Synthase-2 Delay in Cells Overexpressing Prostaglandin ...

G1 Delay in Cells Overexpressing Prostaglandin Endoperoxide Synthase-2 Raymond N. DuBois, Jinyi Shao, Masahiko Tsujii, et al. Cancer Res 1996;56:733-737. Published online February 1, 1996.

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CANCERRE.SEARCH 56. 733-737.February15, 9961

Advances in Brief

G1DelayinCellsOverexpressing Prostaglandin Endoperoxide Synthase-2' RaymondN. DuBois,2Jinyi Shao,MasahikoTsujii, HongmiaoSheng,and R. DanielBeauchamp Departments of Medicine fR. N. D., J. S., M. TI, Surgery fH. S. , R. D. B.J, and Cell Biology (R. N. D., R. D. B.), Vanderbilt University Medical Center, and Veterans Administration Medical Center (R. N. D.J, Nashville, Tennessee 37232-2279

(15â€”18).We and others have shown an increased level of COX-2 expression in human colorectal adenocarcinomas (19 â€”21).Our labo Colorectal cancer is the secondleading causeof death from cancer in ratory also has demonstrated recently that adhesion to selected extra the UnitedStates.Continuoususeof aspirinand othernonsteroidalanti cellular matrix proteins is enhanced and apoptosis is inhibited in inflammatorydrugs (NSAIDs) has been shownto reducethe risk of colorectal cancer in humans by 40â€”50%. Patients with familial adenom intestinal epithelial cells that are programmed to overexpress the COX-2 gene (22). These phenotypic changes, which are related to atous polyposis who take NSAIDs, such as sulindac, undergo a regression of intestinaladenomas.Rodentsexposedto carcinogens that causecolon overexpression of COX-2 and reversible by treatment with NSAIDs, cancerhavea 50â€”60% reduction in the sizeand number ofcolonic tumors may alter the tumorigenic potential of intestinal epithelial cells. Regulation of eukaryotic cell cycle progression as it relates to when treated continuously with NSAIDs. One common target for these drugs is prostaglandin endoperoxide synthase,also referred to as cy malignant transformation has become the focus of intense interest clooxygenase (COX). We and others have shown recently that COX-2 over the past decade (23â€”25). Late in G@,the cell commits to undergo levelsare increaseddramaticallyin 85â€”90% of humancolorectaladeno chromosomal DNA replication. There is a restriction point late in G1, carcinomasand in 40â€”50% of colonicadenomas.We preparedIntestinal at which time the cells no longer require growth factorsto enter the epithelialcellsthat expressthe COX-2 genepermanentlyand foundthat S-phase.An accumulation of unstable proteins is required to pass the theyhavealteredadhesionpropertiesandresistundergoIngapoptosis. We restriction point (26). It has been hypothesized that mammalian G1 report here that these cells also have a 3-fold increase in the duration of cyclins, which regulate one or more cdks, drive cell cycle progression G1, lower levels of cyclin Dl protein, and a marked decreasein retino blastoma kinase activity associatedwith cyclin-dependentkinase 4. The in a rate-limiting manner. It has been shown clearly that overexpres delayin G1 transitmay relateto the resistanceof thesecellsto undergo sion of D-type cyclins can shorten the G1 interval (27â€”30),and these programmed cell death, which could affect their tumorigenic potential. studies provide direct evidence of the importance of D cyclins in the regulation of G@progression. We report here that in intestinal epithe Introduction hal cells programmed to overexpress COX-2, there is a marked delay in progressionthrough G1. Additionally, we observe a marked de Two COX3 isoforms have been identified, which will be referred to creasein cyclin Dl expression and Rb kinase activity associatedwith as COX-l and COX-2 in this report. COX-l is expressed constitu the G1 delay in COX-2-overexpressing cells. tively in a number of cell types, whereas COX-2 is inducible by a variety of factors, which include cytokines, growth factors, and tumor promoters (1). COX-2 was identified initially by many groups as a Materials and Methods member of a class of genes referred to as â€œimmediate earlyâ€• or â€œearly CeHCulture. Ratintestinal epithelial(RIE-l) cellswerea gift fromK. D. growth responseâ€• genes (2â€”6).These genes are induced rapidly and Brown (CambridgeResearchStation, Babraham,Cambridge,United King transiently following growth factor or phorbol ester stimulation of dom).Cells weregrown on plasticdishesin DMEM supplementedwith 10% quiescent cells (7â€”9).The functions of these genes are quite diverse fetal bovine serum(lot 11151032;HycloneLaboratories,Logan,UT), 2 mM L-glutamine,100,000units/literpenicillin G, and 100,000.tgfliterstreptomy and range from nuclear transcription factors (c-fos and jun-B) to cm sulfate. cytokines. The precise role many of these genes play in regulation of StableTransfection.A 2.1-kbSmaI-EcoRVfragmentcontaining theopen cellular responsesto growth stimuli and tumor promoters has not been readingframefor a polypeptideof 604aminoacidsof rat COX-2 wasisolated defined clearly. Abstract

