[CANCER RESEARCH55, 1531—1539,April 1, 1995J

One of the familial syndromes of colon cancer, adenomatous polyposis coli or APC7 (10), was instrumental in the identification of achromosome 5-specific, putative tumor suppressor gene. Individualswith APC, an autosomal dominant disorder, develop hundreds tothousands of adenomatous colonic polyps, the precursors of coloncarcinoma, with a subsequent high risk of colon carcinoma (1 1).Linkage analysis mapped the APC phenotype to the long arm ofchromosome 5 (12, 13). Physical mapping of the locus led to thepositional cloning of the APC gene; mutational analyses showed thatinactivating germline mutations have occurred in affected individualsand that somatic mutations have occurred in colorectal tumors(14—17).These observations suggest that the APC gene functions asa tumor suppressor in colonic cells.

The APC gene (exons 1—15)contains an open reading frame of over8500 nucleotides; it produces at least 2 alternatively spliced RNAsfrom this region of the gene and is predicted to encode a large proteinof 2843 amino acids (14—17).Four exons, isolated subsequently, are5' to the originally published exon 1 (18, 19). These 4 exons, as wellas exon 1, also show alternative splicing.

In order to obtain direct biological evidence that the APC gene, andhence its protein product, functions as a tumor suppressor and toassess whether it is this gene that is responsible for the suppressioneffects observed with whole-chromosome transfers, a vector containing full-length APC eDNA was constructed and introduced into threecolon carcinoma cell lines. Each of these cell lines was characterizedfor mutations at the APC locus and for genomic instability. Theseexperiments have shown that at least one function of the normalfull-length APC gene is tumor suppression.

MATERIALS AND METHODS

Cell Culture. All cell lines were obtained from the ATCC collection andcultured according to the accompanying specifications at 37°Cand 5% CC)2;no antibiotics were used. All media and supplements were obtained fromGIBCO-BRL(Grand Island, NY), with the exception of the fetal bovine serum(Hyclone, Inc., Logan, UT). Puromycin (Sigma, St. Louis, MO) was used forselection of drug-resistant cell clones at the following concentrations: 1.0(HCF116); 25 (DLD-1,SK-MES-1,and W138-2RA); and 10 (SW4SO) @gml.

Minigene Construction. Insert fragments from the APC eDNA and cosmid were purified from agarose gels using GeneClean (Bio 101, Inc.). The twoDNA segments were ligated simultaneously into the insertion vector A-Zap(Stratagene, LaJolla, CA). The use of three different restriction enzymesreduced nonproductive ligations. Both hybridization and PCR amplificationwere used to verify that the correct assembly of APC sequences was present.Several independent phage clones were excised in vivo as Bluescnipt plasmids.One clone was sequenced completely to verify that the APC sequence wascorrect. The multiple fragments were ligated into the A-Zapvector as follows.Insert and vector fragments were mixed at equal molar concentrations. Aligation consisted of 250 ng of total DNA in a 20-gd reaction volume incubatedovernight at room temperature. Five gd of a ligation were packaged into Aphage using Gigapack Gold (Stratagene). The resulting phages were plated andtwo membrane lifts were made from each plate. Clones that hybridized with

7 The abbreviations used are: APC, adenomatous polyposis coli; SSCP, single strandconformational polymorphism; ATCC, American Type Culture Collection.

1531

Response of Colon Cancer Cell Lines to the Introduction of APC, a Colon-specific

Tumor Suppressor Gene'

Joanna Groden,2 Geoff Joslyn,3 Wade Samowitz, David Jones,4 Nitai Bhattacharyya, Lisa Spirio, Andrew Thilveris,Margaret Robertson, Sean Egan,5 Mark Meuth, and Ray %I@k@6Howard Hughes Medical Institute (J. G.. G. J., L S., A. T., M. R., R. WI; Departments of Pathology (W. S.J and Human Biology and Genetics (D. J., N. B., M. MI, The EcciesInstitute of Human Genetics; Section of Experimental Oncology, The University of Utah, Salt Lake City, Utah 84112 (N. B., M. MI; and The WhiteheadInstitute, MassachusettsInstitute of Technology. Cambridge, Massachusetts 02142 (S. E.J

ABSTRACT

The APC gene, mutations in which are responsible for the inheritedcolon cancer syndrome adenomatous polyposis coil (APC), Is described asa tumor suppressor gene. A ftill-length, wild-type APC gene was introduced by transfection into three human colon carcinoma cell lines, eachcharacterized for mutations atloci involved In colon tumor formation. Theresponse of each cell line to the Introduction of APC differed with thegenotype of the cell line. Some of the cell dones derived from thesetransfections displayed altered morphologles; some showed suppression oftumorigenicity based on growth in soft agar and tumor formation in nudemice. One cell line, SW4SO,could not be stably trnnsfected with the APCgene. These results provide the first direct evidence that the APC gene canalter the transformation properties of colon carcinoma cells.

INTRODUCTION

The development of colon carcinoma is associated with an accumulation of mutations in specific tumor suppressor genes and protooncogenes (1), as well as in genes that affect the ability of cells torepair DNA (2—5).The correction of tumorigenicity in colon carcinoma cell lines has been achieved by the reintroduction of some ofthese tumor suppressor genes by whole-chromosome and gene transfer (6—8), as well as by the inactivation of oncogenic Ki-ras byhomologous recombination (9). Therefore, the correction of specificmutations in tumors, whether by reintroduction or removal, can altergrowth capabilities or tumorigenicity and illustrate the role of aparticular gene in the formation of a colorectal tumor.

Two independent groups have reported inhibition of tumorigenicityby chromosome 5 in human colon carcinoma cell lines (7, 8). Singlenormal human chromosomes were introduced into two different coloncarcinoma cell lines by microcell-mediated chromosome transfer. Ineach case, chromosome 5 was introduced (as well as other singlechromosomes) to test tumor-suppressing ability. Both groups concluded that the introduction of normal chromosome 5 eliminated thetumorigenic phenotype of the parental cell lines, as judged by alteredmorphology, reduced cloning efficiency in soft agar, and suppressedtumongenicity of the hybrid cells in athymic mice and suggested thata gene (or genes) mapping to this chromosome was (were) responsiblefor this correction.

