Overexpression of DAN Gene Product in Normal Rat Fibroblasts ...

[CANCERRESEARCH 55,895-900,February 15,1995]

Overexpression of DAN Gene Product in Normal Rat Fibroblasts Causes a Retardation of the Entry into the S Phase' Toshinori

Ozaki, Yohko Nakamura,

Hideki Enomoto,

Masayoshi

Hirose, and Shigeru Sakiyama2

Division of Biochemistry IT. 0., 1'.N., H. E., S. S.! and Division of Clinical Laboratory (M. H.], Chiba Cancer Center Research institute, 666-2 Nitona, Chuoh-ku, Chiba

260, Japan

ABSTRACT Differential screening-selectedgeneaberrative in neuroblastoma(DAN) gene(previouslynamedN03 gene),whoseexpressionIs significantlyre duced in transformed cells, has recently been demonstrated to have a tensor-suppressive activity in vitro. In order to investigate biological roles

of DAN gene product in normal rat fibroblasts (3Y1), marker-selected transfectaats that expressedthe high amount ofDAN geneproduct were generated from 3Y1 cell lines. These clones did not exhibit morphological changes compared with parental 3Y1 cells; however, they showed a de

creasein growth rate and a remarkable reduction in saturation density. Cell cycle analysisrevealedthat the overexpressioaOfDAN geneproduct causes the retardation

of the entry into the S phase. These results suggest

that DAN geneproduct may have an Important role in regulation of the entry of cells into the S phase.

INTRODUCTION

chromosomemappingrevealedthatDAN geneis mappedto lp36.l 1— p36.13, which resideswithin the putative neuroblastomatumor-sup pressor locus (10). Southern blot analysis has suggested that there exists the genomic alteration in the DAN gene locus in neuroblastoma (10). Accordingly, we renamedN03 gene as DAN. In the presentwork, we have investigatedwhether the overexpres sionofDANgene productin 3Y1 cellscausesany effectson cell cycle progression. 3Y1 is a rat cell line established from Fischer 344 rat embryo and its karyotype is normal diploid (11, 12). Thus 3Y1 cell lines serve as a normal immortalized cell system for studiesof the mechanismsunderlyingcell transformationand cellular signal trans duction(13, 14). 3Y1 transfectants,which expressa large amountof DAN gene product, exhibit a retardation of the entry into the S phase. This observation suggests that DAN gene product may have some important roles in determining the timing of the transition from the G1 into the S phase.

As the deregulation of the normal cell cycle control raises the MATERIALS AND METHODS abnormal cycling of cells, which is one of the most common charac teristics of cancer cells, elucidation of molecular mechanisms respon Cell and Culture Conditions.The rat fibroblastline 3Y1 was kindly sible for the cell cycle control may provide a clue to help understand provided by Dr. H. Sakiyama (National Institute of Radiological Science,

the mechanism of oncogenesis. The major regulatorypointsof the eukaryoticcell cycle occurat the G1-S and the G2-M transition. The transition from 02 into M is known

to be controlledby the activityof the maturation-promoting factor, consisting of the cdc2 protein kinase and mitotic cydlin (1—3). Meanwhile, accumulatingevidenceregardingthe biological activ ities of nuclear proto-oncogene and tumor-suppressor gene products emphasizesthe functional importance of G1 for the cell division cycle. Cells overexpressing c-myc proto-oncogene exhibit an increased

growth rate which is characterizedby a shortenedlength of G1 (4). While the growth-suppressive property of pRB (the retinoblastoma susceptibility gene product) is regulated by the cell cycle-dependent phosphorylation, the underphosphorylated forms of pRB found during G1 have a growth-suppressiveactivity (5, 6). Microinjection studies revealedthat pRB inhibits the progressioninto the S phase(7). DAN3 gene has been cloned from a cDNA library derived from normal rat fibroblasts (3Y1) by a differential screening procedure. Structural analysis revealed that DAN gene product does not exhibit any sequencesimilarity with proteins filed in the data base. The expression of DAN gene was significantly reduced in a wide variety of transformed cells (8). The transformed phenotypes were markedly reduced or lost when DAN gene was overexpressed in v-src-trans formed 3Y1 cells (9). These observations suggest that DAN gene productcontainsa tumor-suppressiveactivity in vitro. Furthermore, we have recently obtained the human counterpart of DAN gene and Received9/27/94;accepted12/16/94. The costsof publicationof this article weredefrayedin part by the paymentof page charges. This article must therefore be hereby marked advertisement in accordance with