Continuous NSAID use in humans has been shown to reduce the risk of colorectal cancer by â€”50%(10â€”14),but the molecular basis for the chemoprotective effects of these drugs remains unknown. Studies carried out in rodents have shown a marked reduction in carcinogen-induced colon cancer in animals treated with NSAIDs

of this article

were defrayed

in part by the payment

18U.S.C.Section1734solely to indicatethis fact. work

was

supported

in

part

University).

In this vector, tran

vector also containsa bacterialhygromycinresistancegeneexpressioncas sette, which allows for selection in hygromycin B. 5- and AS-oriented expres sion vectors were prepared. These expression vectors were transfected into a

selected in medium containing hygromycin B (225 units/ml). Total RNA was

of page

charges. This article must therefore be hereby marked advertisement in accordance with I This

Vanderbilt

scription of the cDNA is controlled by the cytomegalovirus promoter. This

nontransformedrat intestinalepithelial cell line (ME-I) using a Lipofectin (GIBCO-BRL,Gaithersburg,MD) transfectionmethod(2), andthecells were

ReceivedI2/1/95;accepted1/2/96. The costs of publication

and blunt-end cloned into the SmaI site of the eukaryotic expression vector

pCB7 (a gift from Bob Coffey,

from

the

United

States

Public

Health

Services

GrantsDK-47297-OlAl (R. N. D.), 5P30ES 00267-29(R. N. D.), GM-53319-l (R. D. B.), and CA-69457 (R. D. B.). R. N. D. is a recipient of a VeteransAdministration

preparedfrom the cells transfectedwith sense-oriented expressionandevalu atedusingNorthernslot blot analysis.Five clones(510, 599,5123,5132,and 5146)expressingthe highestlevel of COX-2 RNA wereidentifiedfrom 225 initial

clones and expanded. We characterized

all five clones and found that

Research Associate Career Development Award and a Boehringer Ingelheim New lnves

theyall exhibitedthe identicalphenotypicandbiochemicalalterations.There tigator Award and is an American GastroenterologicalAssociationIndustry Research fore,theresultsof our studiesusingtheRIE-SlOclonearepresentedhere.The Scholar. 2 To

whom

requests

for reprints

should

be addressed.

at Department

RIE cells transfected

of Medicine/GI,

MCN C-2l04, VanderbiltUniversityMedicalCenter,Nashville,TN 37232-2279.Phone: (615)343-8989;Fax:(615)343â€”6229. 3 The

abbreviations

used

are:

COX,

cyclooxygenase;

Northern RIE,

rat

intestinal

epithelial;

FAP, familial adenomatouspolyposis;NSAID, nonsteroidalantiinflammatorydrug;cdk, cyclin-dependent kinase; Rb, retinoblastoma; 5, sense; AS, antisense; P, parental; TGF, transforming growth factor.

with

the antisense expression

vector

(RIE-AS)

were

evaluatedin a similar manner. Blotting.

RNA and Northern blotting

was carried out essentially

asdescribed(19, 31). Immunoblotting. munoprecipitation

The cells were lysed by for 30 mm at 4Â°Cin radioim assay buffer [150 mM NaC1, 1% Nonidet

733


P-40, and 50 mM

CELL CYCLE PROGRESSIONAND CYCLOOXYGENASE.2

A

Rb Kinase Assays.The cells were dispersedinto immunoprecipitation buffer [50 mMHEPES(pH 7.5), 150mMNaC1,1 mMEDTA, 2.5 mMEGTA, 0.1% Tween 20, 10%glycerol, 1 mM DTT, 10 @.tg/mi leupeptin,25 @.tg/ml

E

aprotinin, 5 @Lg/ml pepstatin, 10 mM f3-glycerophosphate, and 0.1 mM sodium vanadate] and lysed by sonication.