Received 8/24/94; accepted 1/25/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solelyto indicatethisfact.

I This work was supported in part by grants to J. G. from the American Cancer Society

(Aca-IRG-189), the American Gastroentemlogical Association, the American LungAssociation, the Tobacco Research Council, and the NIH (Grant CA63507). R. W. is aninvestigator with the Howard Hughes Medical Institute.

2 Present address: Department ofMolecular Genetics, Biochemistry and Microbiology,

The Universityof CincinnatiCollegeof Medicine,Cincinnati,OH45267-0524.3 Present address: Sequana Therapeutics, Inc., San Diego, CA 92037.

4 Present address: Cardiovascular Research, 7243-209-3, The Upjohn Company, 301

Henrietta Street, Kalamazoo, Ml 49007-4940.5 Present address: Division of Immunology and Cancer Research, The Hospital for

SickChildren,555UniversityAvenue,Toronto,OntarioM5G1X8Canada.6 To whom requests for reprints should be addressed, at University of Utah, Health

Sciences Center, Building 533, Suite 7410, Salt Lake City, UT 84112.

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TUMOR SUPPRESSION BY THE APC GENE

probes derived from two different insert fragments were isolated, and thefidelity of the cloning was determined by PCR analysis. Final A-Zap cloneswere converted into pBluescript clones using the in vivo excision protocolsupplied by Stratagene.

The APC construct contained only the APC-coding sequence flanked by theBamHI restriction sites, a NotI site at the 5'-end, and a Sad site at the 3'-endto make subcloning into other vectors easier. The final cloning was assessed inthree ways: (a) all restriction sites used in the minigene construction weredetermined to be present by restriction digestion; (b) the PCR-generatedsequence and the cloning-junction fragments were sequenced; (c) the constructwas cut into three fragments by digestion with BamHI and HindIII and thencloned into pGEX gst fusion protein vectors. All three fragments produced afusion peptide of the expected size, indicating an absence of frame-shift

mutations.

All restriction digests and ligations were performed using buffers suppliedby the manufacturer.

PCR and DNA Sequencing. DNA templateswere amplifiedfor analysisusing PCR with PCR primers published previously (14, 20, 21). Reactions forsequencing were purified by a centrifugation wash with a Centncon 100column (Amicon, Inc.). These samples were stored until ready for sequencingon the Applied Biosystems model 373A DNA sequencer(Applied BioSystems,Foster City, CA). DNA sequencing ofthe APC minigene was performed usingthe dideoxy chain-termination method with Taq polymerase and fluorescentlytagged M13 universal or reverse sequencing primers. Direct sequencing of theAPC alleles of HCT 116 was performed on biotinylated PCR products using amodification of ABI user bulletin 21 for use with the ABI PRISM 11terminators kit (Applied Biosystems), as described by Spirio et a!. (21).

PCR-SSCP Analysis. PCR-SSCP and sequencing analyses of DNAconformers were performed as described by Groden et a!. (14, 20).

PCR-Dinucleotlde Repeat Analysis. One @gof DNA purified fromHCT1 16 and its subclones was analyzed for variation in repeat length at fiveloci (D11S527, D16S289, D16S295, D16S303, and D19S178) as describedelsewhere (22). PCR products were fractionated on a 6% polyacrylamide geland transferredto Hybond-N+ (Amersham).Blots were then probedwith one ofthe primers that was end-labeledwith terminal transferase(Life Technologies).

Western Blotting. Cytoplasmic protein was extracted from cell lines asdescribed elsewhere (23). Protein extracts representing approximately 5 X 106cells were electrophoresed on a 4.5% Thcine-SDS-polyacrylamide gel andtransferred for 2 h at 100 V onto nitrocellulose in transfer buffer [25 mMTris-192 mM glycine-20% v/v methanol (pH 8.3)] (24). Membranes wereprobed with anti-APC antibody (AB-1), and binding was detected by chemiluminescence as described previously (25).

DNATransfections. DNAtransfectionswere performedusing the transfection reagent DOTAP according to the directions of the manufacturer (Boehringer Mannheim). Cells were plated 2 days before transfection and hadreached a density of approximately 80% confluency. DNA (2.5 @.tg)was usedfor each transfection/60-mm plate. Transfections were performed in mediumwithout serum; complete medium was added 24 h after transfection. Selectionwas begun 1—2days after that, either by the addition of selective medium to theplate or by the dilution of the cells into four 96-well tissue culture plates at 4dilutions (1:5, 1:10, 1:50, and 1:100) in selective medium. After cells weregrown at 37°Cand 5% CO2 for 2—4weeks, actively growing cells wereisolated from the dishes with cloning cylinders or by trypsinizing the cellsdirectly from the individual wells of the 96-well plates. Cell lines then wereexpanded, cultured for further experiments, and stored at —135°C.

Soft Agar Cloning Assay. Cells were trypsinized into suspension, countedwith a hemocytometer, and resuspended at the appropriate concentration foreach plating experiment (10,000, 5,000, 1,000, or 500 cells/plate). The cellswere mixed with 2 ml of plating agar (0.33% plating agar and 1X regular cellculture medium) and overlaid on 5 ml of a base containing 0.5% agar and 1Xregular cell culture medium. The agar was allowed to solidify at room temperature for 30 mm; the plates were incubated at 37°Cand 5% CO2. Cultureswere fed once a week by the addition of 1.5 ml of plating agar. Colonies werescored at 4 weeks after the initial plating by photographing the plates andcounting the visible colonies from the photographs.

Nude Mouse Injections. Five-week-oldfemale CD-i nude mice wereobtained from the Charles River Laboratories and given injections of either i07or 2 X 106 cells in 0.2 ml of sterile PBS/injection site. Mice were housed inmicroisolators in the University of Utah Animal Resource Center for the

duration of the experiments. All mice were cared for and treated in accordancewith Institutional Animal Care Use Committee guidelines.