18 U.S.C.Section1734solely to indicatethis fact. I Tl@@ work

was

supported

by a Grant-in-Aid

from

the Ministry

of Health

3 The

whom

requests

abbreviations

for

reprints

used

are:

should DAN,

be

screening-selected

Transfection DAN

cDNA

and Selection of Stable Cell TranSfectants. A full-length

was inserted

into the EcoRI

restriction

site of the pMEXneo

expression vector (15, 16), which was generously provided by Dr. M. Bar

bacid. For transfection, cells were washed twice with DMEM without serum and the transfection mixture consisting of 10 @g pMEXneo containing DAN

cDNA or expressionvector aloneand 30 @.tg lipofectin (GIBCO-BRL) were overlaid onto cells. After 6-h incubation, cells were replacedin DMEM supplemented with thenormalpercentageof serumandmaintainedfor another 48 h. Then cells were grown in the presenceof 0418 (Sigma; effective concentration, 400 @tg/m1) for 2 weeks and colonies resistant to 0418 were

isolated. Production and Purification ofMBP-DAN Fusion Protein. A full-length rat DAN cDNA was sequentially deleted from its 5' terminus with exonuclease

III. Among these 5' deletion mutants,we chose the DAN 5'-238, which lost 238 basepairs from the 5' terminus ofthe full-length DANcDNA. The plasmid pMAL-cRl (New England Biolabs) was linearized with EcoRI and filled in

with Klenow enzyme.Then the DAN 5'-238 DNA was subclonedinto the enzymatically treated EcoRI site of pMAL-cRI.

For MBP fusion protein

production,overnightcultureof Eschthchia coli JM1O9containingthe above recombinantplasmid was diluted 1:10 in Luria broth supplementedwith glucose (final concentration, 0.2%) and grown for 2 h with shaking at 37°C.

lsopropyl-@-D-thiogalactopyranoside wasaddedto a final concentrationof 0.3 mM and culture was incubated at 37°Cfor 4 h. Bacteria were pelleted and resuspended in 1/100 the original culture volume in the lysis buffer composed

of 10 [email protected] Na2HPO4(pH 7.2),30 m?s@ NaG, 0.25%Tween20, 10mt@i EDTA, 10m@tEGTA, and10mt@i@3-mercaptoethanol. After the additionof lysozyme (1 mg/mI), cells were lysed on ice by mild sonication and subjectedto centrifugation.The supernatantwas loadedon the amyloseresincolumnand the boundproteinwaselutedin the presenceof 10 m@imaltose. Antibody

Productloa.

MBP-DAN

fusion protein was digested

with factor Xa (New EnglandBiolabs) and loadedon the Q-Sepharoseion

addressed.

differential

5% CO2 atmosphere.

Polyclonal

and Welfare

for a new 10-YearStrategyfor CancerControl,Tokyo, Japan. 2 To

Chiba, Japan). Cells were grown in DMEM supplemented with 10% heat inactivated F@Sand antibiotics. Cells were maintained at 37°Cin a humidified

gene

aberrative

exchange

in

neuroblastoma;pRB, retinoblastomageneproduct;MBP, maltose-bindingprotein;cdks, cydlin-dependentkinases. 895

chromatography

to separate DAN polypeptide

from

MBP.

Seven

hundred thirty @g of the purified DAN protein were injected into the lymph nodes of one rabbit. The rabbit received three booster shots with 730 @g DAN

CELL CYCLE-REGULATORYFUNCI'ION OF DAN GENE PRODUCF

proteinat 21-dayintervals.On the 10thday after the third booster,the rabbit was bled. Subsequently, this serum was affinity purified using a MBP-DAN

1

2

3

4

5

6

7

bound Sepharose 4B affinity column. Western Blot Analysis. Total cell lysates were boiled in Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose filters

(SchleicherandSchnell).Filterswereblockedfor 1h atroomtemperaturewith Tris-bufferedsaline with Tween 20 buffer containing5% dried milk, and

“DAN

incubated with a 1:1000 dilution of the affinity-purified antibody for 1 h at room temperature. After washing, the filters were incubated with a 1:1000

dilution of the horseradishperoxidase-conjugatedgoat anti-rabbit IgO (GIBCO-BRL) antiserum for 1 h at room temperature. Signals were visualized with the enhanced chemiluminescence system (Amersham Corp.) according to the manufacturer's instructions.