Cl) Cfl4 LU IU I

4.5kb

o@

I

lii

LU

subjected to immunoprecipitation

by micro

with the cdk4 antibody. After preclearing

the

lysateswith Staphylococcusaureus(Cowan strain), antiserumto cdk4 was added,and the sampleswere incubatedat 4Â°Cfor 2 h, after which protein A-Sepharosewasaddedandmixedfor 1h at4Â°C. Followingcentrifugationfor 1 mm at 14,000rpm, the Sepharosepellets were washedfour times in immunoprecipitationbuffer, thentwice with 50 mMHEPES(pH 7.5) contain ing 1 mMDli'. The Rb kinaseassaywasthenperformedfor 30 mm at 30Â°C in Rbkinasebuffer [50 mMHEPES(pH 7.5), 1mMNaF,2.5mMEGTA, 1mM DTF, 10 mM @3-glycerophosphate, 0.1 mM sodium vanadate,and 10 mM MgCl2] with the additionof 0.1 @tg glutathioneS-transferase-Rb, 20 @M cold AlP, and 10 @.tCi [â€˜y-32P]ATP in a final volumeof 30 p1 The reactionswere

@IL

2.2kb

The lysates were then clarified

centrifugationfor 5 mm at 14,000rpm at 4Â°C.The proteinconcentrationsof eachlysate were then determined,and equivalentamountsof protein were

â€¢..

stopped with the addition

of 30 @.d 2X Laemmli

buffer

[1 X buffer

= 50 mM

Tris Cl (pH 6.8),0.5%j3-mercaptoethanol, 2% SDS,0.1%bromophenolblue, 10% glycerol] and boiled for 5 mm, then analyzedby SDS-PAGE and

0.

0.

i(

(I)

LU

E

LU

E

LU

LU

I

@

I

@

I

autoradiography.

The glutathione

S-transferase-Rb

fusion protein was obtained

from SantaCruzBiotechnology,andtheRb fusionfragmentconsistsof amino

I

acids 769â€”921of the carboxyl-terminal domain of the mouse pRb. The cdk4

antiserawasa kind gift from Ed Leof (Mayo Clinic, Rochester,MN). Results

::@

To investigatethe role of COX-2 expressionin intestinalepithelial cell cycle regulation, RIE cells were transfected permanently with the eukaryotic expression vector pCB7 containing the COX-2-coding region (2.2 kb) in either the S or AS orientation. Northern and

Fig. I . Analysis of COX-2 mRNA and protein levels in RIE cells. A, Northern blot analysis of 5 ,.tg polyadenylated RNA isolated from the RIE cells indicated at the top of

each lane. RIE-S (Sense)and RIB-AS (Antisense)RNA was isolated 3 h following stimulationof quiescentcells with 10%serum.In theRLE-P(Parental)Q lane,RNA was

isolated fromparental RIEcells72hfollowingcultureinDMEMlackingserumorgrowth factors. In the RIE-P +Serum lane, RNA was isolated 3 h following addition of 10%

U

(Â±addition of serum for 3 h), RIE-S, and RIE-AS cells.

Tris (pH 8.0)] containing pepstatin,

and 100

10 @ag/m1aprotinin,

10 @tg/ml leupeptin,

@g/mlphenymethylsulfonylfluoride.