Northern Blot Analysis. Total RNA was isolated from HCF116, lOBCli, and 1OA-BB4cell lines as described by Chomczynski and Sacchi (26).Ten @.tgof total RNA were electrophoresed through formaldehyde-containingagarose gels and pressure blotted onto Biotrans (+) nylon membrane (ICN). A500-base pair c-myc probe was generated by reverse transcription-PCR as

described by Watt et a!. (27) and labeled to a specific activity of >iO@cpm/,.&gDNA with [32P)dCFP by the random primer-labeling method. Membraneswere prehybridized in Quikhyb (Stratagene) for 2 h at 42°C.The cDNA probewas added directly to the hybridization solution to a concentration of 106cpm/mI and incubated at 42°Cfor 16 h. Excess probe was removed by two20-min washes in 1X SSC-0.l% SDS at 25°Cfollowed by two more 20-mmwashes in 0.1x SSC-0.i% SDS at 65°C.Autoradiography was performed for1—6days using intensifying screens at —70°C.The blot was then stripped byboiling on 0.1% SDS and probed as above using a probe specific for the controlRNA, glyceraldehyde-3-phosphate dehydrogenase.

Determination of Growth Rates. Cells of each cell line were seeded in 2ml of selective medium in 30-mm tissue culture dishes at an initial number of2000/plate. Three plates were counted for each cell line at each 24-h time point

for 7 days, and a mean number was calculated and plotted.

RESULTS

Mutational Analysis of Human Colon Cancer Cell Lines at the,tPc Locus Using PCR-SSCP Analysis, DNA Sequencing, andWestern Blotting. A group of cell lines derived from colorectalcarcinomas was assembled and their morphology, growth characteristics, and APC mutations were characterized. These cell lines included DLD-1, HCT116, and SW480, as well as two cell lines derivedfrom lung, SK-MES-1 and W138—2RA.SK-MES-l originated from alung adenocarcinoma; W138—2RA is a cell line derived by SV4Otransformation of a normal lung fibroblast cell line (W138) but whichdisplays an epithelial-like morphology.

DNA, RNA, and protein were purified from each of these cell linesand examined for APC mutations using the PCR-SSCP analysisand/or Western blotting (14, 20). These cell lines were also genotyped

with a dinucleotide repeat marker (D5S346) that maps physicallywithin 70 kilobases of the 3'-end of the APC gene (22).

One of these cell lines, SW480, had been shown previously to carrya premature stop codon in the APC open reading frame at position4017 (17). No other PCR-SSCP anomalies were observed in this cellline during the analysis ofthe APC gene. SW480 appeared to be hemior homozygous for this mutation, because no normal conformers wereobserved, and only one allele was present at the adjacent, highlyinformative D5S346 locus.

Colon carcinoma cell line DID-i carries a single nucleotide ddetion at position 4251 of the APC gene. This was originally detected byPCR-SSCP analysis and confirmed by direct sequencing (data notshown). The pattern of SSCP conformers did not include those bandsexpected from a normal allele, suggesting a hemizygous or homozygous mutant APC genotype. However, heterozygosity at D5S346suggested that one allele may be partially deleted.

No APC mutations were detectable by PCR-SSCP in cell lineHCF116. Direct sequencing ofAPC in this cell line also did not revealany mutations in the open reading frame. Interestingly, heterozygositywas not observed at any of the known polymorphic nucleotides of theAPC gene, its introns, or within the 3'-untranslated region. However,D5S346 was heterozygous in HCT1 16.

APC protein from these cell lines was examined using Westernblots. Fig. 1 shows that cell lines SW480 and DLD-1 contain nofull-length APC protein but do contain truncated protein productscorresponding in size to those predicted by the locations of the

respective DNA mutations. No truncated proteins were observed in

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TUMOR SUPPRESSION BY THE APC GENE

1 2 3 4 ofKi-ras;andasecondthatisassociatedwithgenomicinstabilityanda low incidence of ras and p53 (32). Therefore, three geneticallydistinct colon carcinoma cell lines were chosen for the following APCtransfection assays.

‘IAn KD These three colon cancer cell lines and the two lung cell line

@w were transfected, by means of lipofection, with the APC minigene

construct in the pBabe vector. Each cell line also was transfectedwith the vector containing no insert, as a control for the efficiency

97.0KD— ofthegenetransfer.Transfectionofeachcelllinewasrepeatedat69.0KD leastonceandusuallytwice,withdifferentamountsofDNA,toallow

establishment of puromycin-resistant clonal cell lines from independenttransfections.

Following the lipofection, cells were allowed to grow for 2 days inregular growth medium. Then, selection with puromycin was begun,either in the original dish or by trypsinizing the cells; diluting them1:5, 1:10, 1:50, or 1:100; and plating them with selective medium in96-well dishes. Each transfected cell line was grown in a unique,previously determined concentration of puromycin designed to killnontransfected cells. Dishes were incubated until individual cell colonies were visible in the dishes or until some of the wells in at leastone of the four 96-well dilution plates exhibited a pH change thatindicated actively growing cells.

cell line HCF116; this cell line does contain full-length (or nearlyfull-length) APC protein.

The three colon carcinoma cell lines also were analyzed for instability of microsatellite DNA (dinucleotide repeats and other polymorphic DNA sequences). High frequency variation in the length of theserepeats has been reported in some colorectal carcinomas (28—30).Noevidence for this type of genomic instability was found for SW480 orDLD-1. In contrast, HCT116 exhibited pronounced instability ofdinucleotide repeats. Repeat lengths at six loci on four chromosomeswere analyzed in subclones of HCT116 derived from single cells.Length variation was found at all loci tested, and all of the subclonesexhibited instability for at least one locus. Both expansion and ddetion of repeat units were observed. Fig. 2 shows the DNA repeatpatterns observed in subclones of HCF116 at one locus (D16S195). Asimilar analysis of subclones of another colorectal carcinoma cell line,SW620 (derived from the same tumor as SW480), showed no suchvariation (Fig. 2).

Transfectlon of Colon Cancer Cell Lines with an APC Minigene. The genomic structure of the APC gene facilitated minigeneconstruction. The APC open-reading frame is contained within 15exons; the first 14 exons comprise 2 kilobases of coding sequencewhile the remaining 6.5 kilobases of coding sequence constitute asingle exon. One previously isolated cDNA contained the first 14exons and a small portion of exon 15 (15). This cDNA also containedexon 9A which is absent in an alternative-splice form of the APCmRNA. Exon 15 was isolated from a single genomic cosmid clone. Itwas possible, therefore, to reconstruct the complete APC codingsequence by joining these two cloned DNA segments. PCR was usedto generate BamHI restriction sites at each end of the APC codingsequence to facilitate cloning.