RNA Analysis, Total RNA was extracted using the phenol-SDS method, separated by 1% agarose gels containing formaldehyde, transferred to nylon membrane in 20X SSC and immobilized

by UV cross-linking (17). Hybrid

ization probes were made from gel-purified DNA fragments that were labeled

with [a-32P]dCTPby the random-primingtechnique(18). After hybridization at42°C for 20h in 6X SSCcontaining5X Denhardt'ssolution(19),0.1%SDS, 50% formamide,and 100 @tg heat-denaturedsalmonspermDNA/rn!, the filters

werewashedsequentiallyin 2X SSC-0.1%SDSat room temperaturefor 30 mm and in 0.1X SSC-0.1%SDS at 50°Cfor 30 mm. Autoradiographywas carriedout for 2 daysat —70°C with an intensifyingscreen(17). Southern Blot Analysis. High molecular weight genomic DNA was iso

latedfrom culturecells by the standardprocedure(17). Ten ,.@g of genomic DNA weredigestedto completionwith BamHl,resolvedby 0.8%agarosegel electrophoresis, and transferred to nylon membrane. DNA was detected by

Fig. 2. OverexpressionofDANmRNA in 3Y1 cells.Total RNA(10 @g)was separated in a 1% agarose gel containing formaldehyde and transferred to nylon membrane. Hybridizationwas carriedout usingDAN eDNA as a probe.The cells analyzedwere 3Y1 (Lane 1), V2 (Lane 2), V3 (Lane 3), 51 (Lane 4), 59 (Lane 5), 510 (Lane 6), and 512

(Lane 7). Bottompanel, ethidiumbromidestainingof RNA.

probing with DAN cDNA labeledwith [a-32P)dCTPby randompriming. Hybridizationwasperformedasdescribedin “RNA Analysis―. CellSynchronization andAnalysisofCell Synchrony.Cellswereseeded 512) that expressedhigh levels of DAN mRNA (Fig. 2). The expres sionlevel of DANmRNA in thesecellswas morethan 10 timeshigher in 60-mm dishes and grown in DMEM containing 10% FCS. Then cells were washedthreetimeswith PBSand maintainedin DMEM supplementedwith than those in parental 3Y1 cells and the vector control clones 0.5% FCS for 48 h. After that, cells were transferredto complete medium (V2 and V3). containing10%FCS.Thesynchronyof cellsweremonitoredby assessingthe The presence of the transgenes in the transfected cells was con cellular DNA content by flow cytometry, using a Becton Dickinson FACSCan. firmed by Southern blot analysis (Fig. 3). In addition to the endoge Forflow cytometricanalysis,cellsweretrypsinizedevery3 h aftertheaddition neousbands (2.5 and 3.5 kilobases), extra signals corresponding to the of FCS(final concentration,10%),washedin PBS,fixed in a solutionof 95% transgene were observed only in the clones which expressed a large cold ethanol,andstoredat 4°C. The fixed cells werestainedwith propidium amount of DAN mRNA. The copy number of the integrated DNA did iodide as described (20). Then fluorescence intensity of propidium iodide, not always correlate with the level ofDAN mRNA in the transfectants, which is proportional to the cellular DNA content, was measured. as observedin anotherexperimentalsystem(21). In order to investigatethe level of DAN protein in thesetransfec RESULTS tants, we have raised a rabbit antiserum directed against the bacterially expressedrat DAN protein.Although the affinity-purified antibodies Transfection of 3Y1 Cells with DAN cDNA. An expression vec recognized several bands in total cell lysate prepared from 3Y1 cells tor of rat DAN cDNA drived by long terminal repeat of Moloney murine sarcoma virus was constructed and introduced into 3Y1 cells (Fig. 4, Lane 1), a competitive experiment revealed that the intensity using the lipofectin method (Fig. 1). Control transfection was carried of the signalcorrespondingto the Mr 27,000 proteinwas significantly out with theexpressionvectorwithoutcDNA. After theselectionwith decreased in the presence of the antigen, but not in the presence of 0418 for 2 weeks, 12 independent colonies resistant to G418 were isolated. Among them, we have obtained four clones (Si, 59, 510, and