Centrifuged

1 @.tg/ml cell ly

sates(50 ILg)weredenaturedandfractionatedon 11.25%polyacrylamidegels containing

SDS, then transferred to a nitrocellulose

:2

membrane using a semidry

cell (Bio-Rad, Richmond,CA). Filters were incubatedovernight at room temperaturein blocking solution(Tris-bufferedsalinecontaining5% nonfat, dried milk and0.05%Tween20) followed by a 4-h incubationwith primary rabbit antiserain blocking solution. Filters were washedthree times and incubatedwith a horseradishperoxidase-conjugated, goatantirabbitimmuno globulin as a secondaryantibody(1:5000dilution) for I h. After threeaddi tional washes,filters were developedby the enhancedchemiluminescence system(Amersham,Arlington Heights,IL) andexposedto XAR5 film (East man KOdak,Rochester,NY). Primaryantibodieswereas follows: polyclonal rabbitantiserato humancyclin Dl (UpstateBiotechnology,Inc., LakePlacid, NY) was used at a 1:500 dilution;

polyclonal

A

E a.

serumto thequiescentRIE-Pcells.PanelB: Immunoblottingof COX-2 proteinin RLE-P

40BI:IiÂ°

rabbit antisera to mouse cyclin

D3 (a gift from Aubrey Thompson,University of Texas Medical Branch, Galveston,TX) andhumancdk4(SantaCruz Biotechnology,Inc., SantaCruz, CA) was usedat a 1:2000dilution; andp21 (OncogeneScience,Uniondale, NY) was used at a dilution of 1:1000.

DNA Synthesis.DNA synthesis wasmeasured by [3H]thymidine incorpo rationinto trichloroaceticacid-precipitablematerial(31).RIE cellswereplated @

in multiwell growth

dishes, grown to 80% confluence,

factor-free

medium

n

and then placed in serum- and

4@

for 72 h. Then, the cells were exposed to DMEM

1216 2'OTime

(hrs)

containing 10% fetal bovine serum, and the studies were carried out in triplicate.

After various times following

serum addition,

1

@Ci[3Hjthymidine

wasaddedto eachwell for 2 h. Trichloroaceticacid-precipitablematerialwas solubilized

in 300 ,d 0.2 N NaOH containing

40 @.tg/m1salmon sperm DNA;

100 @al were removedfor liquid scintillation counting,and the resultswere normalized

to 1 X 106 cells.

Fig. 2. Progression of RIE cells through G1 as measured by [3HJthymidine and cell

sorting.A, [3Hjthymidine incorporationinto DNA. RIE-AS cells (solid circles); RIB-P cells (solid squares);RIE-Scells (solid triangles).PanelB: Cell sortinganalysis.Datais plotted as percent of cells in S phase. RIE-AS cells (solid circles); RIB-P cells (solid

squares);RIE-Scells (solid triangles).

734


@-

-@ CELL CYCLE PROGRESSIONAND CYCLOOXYGENASE.2

RIEP

cyclinDi

RIE-AS

than the RIE-AS and RIB-P cells. Then, we examined progression through G1 in the RIB-P. RIE-AS, and RIE-S cells plated on plastic dishes. Our results, shown in Fig. 2A, demonstrate that the length of time required to enter the S-phase, as measured by [3H]thymidine incorporation into DNA, was reduced from 12 h in RIE-P cells to 8 h in the RIE-AS cells and occurred between 20 and 24 h in the RIE-S cells. These results were confirmed by cell sorting analysis (Fig. 2B). This series of experiments demonstratesclearly a 2â€”3-foldincrease in the time required for progression through G1 in the cells that overex press the COX-2 gene constitutively. Work by several investigators has demonstrated that the D-type cyclins are â€œdelayed-earlyâ€• genes that regulate the rate of progression through G1 (27â€”30).Cyclin Dl is a critical target of proliferative signals following growth factor stimulation of quiescent cells, and inhibition of its expression can cause a G1 arrest. Inhibition of intestinal epithelial cell growth by TGF-@ is associated with down regulation of cyclin Dl , with little effect on cdk4 or cyclin D3 levels (32). Therefore, cyclin Dl seemsto perform a crucial role in regulat ing the progression of intestinal epithelial cells through the first gap of

RIE-S

@::@::

cycIinD3 â€”

cdk4@@ p27 p21 Fig. 3. Western blot analysis for cell cycle regulated proteins in exponentially-growing RIE cells. Immunoblotting was performed on cell lysatesusing polyclonal antisera to Cyclins Dl, D3, cdk4, p27 and p21.