The final APC “BamHIcassette―was inserted into pBRBabe-puro,a modified version of the mammalian expression vector pBabe-puro(31). pBabe-puro had been modified by replacement of the pINSreplication origin by a pBR322 origin to derive pBRBabe-puro. Bothvectors carry a puromycin resistance gene for selection, Moloneymurine leukemia virus long terminal repeats, and a multiple cloningsite with a BamHI site.

Recent work has suggested that at least two pathways in thedevelopment of colon cancer exist: one that proceeds through theinactivation of a number of tumor suppressor genes and the activation

46.030.021.514.3

KD —KDKDKD —

.*.@@@@

Fig. 1. Western blot of APC protein in four cell lines detected with a mAb (AB-1)directed to the first heptad repeat of the APC protein. Lane I, protein from HCI'1 16; Lane2, proteIn from SW480; Lane 3, protein from DLD-1; Lane 4, protein from a lymphoblastoidcelllinederivedfromanAPCpatient.Lefiordinate,sizemarkeNKD,molecularweightin thousands

A

I 2 3 4 5 6 7 8 9 1O11121314@C@,,@c'

ftI.II*ts4iw

B I1 2 3 4 5 6 7 8 91011 1213 c@

Fig. 2. Analysis of dinucleotide repeat variation in subclones derived from HCT116(A) and SW620 (B). The microsatellite marker D16S295 was amplified by PCR andproducts were fractionated on 6% polyacrylamide gels. Subclones of HCT116 and SW620are numbered. D16S295 is polymorphic in both HCF116 and SW620; new alleles areevident in subclones derived from HCF116 while subclones of SW620 show no variation.

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Table 1Drug-resistant cell lines generatedfromeachtransfectionExperiment

no. and DNAusedDLD-1HCfl16SK-MES-1SW4SOW138-2RA1.

pBRBabe1. pBRBabe + APC2. pBRBabe2.pBRBabe+APC3.pBRBabe3.pBRBabe+APC4. pBRBabe4. pBRBabe + APC5. pBRBabe5. pBRBabe+ APCM@a

NDND6(1)012(1)NDNDNDND6

13 (4)bND

23(5)541(12)

NDNDNDNDND

NDNDNDND18(0)ND

>30NDNDND

NDNDND30

13060ND

NDNDNDND19(0)ND

>30ND

NDa

ND, not yet determined.

b Numbers in parentheses show the originalnumber of cell lines generated with an altered phenotype.

TUMOR SUPPRESSION BY THE APC GENE

The successfully transfected cells then were cloned, either withcloning cylinders or by dilution into multiwell dishes (the cells growing in the few wells that showed growth were clonal, having resultedfrom inoculation with a single cell that had taken up the drug resistance gene). These drug-resistant clones were expanded into individualcell lines and used as a source for DNA, RNA, and protein analyses.Each of the derived cell lines was maintained in the tissue culturelaboratory so that any obvious changes in growth characteristics, life

span, or morphology could be observed.The number of drug-resistant cell clones obtained from each gene

transfer can be seen in Table 1. Cell lines HCF116, SK-MES-1, andWI-38-2RA transfected efficiently and generated numerous clones;

transfection of cell line DLD1 generated a small number of clones. Nocell lines were established from multiple attempts to transfect SW480with APC, although drug-resistant cell lines were obtained followingtransfection with the control plasmid.

Transfection of Colon Cancer Cell Lines DLD-1 and HCT116

with the APC Minigene Results in Morphological Changes inSome Cell Clones. Dramatic changes in morphology were observedin some of the drug-resistant cell clones derived by transfection of twoof the parental cell lines derived from colorectal carcinomas with theAPC minigene (Figs. 3 and 4). No morphological changes wereobserved in more than 100 clones derived from either of the two lungcell lines or in any of the drug-resistant cell lines derived bytransfection with the selectable marker alone.

The morphological differences associated with APC gene transferwere extreme and similar, whether the cell lines were derived fromDLD-1 or from HCT116. Cells were larger and flatter than theirparental counterparts; their nuclei were larger and more distinct.Transfected cells often contained micronuclei. Numerous cells in amicroscopic field exhibited small granuoles; others contained manylarge vacuoles; often, a third cell type could be found that wasenlarged and filled with numerous, somewhat refractile vacuoles thatobscured the nucleus. Some of these characteristics are evident inFigs. 3 and 4.

In Vitro Characterization of APC-transfrcted Cell Clones.Three of the cell lines derived from APC transfection of HCF116 were

examined for differences other than the altered morphologies described above. Added to this group as a control was the parental cellline, HCF116. The three transfected cell lines were lOB-Cu (transfected with pBRBabe-puro), 10-1-5, and 1OA-BB4 (transfected withpBRBabe-puro+APC). Both 1OA-BB4 and 10-1-5 exhibited thealtered phenotype.

The parental cell line HCF116 had been shown previously to havea population-doubling time of 20—22h (33). To assess the effect ofAPC transfection on cell growth, the population-doubling time foreach of the cell clones selected above was determined by seeding 7pairs of tissue culture dishes with 1000 cells each and counting the

number of cells present in each pair of dishes at the same time everyday. The results of these cell counts can be seen in Fig. 5 and show a

decrease in the population doubling time for cell line 10-1-5 ascompared to the parental HCF 116 line and lOB-Cu.

No change is apparent in the life span or immortality of any of thecell lines investigated. After 14 months in culture, none has exhibitedany sign of senescence or cell death, regardless of the morphologicalphenotype.

Southern blotting was used to characterize each cell line transfectedwith the APC construct, to determine which contained the 8.5-kilobase APC insert. DNAS were digested with BamFll restriction endonuclease, which cleaves each end of the APC cassette; electrophoresedin agarose gels; and blotted onto nylon membrane. Blots were probedwith a PCR-generated, radiolabeled DNA probe amplified from 450nucleotides of exon 15 of the APC gene. The bands observed on thefilms showed a complicated pattern of hybridization that included inalmost all cases an 8.5-kilobase band; these results suggested that theinsertion events were complex for all the cell lines (data not shown).All samples also showed a band of >20 kilobase, derived from agenomic exon 15 sequence. However, PCR amplification with prim

em placed in exons 1 and 9 generated a product (which requires exonconnection) in allAPC-transfected cell lines, suggesting that a portionof the APC minigene was present in each, regardless of morphologicalphenotype.