MBP (Fig. 4). In addition,the Mr27,000 proteinwas not detected in total cell lysate from v-src-transformed3Y1 cells (data not shown). As reported previously, DAN mRNA is not detectable in v-src transformed 3Y1 cells and rat DAN cDNA has a capacity to encode a

proteinproductof approximateMr 23,000underthein vitro transcrip tion and translationsystem (8). The observedmolecular weight in

Western blot analysis was slightly larger than that of the in vitro translation product. Our preliminary result suggests that it could be due to the modification of DAN gene product.Therefore, we tenta tively concluded that the Mr 27,000 protein detected in Western blot analysis corresponds to DAN gene product. Then total cell lysate was prepared from each transfectant and analyzed by Western blotting

using this polyclonal anti-rat DAN antibody. As shown in Fig. 5, four clones (Si, 59, 510, and 512) which contain the transgene in their genomes express a large amount of DAN protein. The intensity of the Mr 27,000 band in these clones was more than 10-fold higher than that Fig. 1. Structure of plasmid pMEXneoDAN. A full-length rat DAN cDNA was subclonedinto the uniqueEcoRl sitewithin a multiple cloningsite flankedby a Moloney murine sarcomavirus long terminal repeat(MSV-LTR). The plasmid is 8.4 kilobases.

of the control transfectants (V2 and V3) or the parental 3Y1 cells.

Growth Propertiesof the DAN-overexpressing Cells.Several

parameters that correlate with cell growth were examined in the 896

@

.

CELL CYCLE-REGULATORYFUNCtiON OF DAN GENE PRODUCF

1

2

3

5

4

7

6

. —6.5 -4.4

S

.

@

—@-

iE@-*

r

Till :ii:a•

@

-2.0

:::::

. Fig. 3. Genomic integrationof the transgenes.High molecular weight DNA (10 p@g)isolated from each cell line was digestedwith BamHl and analyzed by the Southem blot

hybridization technique using32P-labeled DANcDNAasa probe.Arrowheads, endogeneous DANDNA.ADNAdigested withHindlll wasusedasa molecular marker.Thecells analyzedwere 3Y1 (Lane 1), V2 (Lane2), V3 (Lane3), 51 (Lane4), S9 (Lane5), SlO (Lane 6), and512 (Lane 7).

@

@

69

showed a doubling time (14 h) and a saturation density (1.6—

46

@

p

1.7 X 106 cells/35-mm dish) which were similar to those of the parental 3Y1 cells (15 h and 1.7 X 106 cells/35-mm dish, respec tively). On the other hand, four independentDAN-overexpressing cells (Si, S9, 510, and 512) displayed a prolonged doubling time

@ @

1234567

DAN-overexpressing cells and the control transfectants. The cells overexpressing DAN gene did not display obvious morphological changes,but they showed marked differences in growth properties when compared with the control transfectants (Table 1). When grown in the standard medium containing 10% FCS, the control transfectants

1234567 69

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46

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— ..••

21.5 — ii@

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Fig. 5. Western blot analysisof the DAN-overexpressingcells. Equal amountof total

cell lysatefromeachtransfectant wasseparated by electrophoresis on a 10%SDS polyacrylamidegel, transferredto nitrocellulose,andprocessedasdescribedin “Materials

@

-

@

and Methods.― The cellsanalyzedwere 3Y1 (Lane I), V2(Lane 2), V3 (Lane 3), 51 (Lane 4), 59 (Lane 5), 510 (Lane 6), and 512 (Lane 7). Arrowhead, DAN protein. Left, molecular

massmarkersin kDa. 21.5— ‘@

@

_

- -

Fig. 4. Westernblotting usingaffinity-purified antiratDAN polyclonalantibody.Total cell lysatesfrom 3Y1 cells were subjectedto 10% SDS-PAGEand separatedproteins were transferredto nitrocellulosefilter. Affinity-purified antiratDAN polyclonalantibody

