitors sporadicmammaryneoplasmsinwhichnopredis- propermicrotubuleattachmenttochromosomes posinggermlinemutationsexist.Examinationof priortoprogressionthroughmitosistoyieldeu- mutationratesinsomaticcellsimpliesthatthe ploiddaughtercellsthroughregulationofthean- clonalacquisitionofsufficientmutationstopro- aphasepromotingcomplex(APC)(Lengaueretal., motemultisteptumorigenesisisimprobableduring 1998).Yeastgenesimportantinthespindlecheck- ahumanlifetime(Loeb,1991;Orr-Weaverand pointincludemembersoftheBUB(buddingun- Weinberg,1998).Additionalmolecularmecha- inhibitedbybenomyl)andMAD(mitoticarrest- nismsmayberequiredtodrivethecelltowarda deficient)families.Earlyevidencesuggeststhat malignantphenotype.Insomesolidtumors,espe- alterationsofthehumanhomologsofthesegenes ciallysomecoloncancers,malignantprogressionis acceleratedwhenthecells’genomesarerendered unstablebyalterationsingenesthathavedefective Supportedby:NIH;ContractGrantnumber:KO8CA66613-01; DNAmismatchrepairability,allowingtherapid WendyWillCaseCancerFund(toEMP);ROI(toEMP);Grant accumulationofmutationstodrivetumorigenesis number:CA72877-02. *Correspondenceto:ElizabethM.Petty,DepartmentofInternal (Lengaueretal.,1998).Mostbreastcancersdonot Medicine,DivisionofMedicalGenetics,4301MSRBIII,University harbormismatchrepairdefectstoaccountforin- ofMichigan,1150WestMedicalCenterDrive,AnnArbor,MI 48109.E-mail:[email protected] creasedgenomicinstability;however,amajority Received11May1999;Accepted6June2000 exhibitsomedegreeofaneuploidy,especiallyat ©2000Wiley-Liss,Inc. HUMAN MAD2L1 GENE IN BREAST CANCER 357 may in fact be associated with human malignancy. indeed, alterations of this gene were important in Reduced expression of the human MAD2L1 tran- aneuploidy and breast cancer. script was previously reported in T47D, a human Ten SUM breast cancer cell lines, 12 American breast cancer cell line that demonstrated a defec- Type Culture Collection breast cancer cell lines, tive mitotic arrest response (Li and Benezra, 1996). and 5 human papilloma virus (HPV) immortalized However, further studies exploring the potential normal breast cell lines were initially available for role of MAD2L1 in breast cancer are lacking. study. SUM breast cancer and normal breast cell MAD2L1, located on 4q27, encodes a 205-amino- lines were developed at the University of Michigan acid protein that is associated with the kinetochore Comprehensive Cancer Center by Stephen P. of unattached chromosomes and inhibits APC, but Ethier and are well characterized with regard to is absent when chromosomes are correctly aligned their cytogenetic phenotype (Ethier et al., 1993, on the metaphase plate (Chen et al., 1996). MAD2 1996; Garcia et al., 1997; Sartor et al., 1997; Flana- protein exists as a monomer or as a tetramer, with gan et al., 1998; Forozan et al., 1998; Ignatoski and the latter state likely being the active form that Ethier, 1998). These cell lines were cultured in inhibits APC (Fang et al., 1998a, 1998b). Oligomer- Ham’s F-12 medium, either serum-free or with 5% ization of MAD2 or a conformation associated with FBS, in the presence of Fungizone and gentamy- the MAD2 tetrameric state may transduce the mi- cin. In addition, insulin, hydrocortisone and epider- totic checkpoint signal (Wolf and Jackson, 1998). mal growth factor or choleratoxin were included in Mutations in BUB1, a human homolog to another the medium (see http://p53.cancer.med.edu/clines/ yeast mitotic arrest gene, demonstrated functional clines/elab.html). The breast cancer cell line importance in human colorectal cancers exhibiting CAL51 was kindly provided by Dr. J. Gioanni, chromosome instability. Specifically, dominant Nice, France (Gioanni et al., 1990) and grown in Dulbecco’s modified Eagle medium (DMEM) sup- negative mutant BUB1 alleles in colorectal tumor plemented with 10% fetal bovine serum (FBS). cell lines promoted CIN and were associated with The remaining breast cancer cell lines were main- abnormal mitotic checkpoint responses (Cahill et tained according to the American Type Culture al., 1998). Lee et al. (1999) recently demonstrated Collection instructions. that mutations in Bub1 and Mad3L mitotic spindle Total RNA was isolated from all cell lines at ap- checkpoint genes can potentiate growth and cellu- proximately 80% confluency using Trizol reagent ac- lar transformation in a study of Brca2-deficient mu- cording to the manufacturer’s instructions (Gibco- rine cells. It would appear that defects in Brca2 and BRL, Grand Island, NY). For Northern blot analysis, mitotic spindle checkpoint genes could work to- 10 ␮g of total RNA from 13 breast cell lines was gether to overcome checkpoint controls in the cell fractionated on 1.25% agarose gel and transferred to cycle, thereby promoting cell division, resulting in Hybond Nϩ (Amersham Life Science, Piscataway, aneuploidy, and driving tumorigenesis. These find- NJ)in10ϫ SSC. A 1.3-kb MAD2L1 full-length RT- ings suggest that similar alterations in other mitotic PCR product (5-TGTCCGCGGAGTGGAAGC-3; arrest genes may play roles in breast cancer by 5-ACTTTATTTCCTCACTTTCA-3) and a 407-bp abolishing the normal mitotic checkpoint control GAPDH RT-PCR product (5-GGGAGCCAAAA- mechanism. Loss of normal control would allow GGGTCATCA-3; 5-TTTCTAGACGGCAGGTC- mammary epithelial cells to divide even when AGGT-3) were labeled by random priming with chromosomes are not correctly attached to the spin- [␣32P] dCTP. Hybridization was performed at 60°C dle, giving rise to aneuploidy and chromosomal overnight in Church buffer (1-mM EDTA, 0.5-M instability that may accelerate tumor progression. NaHPO4, 7% SDS). Filters were washed at 60°C in Because decreased expression of MAD2L1 was 2 ϫ SSC, 0.1% SDS, and exposed to Kodak XAR-5 previously described in one breast cancer cell line film. (Li and Benezra, 1996), we elected to explore fur- By Northern blot analyses, all MAD2L1 tran- ther involvement of MAD2L1 in breast cancer. We scripts were of the anticipated 1.4-kb size, and no analyzed MAD2L1 transcript expression in 13 aberrant transcripts were detected. MAD2L1 ex- breast cancer cell lines to help determine the fre- pression levels were visually assessed as compared quency of altered expression of MAD2L1 in breast to ethidium bromide-stained gels for loading and cancer. Given that many of these well-character- also standardized against glucose-3-phosphate-de- ized cell lines are markedly aneuploid, we hypoth- hydrogenase (GAPDH) gene expression levels by esized that they would be likely to have the highest calculating the ratio of MAD2L1 to GAPDH for all probability of harboring alterations in MAD2L1 if, cell lines as quantified by densitometry (data not 358 PERCY ET AL. script expression, but all attempts were made to minimize this possibility by harvesting the cells at 80% confluence. Southern blot analysis did not reveal any structural changes that could explain the variability in MAD2L1
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