Effect ofAPC Transfection on Tumorigenicity. The same groupof three cell lines derived from HCT116 were characterized withrespect to tumorigenic potential. At the time of its submission to theATCC, cell line HCT116 had been found to grow colonies in soft agarand to generate tumors in 100% of the animals into which it wasinjected (ATCC catalogue). We reexamined this property in the transfected derivatives by: (a) characterizing the ability of each cell line togrow in a soft agar cloning assay, often considered representative oftumor-forming ability; and (b) by testing each cell line for its abilityto form tumors in athymic mice after sc. injection of cells.

Results from the soft agar cloning assay can be seen in Table 2.The failure of cell lines 1OA-BB4 and 10-1-5 to grow any coloniessuggested that their ability to form tumors had been suppressed.The control cell lines, HCT116 and lOB-Cu, formed colonieseasily.

Each of the cell lines was then tested directly for its ability to elicittumors in mice. Injections consisted of either 2 X 106 or 1 x iO@cells.The results can be seen in Table 3. The injected mice that did not formtumors were still tumor-free after 3 months. Mice that did form tumorswere sacrificed when their tumors were of sufficient size to score aspositive. Each tumor was grown in tissue culture and was shown to bepuromycin resistant. PCR verified that the presence of contiguous

DNA sequences from the first 14 APC exons and that the origin ofeach tumor was human.

Expression of c-myc Is Unmodified in the APC-transfected CellLines. Certain colorectal tumors display disregulated expression ofthe transcription factor c-myc (9, 34, 35). To investigate whether arelationship exists between APC mutation and c-myc disregulation, we

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TUMOR SUPPRESSIONBY ThE APC GENE

Fig. 3. Micrographs of HCT116 cells and cells derived from HCT116;A, cell line HCF116; X 160. B, cells derived from transfection with pBabe-puro only; X 160. C, cells derivedfrom transfection with pBabe-puro plus APC; X 160. D and E, cells derived from transfection with pBabe-puro plus APC; X 400.

examined RNA levels in control cells and in cells containing the APC DISCUSSIONminigene. Northern analyses demonstrated similar levels of c-mycRNA in the HCT116, lOB-Cu (vector only), and 1OA-BB4 (vectorplus APC) cell lines. A control RNA, glyceraldehyde dehydrogenase,also remained constant in the three cell lines, verifying equivalentamounts of RNA on the blot (Fig. 6). These experiments wererepeated three times.

These experiments provide the first functional evidence that theAPC gene can act as a tumor suppressor and can even be cytotoxic orcytostatic when overexpressed in some colon cancer cell lines. Atumor suppressor role for APC has been suggested by the associationof the inherited cancer syndrome with mutations in the gene, by the

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aS

TUMOR SUPPRESSION BY ThE APC GENE

‘- .,

@,@

V

@IYz\ ‘I

/ /

C

Fig. 4. Micrographs of DLD-1 cells and cells derived fromDLD-1; X 160. A, cell line DLD-1; B, cells derived fromtransfection of DLD-1 with pBabe-puro plus APC.

.,4

losses of heterozygosity affecting 5q, and through direct mutationalanalyses of APC in colorectal tumors. Our data indicate that if theAPC gene is reintroduced into either of two colon carcinoma cells, adramatic morphological alteration can occur. This alteration also canaffect cell growth and the ability of the transfected cells to formtumors. These transfection studies also suggest that the introduction ofAPC into one cell line, SW480, which carries only mutant APC and

is not characterized by any genomic instability, results in cytotoxicityor cell death.

In the experiments described here, we have chosen cell lines established from three colorectal carcinomas as recipients of APC.However, only two of these cell lines, DLD-l and SW480, carryinactivating APC mutations. Cell lines SW480 and DLD-l sharetypical characteristics of colorectal carcinomas: both contain chainterminating APC mutations; both are hemi- or homozygous for these

mutations; neither contains full-length APC protein; and both containtruncated APC protein. In contrast, cell line HCT1 16 containsapparently full-length APC protein; no truncated APC product wasobserved, nor were APC mutations detected with PCR-SSCP analysis or by direct sequencing of the APC open reading frame. It isnot known whether the APC protein detected in HCT1 16 at theapproximate size of 300 kilobases is functional. Although notruncated APC product is seen in HCT1 16, it remains possible thatit carries one mutant APC allele. Numerous cell lines and tumorswith mutations near the 5'-end of the gene lack a detectable mutantAPC protein. The negative results obtained with PCR-SSCP anddirect sequencing likewise cannot rule out the presence of a mutation in one of the APC alleles that affects the more 5' orregulatory regions of the gene. However, the fact that other colontumor cell lines also contain apparently full-length APC protein

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Table 2Soft agar colony-forming assays for theAPC-transfected celllinesColony

no.10,000

cells5,000 cells1,000 cells500cellsCelllineplatedplatedplatedplatedHCI'1161133931lOB-Cu1694010-1-500001OA-BB40000

000

w

z-j@1

Uia

150

100

50

0

Fig. 5. Growth rate of APC-transfected cell line HCI'l 16-10-1-5 and vector-onlytransfected cell line HCT116-lOB-Cl 1.

Cell line1 X iO@cells2 X 106cellsHCT1I63/3NDalOB-Cu3/32/210-1-52/30/61OA-BB4ND0/6HS578BST0/1NDPBS0/3NDa

ND, not yet determined.