(20—24h) and their saturation densities were lower (0.5—1.1X 106 cells/35-mm dish) than those of the control transfectants. Cell Cycle Analysis of DAN-overexpressing Clones. In order to

examinewhethertheconstitutiveexpressionof DAN geneaffectscell cycle progression and hence cell growth, the flow cytometric analysis was performed. As shown in Table 2, in the exponentially growing 3Y1 cells,thedistributionofcells in G@,5, andG2-M were 24%, 66%,

waspretreatedwith buffer alone(LaneI), MBP (Lanes2—4), or MBP-DAN(Lanes5—7). and 10%, respectively. Similar values were obtained with the control The amountof thecompetitorproteinswas 1.0 @g (Lanes2 and5), 5.0 g@g (Lanes3 and cells. In contrast, the DAN-overexpressing cells displayed an increase 6), or 10 g.Lg(Lanes 4 and 7) per 15 @dreaction volume. The reaction mixtures were

incubated at 37°C for 1 h andusedfor Westemblotanalysis. Arrowhead, M, 27,000 in the G@(40—58%)and a decrease in the S phase (31—44%).These findings indicate that the overexpression of DAN gene might lengthen 897

protein.Left, molecularmassmarkersin kDa.

@

@Fig.

I

CELL CYCLE-REGULATORYFUNCTIONOF DAN GENE PRODUCT

transfectantsCellsGrowth Table1 Growthpropertiesof the

known that histone gene expression is initiated at the entry into the S phase (22), the level of histone H3 mRNA was monitored in this synchronouscell culture. As shown in Fig. 8, A and B, the amount

monolayers―Doubling in time cells)3Y1 V2 V3

Si 59

510 51215.1

Saturationdensity

(h)

of histone H3 mRNA started to accumulate i2 h after serum stimulation both in 3Yi and V3 cells. The onset of histone H3

(106 1.7 1.7 1.6 0.8 0.5 1.1 1.1

14.0 14.2 21.2 20.7 23.9 20.7

mRNA expressionwas delayed until 15 h following serum addition

in the DAN-overexpressing cells (Fig. 8C). DISCUSSION

a Doublingtime was determinedby calculatingthe growthrateof exponentially growingcells. Saturationdensityis the numberofcells per 35-mm dishesafter the culture

hadreachedconfluency.Eachexperimentwascarriedout at leasttwice.

DAN gene has been originally cloned from a cDNA library of rat fibroblast3Yi cellsby a differential screening(8). Overexpressionof DAN gene product in v-src-transformed 3Y1 cells resulted in the

phenotypic reversion (9). Recently, it has been shown that human

Table2 Effectsof DAN overexpressionon asynchronous cell?CellsG1SG2-M3Y1246610V3197655144441259583111510464113512404416

DAN gene is mapped to ip36.ii—p36.i3, which resides within the

putativetumor-suppressive locusin neuroblastoma(10). Furthermore, genomicalterationwithin or closelylinked to theDAN genelocuswas detected in neuroblastoma tissues (iO). These observations strongly suggest that the DAN gene is a newly identified tumor-suppressor gene. To date, several tumor-suppressor genes have been cloned and the a Cellswereseededat2.5 X 10'cells/1O-cm-diameter dish.Themediumwasreplaced biological roles of their protein products have been investigated 48 h later with fresh medium. After an additional 24 h, cells were trypsinized and extensively (6). Among them, pRB and p53 are the best studied and cytometricanalysiswas carried out.

characterizedtumor-suppressorgenes.Genetransferexperimentsre vealed that the overexpression of these genes causes not only pheno @9@w@—

@

12@

typic reversion of the tumorigenic properties of cultivated tumor cells but alsothe G1 arrestin normal cells (24). It is believedthat someof

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151w

I8@w

21@

serumstarvation, 6. Kinetics of cell cycle progression after the addition of serum. After the

FCSat

3Y1 cell growthwasbegunin completemediumsupplemented with 10%

andanalyzed the zero time point. Then cultureswere harvestedat the indicated time points 4N by flow cytometryfor DNA content.The channelpositionsfor 2 N (G1) and G2-M,right). (G2-M) cells are notedby arrowheadsat the bottomof each column(G1, left, population.X-axis, Cells with DNA content between 2N and 4N represent the S phase fluorescence; Y-axis, cell number.