Westernblottinganalyses were carriedout to determinerelativelevels of COX-2 mRNA and protein in these cells. COX-2 expression levels in RIE-S cells were compared with RIE-P cells in the presence or absence of growth stimulation by the addition of 10% serum to the cell culture medium. As shown in Fig. 1A, there are two mRNA bands present in the serum-treated RIE-S cells, which hybridize to the COX-2 cDNA probe: a 4.5-kb mRNA, which corresponds to the endogenous transcript; and a 2.2-kb band, which corresponds in size to the transfected COX-2 gene. In the RIE-AS cells, only the 2.2-kb

the cell cycle. Next, we examined the levels of cell cycle-regulated

antisense mRNA

RIE-AS cells. From theseresults,we concludethat cyclin Dl expres sion was decreasedin intestinal epithelial cells programmed to over express COX-2. Then, we explored the temporal pattern for changes in cyclin DI, cdk4, and p21 levels following serum stimulation of quiescent RIE-P, RIE-S, and RIE-AS cells. These results, shown in Fig. 4, indicate that cyclin Dl is expressed abundantly in serum-deprived RIE-AS cells, and its levels do not change significantly following serum stimulation. The level of cyclin Dl is very low in serum-deprived RIB-S cells, its induction is markedly delayed, and the level is reduced compared with the pattern seenfor the RIE-P cells. Induction of cdk4 and p21 is also delayed in the RIE-S cells compared with the other two cell lines, and

is present, indicating

proteins in exponentially growing RIE-P, RIE-S, and RIE-AS cells. Our results, shown in Fig. 3, demonstrate that cyclin Dl protein levels

are barely detectable in asynchronous RIE-S cells in the midlog growth phase but easily detected in the RIB-P and RIE-AS cells, whereas cyclin D3 levels were equivalent in all three cell lines. The

level of cdk4 was reduced by 50% in the ME-S cells, whereas p21 levels seemed to be slightly elevated compared with levels seen in

that the presence of the anti

sense transcriptinhibits the appearanceof the endogenous COX-2 mRNA. The responseof the RIE-P cells to serum stimulation is shown in Fig. lA (far right panel),

and only the 4.5-kb transcript

is present

in these nontransfected cells. COX-2 protein levels were determined by Western blotting and are shown in Fig. lB. These results indicate that the level of COX-2 protein correlates well with the level of mRNA and that COX-2 protein levels in the RIE-S cells are equiva lent to levels seen transiently following serum induction. Also, these results demonstrate that the antisenseconstruct inhibits the expression of the COX-2 protein and mRNA in RIE-AS cells effectively.

We observed empirically that the RIE-S cells grow more slowly

Time Following Serum Addition @-

@.

.@

q @

@

@

@

@ @

DI, cdk4 and p21 following serumof quiescent RIE cells.RIE cells wereplacedin DMEM lacking serumandgrowth factorsfor 72 h andthentreated with DMEM containing 10% serum for the times indicated at the top of the Fig. Immunoblots were

preparedasdescribedabove.

&.

uc

.@

c*1

0

1@

C@l

-

@--@

C@) -

Cyclin Dl

RUE-AS

@:i

RUE-P RUE-S

-

RUE-AS

-

RIE-P RIE-S RIE-AS

0

cc;

RIE-P RUE-S Fig. 4. Temporal pattern of expression for cyclin

@

o

0

@

L.

.@

@-@--

-------@-

-@ -@

@â€”

â€”

. -

aâ€”@

@-

@J

_1l@

@-=

Ji@

@-I

cdk4

â€”@---

-@

@: -@-@

@â€”

=_â€”::

735


â€”.

â€”

p 21

CELL CYCLE PROGRESSIONAND CYCLOOXYGENASE-2

suggest that overexpression of COX-2 may confer a survival advan tageto cells by slowing cell cycle progression,preventing apoptosis,and enhancing adhesion to the extracellular matrix. Prolonged survival of abnormal cells can favor tumor progressionand facilitate the accumula tion of sequentialgenetic mutations which would result in tumor promo tion. Our results are consistent with this hypothesis and suggest a link

Time (hrs)

048048 @L

between COX-2

overexpression,

prolongation

of G1, and inhibition

of

apoptosisin intestinal epithelial cells. Studiesare underway to determine

RUE-AS

the effect of selective COX-2 inhibitorson cyclin protein levels in

RUE-S

COX-2-overexpressing Fig. 5. Retinoblastoma (Rb) kinase activity in RIE-S and RIE-AS cells. Time following serum stimulation is marked at the top of each lane. The phosphorylated Rb protein is denoted by the arrow to the left of the Fig. and the cell line evaluated is denoted below the Fig.