TUMOR SUPPRESSION BY THE APC GENE

one gene can be assessed, rather than that of many genes, as withwhole-chromosome transfer. The experiments presented here suggestthat the action of the normal APC gene alters the morphology andtumorigenic characteristics of the HCT1 16 cell line and at least themorphological characteristics of DLD-1. We have also shown thatAPC may not directly down-regulate the expression of c-myc in cellline HCT1 16 and suggest that another gene(s) on chromosome 5 maybe responsible for the effect observed in whole-chromosome transfers(8, 31, 34). This observation is in contrast to data demonstrating that

the transfer of chromosome 5 facilitates the down-regulation of c-mycin colon carcinoma cell line SW480 and suggests that genes otherthan, or in addition to, APC may be required for c-myc regulation (8,31, 34). Recent work by Shirasawa et a!. (9) showed down-regulationof c-myc expression when the activated Ki-ras gene was disrupted byhomologous recombination in two of the same colon-carcinoma celllines used in our experiments (HCT1 16 and DLD-1). In sum, experiments suggest that perhaps the down-regulation of c-myc reflects a

8 general, decreased growth potential of cells when activated Ki-ras isdisrupted or normal APC is introduced.

Mutations of the APC, Ki-ras, or p53 genes are considered necessary but individually insufficient for colorectal tumor formation. Thedisruption of activated Ki-ras in cell lines DLD-l and HCF1 16 and

the subsequent correction of tumorigenicity in these cell lines suggested that this single genetic alteration could revert both cell lines toa more normal phenotype (9). Single chromosome transfer of chromosome 5 or 18 also is able to revert tumorigenic cells to a morenormal phenotype (7, 8), as is the reintroduction of a normal p53 gene(37). Our experiments suggest that introduction of APC by itselfaccomplishes a similar alteration in the phenotype of tumorigenic

cells. Additionally, SW480 is characterized by a 12VaI mutation inKi-ras and 273Mg to His and 3°@Proto 5cr in p5.3; DLD-l is characterized by a ‘3Aspmutation in Ki-ras and a 2415er to Phe in p53;HCT116 is characterized by a 13Asp in Ki-ras, no detectable mutationin p53, and a high degree of microsatellite instability (38). Theseresults support the idea that more than one mutation is required fortumorigenicity and that these mutations must accumulate in somecomplementary way to confer tumorigenicity.

The reasons for the different responses among the three cell lines tothe introduction ofAPC are unknown. Addition of full-length APC to

0 2 4 6

DAYS

Table 3 Tumorformation in nude mice by the APC-transfected cell lines

No. of mice forming a tumor/no. injected

(25) first suggested that another mutational pathway to coloncarcinoma might exist and that it represented a significant route totumor formation.

Mutational analyses of colonic tumors have revealed that a smallproportion are characterized by genomic instability at dinucleotide

repeat and other highly polymorphic DNA loci (28—30).These findings have led to speculations that alternative genetic pathways orgenotypes may be associated with colon cancer. The recent identification of four hereditary nonpolyposis colon cancer genes (hMSH2,hMLHJ, PMSJ, and PMS2) as homologues of bacterial DNA mismatch repair proteins has confirmed this idea (2—5,36). In ourexperiments, HCT1 16 displayed microsatellite instability, in additionto the retention of apparently full-length APC protein. Therefore, it isa cell line that is representative of one of these alternative pathways tocolorectal tumor formation.

The results of our experiments with HCT116 and DLD-l suggestthat the introduction of APC into a colon carcinoma cell line cancomplement the abnormalities that cause tumongenicity. Through theintroduction of a single gene into colon carcinoma cells, the effect of

123456

A •ôe..• 2.4kb

c@myc

B@ 1.3kb

GAPDHFig. 6. Expression of the oncogene c-myc in HCF1 16 cells transfected with the APC

minigene. Northern blots are shown that were probed sequentially with cDNAs specificfor human c-myc (A) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (B).Lanes I and 4, RNA from HCI'116; Lanes 2 and 5, lOB-Cu; Lanes 3 and 6, 1OA-BB4.in, kilobases.

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TUMOR SUPPRESSION BY THE APC GENE

cells may elicit different phenotypes depending on the genotype of thecell line at the APC locus and perhaps at other loci as well. The factsthat both SW480 and DLD-1 contain only truncated APC protein andthat these truncated APC proteins are almost identical in size do notseem to explain satisfactorily the difference in their response tonormal APC. The absence of any drug-resistant clones from thetransfection of SW480 with the APC minigene is remarkable, giventhat drug-resistant clones were easily derived from the transfection ofvector alone into this cell line. Perhaps, this is due to apoptosis ortoxicity caused by the overexpressed APC gene. The low number ofclones derived from the transfection of DLD-l, either with the vectoralone or with the APC minigene, may reflect the low transfectionefficiency characteristic of this cell line.

One caveat of gene transfer is that the level of protein expressionmay not be physiologically appropriate for the cell. However, thesimilarity of the results generated previously from whole-chromosome transfer and gene transfer experiments reported here suggest that

tumor suppression is possible with either method. Also, our resultswith WI38-2RA and SK-MES-1, cell lines without mutant APC andwith normal-size protein, suggest that a cell can tolerate at least someoverexpression of APC without phenotypic consequences, even whena normal amount is already present. It also is suggestive of some celltype specificity.

The presence of full-length APC protein in cell line HCT1 16 priorto the transfer of the APC gene confounded our ability to distinguishquantitatively the endogenous from the exogeneous APC protein.Work is in progress to characterize the levels of exogenous APCexpression from the transfected cell clones.

The function of APC is as yet unknown, although the association ofAPC with catenins in the cytoplasm of cells suggests that it has a role

in the maintenance of cell-cell interaction through adherens junctions(39,40).PerhapstheoverexpressionofAPCincell linesderivedfromthe transfection of cell line HCT1 16 results in a complementation ofother abnormalities of this tumorigenic cell line that have to do withthe maintenance of cell-cell contacts. Its cytotoxic and cytostaticeffects in the other cell lines suggest that APC may have other as yetunknown functions in the cell that control cell growth or cell death.How APC fulfills these functions is unknown and awaits furtherexperimentation. Which domains of the APC protein are responsiblefor effecting these types of controls can be addressed by breaking thegene into smaller parts and testing each for its ability to correctuncontrolled cell growth, tumorigenicity, genomic instability and/orits ability to regulate cell differentiation and morphology. The response of cell line SW480 to APC introduction is also of great interest,because its cytotoxic effects in this cell line are dramatic. The resultsof such experiments will be especially relevant for the design ofappropriate gene therapies for colon cancer treatment.

ACKNOWLEDGMENTS

We thank Jay Morgenstern for the pINS and pBabe-puro plasmids and RuthFoltz for the critical reading of the manuscript.