G1. In order to refine this possibility in more detail, the effect of DAN geneoverexpressionin the synchronouscell culturewas studied.3Y1

cellsweregrowtharrestedbyserumlimitationfor48 h(G1arrest)and were then inducedto transitthe cell cycle by serumrestoration.Cells were harvested at successive time points after the serum stimulation

and their cellular DNA content was monitored by flow cytometry. Approximately 90% of 3Y1 cells displayed a G@DNA content at the zero time point (Fig. 6 and Fig. 7A). The percentage of cells in the S phase was 8% and 48% at 9 and 12 h poststimulation, respectively,

indicating that the G1-S transition was initiated at 12 h after the additionof serum.The initiationof the G2-M was observedi8 h after the serum stimulation. As shown in Fig. 7B, almost the samecell cycle

patternwasobservedin V3 control cells. In the DAN-overexpressing cells, the significantenrichmentof the S phasewas not evidentuntil 15 h after the serum stimulation, indicating that the G1-S transition was delayed3 h in cells overexpressingDAN protein.The initiation of the G2-M was observed21 h after the addition of serum in the DAN-overexpressingcells (Fig. 7), indicating that the interval be tween the S and the G2-M was not influenced by the overexpression

Fig. 7. Cell cycle analysis of the DNA content of synchronizedcells. Cells were released from serum starvation-induced growth arrest and then harvested at indicated

of DAN gene product.

times. The DNA contentof cells was analyzedby flow cytometry and stainingwith

0

9

12

D

IS

18

210

9

12

E

15

18

2)

0

9

12

15

1$

21

F

propidium iodide. The percentagesof cells in each phaseof the cell cycle were calculated

Delayed S phase initiation in the DAN-overexpressingcells was from flow cytometricmeasurements of DNA content.U, G,; •, 5; A, 02-M. A, 3Y1; B, confirmed by the analysis of histone gene expression. Since it is well V3; C, Si, D, S9; E, SlO; and F, S12. 898

CELLCYCLE-REGULATORY FUNCTIONOFDANGENEPRODUCT

A

0

9

12

15

identified tumor-suppressorgeneproducts, p2i (WAF1/CIP1) and

18 21

pi6 (multiple tumor suppressor),both contain an inhibitory activ ity against cdks through physical interaction with them (29—32). DAN gene product has no sequence similarity with proteins filed in the data base (Swiss-Prot, Release 29.0). There exist several struc tural features

in the amino

acid sequence

of DAN

protein,

includ

ing casein kinase II target sites, N-linked glycosylation and Nmyristylation motifs and zinc finger-like domain (8); however,

their functional significance is yet to be determined. Recently, it has been reported that 3Y1 cell lines contain p53 point mutation in one of the mutational hot spots (33). Considering that the mutant form of p53 is unable to activate the expression of WAF1/CIPJ gene (30), it is unlikely that the DAN-induced growth retardation

could be mediated by the activity of WAFJ/CIPJ gene product.

B

The level of DAN protein is nearly constant during the cell cycle of 3Y1 cells (data not shown).It is not clear whetherDAN geneproduct retains the growth-suppressive activity throughout the cell cycle or only near the Ga-S transition.Recently, a new cdk inhibitor, p27'――, hasbeen identified and characterizedin detail (34, 35). Overexpres sion of p2lkiiil obstructs cell entry into the S phase. Interestingly, the expression of p27k@@I did not change during the cell cycle either in mRNA or protein levels (35). It is possible that the growth-suppres sive activity of DAN gene product is regulated, as in the case of p27@', by the interaction with key molecules which involved in the cell cycle control.Another possibilitymight be the cell cycle-depen

dent modification ofDAN gene product as in the case of pRB (36—38).

C

Although the precise mechanism for the DAN-mediated growth sup pressionremainsto be determined,the resultsdescribedin this report

indicate that DAN gene product is one of the important participants in the process of the cell cycle progression.

ACKNOWLEDGMENTS We are grateful to Dr. M. Iwabuchi for providing wheat histone H3 DNA.

REFERENCES 1. Dunphy,W.0., andNewport, J.W.Unraveling of mitoticcontrolmechanisms. Cell, 55: 925—928, 1988.

Fig. 8. Expressionof histoneH3 mRNA in DAN-overexpressing cells during the cell cycle.Total RNA wasisolatedfrom eachcell line at theindicatedtime pointsafterserum stimulation.Northernblot hybridizationwas performedusing32P-labeledwheathistone H3 DNA as a probe (23). The cell lines analyzedwere 3Y1 (A), V3 (B), and 59 (C). Ethidium bromidestainingof RNA is alsopresented(lower panels).