p21 levels remain elevated in the RIE-S cells 32 h following serum addition compared with RIE-AS cells, in which p21 levels decreaseafter 12 h. These results show a clear correlation between the reducedcyclin Dl levels and the prolongation of G1 in the RIE cells that expressCOX-2 constitutively. To evaluatethe functional significance ofdecreasedcydlin Dl levels, we carried out experiments to determine Rb kinase activity following immunoprecipitation of the active cyclin-cdk4 complex with an anti-cdk4 antibody, and theseresults are shown in Fig. 5. Rb kinase activity was reduced markedly in the PiE-S compared with the PIE-AS

cells from 0 to 8 h following serum stimulation. These experiments indicate that the kinase activity

associated with the cdk4 complex

is

reduced markedly in the cells that expressCOX-2.

References 1. Eberhart, C. E., and DuBois, R. N. Eicosanoids and the gastrointestinal tract. Gastroenterology. 109: 285â€”301 , I 995. 2. DuBois, R. N., Tsujii, M., Bishop, P., Awad, J. A., Makita, K., and Lanahan, A. Cloning and characterization of a growth factor-inducible cyclooxygenase gene from rat intestinal epithelial cells. Am. J. Physiol., 266: G822â€”G827,1994. 3. Farber, J. M. A collection of mRNA species that are inducible in the RAW 264.7 mouse macrophage cell line by gamma interferon and other agents. Mol. Cell. Biol., 12: 1535â€”1545, 1992. 4. Kujubu, D. A., Fletcher, B. S., Varnum, B. C., Lim, R. W., and Herschman, H. R. 11510, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J. Biol. Chem., 266: 12866â€”12872, 1991. 5. Simmons, D. L., Levy, D. B., Yannoni, Y., and Erikson, R. L. Identification of a

phorbol ester-repressiblev-src-inducible gene. Proc. NatI. Aced. Sci. USA, 86: 1178â€”1182, 1989. 6. Ryseck, R. P., Raynoschek, C., Macdonald-Bravo, H., Dorfman, K., Mattei, M. G.,

and Bravo, R. Identification of an immediateearly gene,pghs-B, whose protein producthasprostaglandinsynthase/cyclooxygenase activity. Cell Growth & Differ., 3: 443â€”450,1992. 7. Nathans, D., Lau, L. F., Christy, B., Hartzell, S., Nakabeppu, Y., and Ryder, K. Genomic response to growth factors. Cold Spring Harbor Symp. Quant. Biol., 53:

893â€”900, 1988. 8. Herschman, H. R. Primary response genes induced by growth factors and tumor

Discussion Our findings provide evidence that overexpressionof COX-2 leadsto prolongation

intestinal epithelial cells.

of G1 through effects on cycin

Dl expression and cdk4

kinaseactivity of the cyclin complex. Cyclin Dl hasbeenshown to serve a key role in progressionthrough G1 in eukaryotic cells (27â€”30, 33). We

have reportedpreviouslythatratintestinalepithelialcells, whichexpress the COX-2 gene permanently, have an altered phenotype (22). They exhibit increasedadhesionto extracellular matrix proteins,and unlike the P-transfected (RIE-P) or AS-transfected (RIE-AS) cells, they do not undergo apoptosisin responseto sodium butyrate treatment.This report is the first demonstration

that COX-2

overexpression

also leads to de

promoters.Annu. Rev. Biochem.,60: 281â€”319, 1991. 9. Rollins, B. J., andStiles,C. D. Serum-induciblegenes.Adv. CancerRca.,53: 1â€”32, 1989. 10. Giovannucci,E., Egan,K. M., Hunter,D. J., Stampfer,M. J., Colditz, G. A., Willett, W. C., and Speizer,F. E. Aspirin andthe risk of colorectalcancerin women. N. Engl. J.Med.,333:609â€”614, 1995. 11. Giovannucci, E., Rimm, E. B., Stampfer, M. J., Colditz, G. A., Ascherio, A., and Willett, W@C. Aspirin use and the risk for colorectal cancer and adenoma in male