REFERENCES

1. Fearon, E. R., and Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell,61: 759—767,1990.

2. Fishel, R., Lescoe, M., Rao, M. R. S., Copeland, N., Jenkins, N., Garber, J., Kane, M.,Kolodner, R. The human mutator gene homologue MSH2 and its association withhereditary nonpolyposis colon cancer. Cell, 75: 1027—1038,1993.

3. Leach, F. S., et a!. Mutations of a mutS homologue in hereditary nonpolyposiscolorectal cancer. Cell, 75: 1215—1225,1993.

4. Papadopoulos, N., et a!. Mutation of a mutL homologue in hereditary colon cancer.Science (Washington DC), 263: 1625-1629, 1994.

5. Bronner, C. E., Baker, S. M., Morrison, P. T., Warren, G., Smith, L G., Lescoe,M. K., Kane, M., Earabino, C., Lipford, J., Lindblom, A., Tannergard, P., Bollag,

R. J., Godwin, A. R., Ward, D. C., Nordenskjold, M., Fishel, R., Kolodner, R., andLiskay, R. M. Mutation in the DNA mismatch repair gene homologue hMJR1 isassociated with hereditary non-polyposis colon cancer. Nature (Land.), 368:258—261,1994.

6. Baker, S. J., Fearon, E. R., Nigro, J. M., Hamilton, S. R., Preisinger, A. E., Jessup,J. M., vanTuinen, P., L.edbetter, D. H., Barker, D. F., Nakamura, Y., White, R., andVogelstein, B. Chromosome 17 deletions and p53 mutations in colorectal carcinoma.Science (Washington DC), 244: 217—221,1989.

7. Tanaka, K., Oshimura, M., Kikuchi, R., Seki, M., Hayashi, T., and Miyaki, M.Suppression of tumorigenicity in human colon cancer cells by introduction of normalchromosome 5 or 18. Nature (Lond.), 349: 340—342,1989.

8. Goyeue, M. C., Cho, K. R., Fasching, C. L, Levy, D. B., Kinzler, K. W., Paraskeva,C., Vogelstein, B., and Stanbridge, E. J. Progression of colorectal cancer is associatedwith multiple tumor suppressor gene defects but inhibition of tumorigenicity isaccomplished by correction of any single defect via chromosome transfer. Mol. Cell.Biol., 12: 1387—1395,1992.

9. Shirasawa, S., Furuse, M., Yokoyama, N., and Sasazuki, T. Altered growth of humancolon cancer cell lines disrupted at activated Ki-ras. Science (Washington DC), 260:85—88,1993.

10. Gardner, E. J., and Richards, R. C. Multiple cutaneous and subcutaneous lesionsoccurring simultaneously with hereditary polyposis and osteomatosis. Am. J. Hum.Genet., 5: 139—147,1953.

11. Burt, R. W. Polyposis Syndromes. In: T. Yamada, D. H. Alpers, C. Owyang, D. W.Powell, and F. E. Silverstein (eds.), Textbook of Gastroenterology, Vol. 2, pp.1674—1696.Philadelphia: J. B. Lippincoti Co., 1989.

12. Bodmer, W. F., Bailey, C., Bodmer, J., Bussey, H., Ellis A., Gorman, P., Lucibello,F., Murday, V., Rider, S., Scambler, P., Sheer, D., Solomon, E., and Spurr, N.Localization of the gene for familial adenomatous polyposis on chromosome 5.Nature (Lund.), 328: 614—616,1987.

13. Leppert, M., Dobbs, M., Scambler, P., O'Connell, P., Nakamura, Y., Stauffer, D.,Woodward,S., Burt,R. W., Hughes,J. P., Gardner,E. J., Lathrop,M., Wasmuth,J.,L.alouel,J-M., and White, R. The gene for familial polyposis coli maps to the longarm of chromosome 5. Science (Washington DC), 238: 1411—1413,1987.

14. Groden, J. Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L, Albertsen, H.,Joslyn, G., Stevens, J., Spirio, L., Robertson, M., Sargeant, L, Krapcho, K., Wolff, E.,Burt, R. W., Hughes, J. P., Warrington, 3., McPherson, J., Wasmuth, J., Lu Paslier, D.,Abderrahim, H., Cohen, D., Leppert, M., and White, R. Identification and characterization of the familial adenomatous polyposis coli gene. Cell, 66: 589-600, 1991.

15. Joslyn, G., Carlson, M., Thliveris, A., Albertsen, H., Gelbert, L, Samowitz, W.,Groden, J., Stevens, J., Spirio, L, Robertson, M., Krapcho, K., Sargeant, L, Wolff,E., Burt, R., Hughes, J. P., Warrington, J., McPherson, J., Wasmuth, J., Lu Paslier, D.,Abderrahim, H., Cohen, D., Leppert, M., and White, R. Identification of deletionmutations and three new genes at the familial polyposis locus. Cell, 66: 601—613,1991.

16. Kinzler, K. W., Nilbert, M. C., Su, L-K., Vogelstein, B., Bryan, T. M., Levy, D. B.,Smith,K.J.,Preisinger,A.M.,Hedge,P.,McKechnie,D.,Finniear,R.,Markham,A.,Groffen, J., Boguski, M. S., Altschul, S. F., Horti, A., Ando, H., Miyoshi, Y., Miki,Y., Nishisho, I., and Nakamura, Y. Identification of FAP locus genes from chromosome 5q21. Science (Washington DC), 253: 661—665,1991a.

17. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horn, A., Koyama, K,Utsunomiya, J., Baba, S., Hedge, P., Markham, A., Krush, A. J., Peterson, G.,Hamilton, S. R., Nilbert, M. C., Levy, D. B., Bryan, T. M., Preisinger, A. M., Smith,K. J., Su, L-K., Kinzler, K. W., and Vogelstein, B. Mutations of chromosome 5q21genes in FAP and colorectal cancer patients. Science (Washington DC), 253:665—669,1991.

18. Horn, A., Nakatsuru, S., Ichii, S., Nagase, H., and Nakamura, Y. Multiple forms ofthe APC gene transcripts and their tissue-specific expression. Hum. Mol. Genet., 2:283—287,1993.