2. Nurse,P. Universalcontrol mechanismregulatingonsetof M-phase.Nature(Lond.), 344: 503—508, 1990. 3. Hunter,T., andPines,J. Cyclins andcancer.Cell, 66: 1071—1074, 1991. 4. Karan,J., Watson,J. V., Love,A. D., Green,S. M., andVedeckis,W. Regulationof cell cycle durationby c-myclevels.Oncogene,4: 773—787, 1989. 5. Bishop, J. M. Molecular themes in oncogenesis. Cell, 64: 235-248, 1991.

6. Marshall,C.J.Tumorsuppressor genes.Cell,64:313-326,1991. 7. Goodrich, D. W., Wang, N. P., Qian, Y-W., Lee, E. Y-H., and Lee, W-H. The retinoblastomageneproductregulatesprogressionthroughthe G@phaseof the cell cycle. Cell, 67: 293—302, 1991.

the tumor-suppressor geneproductsplay physiologicalrolesin normal cells, such as the negative regulation of cell cycle progression. In the case of pRB, the growth

suppressive

activity

8. Ozaki, T., and Sakiyama, S. Molecular cloning and characterizationof a cDNA

could be lost by the

showingnegativeregulationin v-src-transformed3Y1 fibroblasts.Proc.Natl. Acad. Sd. USA, 90: 2593-2597,1993.

genetic lesions including point mutations or deletions, phosphoryla tions, or complex formation with viral oncoproteins(6). In the presentwork, we have examinedthe possibleroles of DAN geneproducton the growthcontrolof rat 3Y1 fibroblasts.Cells which express a large amount of DAN gene product displayed obvious growth-suppressiveproperties,including a prolongeddoubling time and a reduction of the saturation density, when compared with those

of the parental 3Y1 cells. The flow cytometric analysis revealed that

9. Ozaki,T., andSakiyama, S. Thmor-suppressive activityof N03geneproductin v-src-transformedrat 3Y1 fibroblasts.CancerRes.,54: 646—648, 1994. 10. Enomoto, H., Ozaki, T., Takahashi, E., Nomura, N., Tabata, S., Takahashi, H.,

Ohnuma,N., Tanabe,M., lwai, J., Yoshida,H., Matsunaga,T., and Sakiyama,S. Identification of humanDAN gene,mappingto the putative neuroblastomatumor suppressivelocus.Oncogene,9: 2785—2791, 1994.

11.Kimura,0., Itagaki,A.,andSummers, J.Ratcellline3Y1anditsvirogenicpolyoma andSV4O-transformed derivatives.mt. i. Cancer,15: 694—706, 1975.

12.Yamashita, T., Yamamoto, K., Shiroki,IC, Kimura,0., Matsui,K, Osato,T., Shimojo,H., andFujinaga,K. Chromosomalalterationsof rat cell lines transformed by humanadenovirustype-12 vision,whole DNA and leftend DNA fragments.lnt. J.

the elevatedlevel of DAN expressionlengthenedG1. Since the inter val between S and G2-M was not altered in theseDAN-overexpressing

Cancer,26: 435—442, 1980. 13. Shiroki, K., Hamaguchi,M., and Kawai, S. Highly efficient focusformationby Rous

clones, it is possible that DAN gene product negatively regulates the timing of the transition only at the Ga-S boundary. The rate of the progressionthroughG@of the cell cycle in mum malian cells is governedby the sequentialactivationof G1 cyclinsand

their catalytic partners(cdks) (3, 25). The cdk activity can be increased through phosphorylation

sarcomavirus on adenovirustype 12 E1A-transformedrat 3Y1 cells. J. Virol., 66: 1449—1457, 1992. 14. Sternberg,D. W., SchoLz,G., Fukui, Y., and Hanafusa,H. Activation ofa histoneHi

kinaseby tyrosinephosphorylationin v-src-transformedfibroblasts.EMBO J., 12: 323—330,1993. 15. Katzav, S. K., Zanca, D. M., and Barbacid,M. vav, a novel humanoncogenederived

(26—28). In contrast, newly

from a locusubiquitouslyexpressedin hematopoieticcells.EMBO J.,8: 2283—2290,

1989. 899

CELL CYCLE-REGULATORYFUNCTIONOF DAN GENE PRODUCT 16. Homma, Y., Emori, Y., and Takenawa, T. Isolation and characterizationof rat 3Y1

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