healthprofessionals.Ann. Intern.Med., 121: 241â€”246, 1994. 12. Mamett, L. J. Aspirin and the potential role of prostaglandins in colon cancer. Cancer Res., 52: 5575â€”5589,1992. 13. Thun, M. 1., Namboodiri, M. M., Calle, E. E., Flanders, W. D., and Heath, C. W. J. Aspirin use and risk of fatal cancer. Cancer Res., 53: 1322â€”1327,1993. 14. Thun, M. J., Namboodiri, M. M., and Heath, C. W. i. Aspirin use and reduced risk of fatal colon cancer. N. Engl. J. Med., 325: 1593â€”1596,1991. 15. Reddy, B. S., Rao, C. V., Rivenson, A., and Kelloff, G. Inhibitory effect ofaspirin on azoxymethane-induced colon carcinogenesis in F344 rats. Carcinogenesis (Land.), 14: 1493â€”1497, 1993.

creasedexpressionof cycin Dl and prolongation of G1. In intestinal mucosa, in vivo, continuously proliferating stem cells Reddy,B. S., Tokumo, K., Kulkarni, N., Aligia. C., and Kelloff, G. Inhibition of are located in the crypt region. Both cellular differentiation and 16. colon carcinogenesis by prostaglandin synthesis inhibitors and related compounds. proliferative arrest are induced as intestinal stem cells ascend upward Carcinogenesis (Land.), 13: 1019â€”1023,1992. 17. Singh, J., Kulkarni, N., Kelloff, G., and Reddy, B. S. Modulation of azoxymethane on the basement membrane along the crypt-villus axis toward the gut inducedmutationalactivationof ras protooncogenesby chemopreventiveagentsin lumen. When the cells reach the surface of the villi, they become fully colon carcinogenesis. Carcinogenesis(Land.). 15: 1317â€”1323, 1994. differentiated, apoptosis is activated, and the cells are releasedinto the 18. Rao, C. V., Rivenson,A., Simi, B., and Reddy, B. S. Chemopreventionof colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. lumen. In the intestine, epithelial cell turnover is very rapid (3â€”4 Cancer Res., 55: 259â€”266,1995. days) under normal circumstances. By markedly increasing the time 19. Eberhart, C. E., Coffey, R. J., Radhika, A., Giardiello, F. M., Ferrenbach, S., and DuBois, R. N. Up-regulation of cyclooxygenase 2 gene expression in human cob required for transit through G, and inhibiting programmed cell death, rectaladenomasand adenocarcinomas. Gastroenterobogy, 107: 1183â€”1 188, 1994. COX-2 overexpression could increase the length of time that abnormal 20. Kargman, S. L., O'Neill, G. P., Vickers, P. J., Evans, J. F., Mancini, J. A., and Jothy, cells (with a lO mutation) would spend in transit to the villus tip and S. Expressionof prostaglandinG/H synthase-l and -2 proteinin humancolon cancer. CancerRes.,55: 2556-2559, 1995. increase the chance that a second mutation could occur (progression). 21. Sano, H., Kawahito, Y., Wilder, R. L., Hashiramoto, A., Mukai, S., Asai, K., Kimura, Because cell cycle transit and apoptosis are important factors in S., Kato, H., Kondo, M., and HIa, T. Expressionofcycbooxygenase-land-2 in human maintaining the integrity and function of intestinal mucosa, alterations colorectal cancer. Cancer Res., 55: 3785â€”3789,1995. in these processes could have significant

biological

consequences and

could affect the tumorigenic potential of epithelial cells. Recently, links between cell cycle progression and apoptosis have become the focus of a number of groups evaluating the molecular basis for the

development of colorectal cancer (34â€”37).Although several recent reports have demonstrated increased COX-2 expression in human colorectal carcinomas, the role of COXs in colorectal carcinogenesis is unclear. Our findings reported here and from previous work (22)

22. Tsujii, M., and DuBois, R. N. Alterations in cellular adhesionand apoptosisin epithelial cells overexpressingprostaglandin endoperoxide synthase-2.Cell, 83: 493â€”501,1995. 23. Alexandrow, M. G., and Moses, H. L. Transforming growth factor j3 and cell cycle

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