19. Thliveris, A., Samowitz, W., Matsunami, N., Groden, J., and White, R. Isolation ofa promoter and alternatively spliced sequence 5' to exon 1 of the APC gene. CancerRca., 54: 2991—2995,1994.

20. Groden, J., Gelbert, L, Thilveris, A., Nelson, L, Robertson, M., Joslyn, G., Samowits, W., Spirio, L, Carlson, M., Burt, R., Leppert, M., and White, R. Mutationalanalysis of patients with adenomatous polyposis: identical inactivating mutations inunrelated individuals. Am. J. Hum. Genet., 52: 263—272,1993.

21. Spirio, L, Olschwang, S., Groden, J., Robertson, M., Samowitz, W., Joslyn, 0.,Gelbert,L, Thliveris,A.,Carlson,M.,Otterud,B.,Lynch,H.,Watson,P., LaurentPuig, P., Thomas, G., Leppert, M., and White, R. Alleles of the APC gene: anattenuated form of familial polyposis. Cell, 75: 951—957,1993.

22. Spirio, L., Joslyn, G., Nelson, L., Leppert, M., and White, R. A CA repeat 30—70KBdownstream from the adenomatous polyposis coli (APC) gene. Nucleic Acids Rca.,19: 6348, 1991.

23. Lee, K. W., Bindereif, A., and Green, M. A small-scale procedure for preparation ofnuclear extract that support efficient transcription and pre-mRNA splicing. GeneAnal. Tech., 5: 22—31,1988.

24. Schagger, H., and von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gelelectrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal.Biochem., 166: 368-379, 1987.

25. Smith, K. J., Johnson, K. A., Bryan, T. M., Hill, D. E., Markowitz, S., Wilson, J. K.,Pareskeva, C., Petersen, 0., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. TheAPC gene product in normal and tumor cells. Proc. NatI. Acad. Sd. USA, 90:2846—2850,1993.

26. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acidguanidinium thio-cyanate-phenol-chloroform extraction. Anal. Biochem., 162:156—159,1987.

27. Watt, R., Stanton, L W., Marcu, K. B., Gallo, R. C., Croce, C. M., and Rovers, 0.

1538

Research. on January 15, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

TUMOR SUPPRESSION BY THE APC GENE

Nucleotide sequence of cloned cDNA of human c-myc oncogene. Nature (Land.),303: 725—728,1983.

28. Ionov, Y., Peinado, M. A., Malkhosyan, S., Shibata, D., and Perucho, M. Ubiquitoussomatic mutations in simple repeated sequences reveal a new mechanism for coloniccarcinogenesis. Nature (Land.), 363: 558—561,1993.

29. Aaltonen, L. A., Peltomaki, P., Leach, F. S., Sistonen, P., Pylkkanen, L., Mecklin,J-P., Jarvinen, H., Powell, S. M., Jen, J., Hamilton, S. R., Petersen, 0. M., Kinzler,K. W., Vogelstein, B., and de Ia Chapelle, A. Clues to the pathogenesis of familialcolorectal cancer. Science (Washington DC), 260: 812—816,1993.

30. Thibodeau, S. N., Bran, 0., and Schaid, D. Microsatellite instability in cancer of theproximal colon. Science (Washington DC), 260: 816—819,1993.

31. Morgenstern, J. P., and Land, H. Advanced mammalian gene transfer: high titreretroviral vectors with multiple drug selection markers and a complementary helperfree packaging cell line. Nucleic Acids Rca., 18: 3587—3596,1990.

32. Shibata, D., Peinado, M. A., Ionov, Y., Malkhosyan, S., and Perucho, M. Genomicinstability in repeated sequences is an early somatic event in colorectal tumorigenesisthat persists after transformation. Nat. Genet., 6: 272—281,1994.

33. Brattain, M., Fine, W. D., Khaled, F. M., Thompson, J., and Brattain, D. Heterogeneity of malignant cells from a human colonic adenocarcinoma. Cancer Rca., 41:1751—1756,1981.

34. Erisman, M. D., Scott, J. K., and Astrin, S. M. Evidence that the familial adenomatous

polyposis gene is involved in a subset of colon cancers with a complementable defectin c-myc regulation. Proc. Natl. Acad. Sci. USA, 86: 4264—4268,1989.

35. Rodriquez-Alfageme, C., Stanbridge, E. J., and Astrin, S. M. Suppression of deregulatedc-myc expressionin humancolon carcinomacells by chromosome5 transfer.Proc.NatI.Acad.Sci.USA,89: 1482-1486,1992.

36. Nicolaides, N. C., Papadopoulos, N., Liu, B., Wei, Y-F., Carter, K. C., Ruben, S. M.,Rosen, C. A., Haseltine, W. A., Fleischman, R. D., Fraser, C. M., Adams, M. D.,Ventor, J. C., Dunlop, M. G., Hamilton, S. R., Peterson, 0. M., de la Chapelle, A.,Vogelstein, B., and Kinzler, K. W. Mutations of two PMS homologues in hereditarynonpolyposis colon cancer. Nature (Land.), 370: 75—77,1994.

37. Baker, S. J., Markowitz, S., Fearon, E. R., Willson, J. K. V., and Vogelstein, B.Suppression of human colorectal carcinoma cell growth by wild-type p53. Science(Washington DC), 249: 912—915,1991.

38. Bhattacharyya, N. P., Skandalis, A., Ganesh, A., Groden, J., and Meuth, M. Mutatorphenotypes in human colorectal carcinoma cell lines. Proc. Nail. Acad. Sci. USA, 91:6319—6323,1994.

39. Rubinfeld, B., Souza, B., Albert, I., Muller, 0., Chamberlain, S. H., Masiarz, F. R.,Munemitsu, S., and Polakis, P. Association of the APC gene product with @-catenin.Science (Washington DC), 262: 1731—1734,1994.

40. Su, L-K., Vogelstein, B., and K.inzler,K. Association of the Ak tumor suppressorprotein with catenins. Science (Washington DC), 262: 1734—1737,1993.

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1995;55:1531-1539. Cancer Res   Joanna Groden, Geoff Joslyn, Wade Samowitz, et al.   , a Colon-specific Tumor Suppressor Gene

APCResponse of Colon Cancer Cell Lines to the Introduction of

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