Vol. 5, 1093-1103, October 1994 Cell Growth & Differentiation 1093

Similarities between Somatic Cells Overexpressingthe mos Oncogene and Oocytesduring Meiotic lnterphase1

Kenji Eukasawa, Monica S. Murakami, Donald C. Blair,Ryoko Kuriyama, Tim Hunt, Peter Fischinger, andCeorge E. Vande Woude2

ABL-Basic Research Program 1K. F., M. S. M., G. F. V. W.l and Laboratoryof Molecular Oncology ID. G. B.l, NCI-Frederick Cancer Research and

Development Center, Frederick, Maryland 21 702-1 201 ; Department ofCell Biology and Neuroanatomy, University of Minnesota Medical School,

Minneapolis, Minnesota 55455 IR. K.l; Imperial Cancer Research Fund,Clare Hall Laboratories, South Mimms, Herts EN6 England 3LD (T. HI;

and Medical University of South Carolina, Charleston, South Carolina29425-1020 lP. F.l

Abstract

The mos protooncogene encodes a serine/threonine kinaseand is a key regulator of oocyte meiotic maturation. Afteracute infedion of Swiss 3T3 cells with virus containing thev-mos oncogene, cells expressing high levels of v-Mosround up and detach from the monolayer (floating cells),while cells that remain attached express 10-fold lowerlevels of v-Mos and are transformed. The floating cells aregrowth arrested with their chromosomes partially con-densed in the absence of histone Hi kinase adivity, whilemitogen-adivated protein kinase adivity is very high. Col-lectively, these properties are similar to properties ob-served in maturing oocytes between meiosis I and II. Inv-mos-transformed cell populations, mitogen-adivatedprotein kinase adivity is also elevated, correlating with thedegree of morphological transformation and the level ofMos expression. Moreover, phosphoprotein modificationsspecific for M are found in both the floating cells and inv-mos-transformed cells, regardless of their cell cyclestage. One explanation for both morphological transfor-mation and the phenotypes of the floating cells is that Mosimposes a meiotic program on different stages of the so-matic cell cycle. The extent of this meiotic phenotype isproportional to the level of v-Mos expression. These resultssuggest that both morphological transformation and thephenotypes of the floating cells induced by Mos in Swiss3T3 cells are related to its normal adivities during oocytematuration.

Introduction

The mos gene was originally identified as an acquiredcellular sequence carried by the acute transforming retro-virus, Mo-MuSV3 (i-4). Recombinant v-mos, under a pro-

Received 5/27/94; revised 7/29/94; accepted 8/9/94.

I Research sponsored in part by the National Cancer Institute, DHHS, under

contract No. N01-CO-74101 with ABL. Research funding for K. F. wasprovided by the G. Harold and Leila Y. Mathers Charitable Foundation.2 To whom requests for reprints should be addressed, at ABL-Basic Research

Program, NCI-Frederick Cancer Research and Development Center, P.O.Box B, Frederick, MD 21702.3 The abbreviations used are: Mo-MuSV, Moloney murine sarcoma virus;MPF, maturation promoting factor; MAPK, mitogen-activaled protein kinase;MTOC, microtubule organizing center; P.1., post infection; DAPI, 4,6-dia-

midino-2-phenylindole; MBP, myelin basic protein; PBS, phosphate-buff-

viral long terminal repeat promoter, was the first oncogeneshown to transform cells efficiently in DNA transfectionassays (5-7). Similarly, the murine cellular homologue,c-mos’�, under long terminal repeat control was shown totransform cells efficiently (4, 5), demonstrating that v-Mosand c-Mos have essentially the same biological activitiesand that inappropriate expression of the mos protoonco-gene in fibroblast cells is sufficient to cause morphologicaltransformation (3, 4, 8, 9).

The c-mos protooncogene product is normally expressedin germ cells (iO) and is required throughout oocyte matu-ration in Xenopus to activate MPF (11-18). Mos has alsobeen identified as an active component of cytostatic factor(1 9), an activity associated with the arrest of oocyte matu-ration at metaphase of meiosis II, the state corresponding toan unfertilized egg (20). Thus, Mos is a key regulator ofmeiosis and the production of unfertilized eggs. Mos hasbeen shown to associate with tubulin in vitro and the spin-dIe and the spindle poles in vivo, suggesting that Moscontributes to meiotic microtubule reorganization (21 , 22).MAPK has also been implicated in meiotic microtubulereorganization (23) and has been shown to localize toMTOCs during meiosis (24). Recently, Mos has been shownto activate MAPK in vivo and in vitro in Xenopus oocytes orcell-free extracts (25, 26).

Acute infection of Swiss 3T3 cells with Mo-MuSV thatcontains the v-mos protooncogene causes a large percent-age of the cells to round-up and detach from the monolayer(floating cells; Refs. 27 and 28), and only the cells thatremain attached grow as morphologically transformedcells. We have shown that approximately 70% of the float-ing cells are growth arrested with 2C or 4C DNA content.4The cells with 4C DNA content are binucleated and haveundergone nuclear division without cytokinesis.4 In thisstudy, we further characterized Swiss 3T3 cells overexpress-ing or transformed by v-Mos and show, in the floating cells,

partial chromosomal condensation in the absence of Hikinase activity. Furthermore, phosphoprotein modificationsthat are specific to M in nontransformed cells are detectedin the floating cells and in v-mos-transformed cells inde-pendent of the cell cycle stage.

Results

Morphological Transformation and v-Mos Expression. Toobtain high levels of Mos expression in somatic cells, weinfected Swiss 3T3 cells with Mo-MuSV containing thev-mos oncogene. Control cells were either mock-infectedor infected with helper Mo-MuLV. As previously reported,60-70 h P.1., the majority of Mo-MuSV-infected cells (70-90%) round up and detach from the monolayer as floating

ered saline; SDS-PAGE, sodium dodecyl sulfate-gel electrophoresis; TBS,Tris-buffered saline; DTT, dilhiothreitol; PMSF, phenylmethylsulfonyl fluo-

ride.4 K. Fukasawa and G. F. Vande Woude, submitted for publication.

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1094 rnos-incluced M-phase Phenotypes in Somatic Cells

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Fig. 1. The immunoblot analysis

of v-Mos expression in cells in-fected with Mo-MuSV and pho-

tomicrographs of cloned v-mostransformed cell lines. A, cell cx-

tracts containing -� 1 00 pg of total

proteins were run on a i0”/� SOS-PAGE and transferred onto Immo-

bilon (Millipore( sheets. The blotswere then prc)bed with anti-Mos”

polyclonal antibody ZC-75. Anti-

body-antigen complex was de-tected with alkaline phospha-

tase-conjugated anti-rabbit IgG

antibody, followed by reactionwith bromochloroinclolyl phos-phate-nitroblue letrazolium (Bio-

Rad(. Lane 1, control Mo-MuLV-infected cells; Lane 2, PTS1

c-rnos” transformed cell line;Lane 3, floating cells; Lane 4, Mo-

MuSV-infectecl cells that remain at-tached to the dish 40 h P.1.; Lane 5,

Mo-MuSV-infected cells that re-main attached to the dish 70 h P.1.;

Lane 6, Tx3 line; Lane 7, Tx2 line;Lane 8, Txi line. B, cells grown inculture dish were directly photo-

graphed using an inverted niicro-

scope. a, control Swiss 3T3 cells; b,Txi cells; c, Tx2 cells; d, Tx3 cells.x 100.

cells.4 Approximately 70% of floating cells exclude trypanblue but do not proliferate, while the remaining cells aredead or dying (27).� Only the cells that remain attachedgrow as foci of morphologically transformed cells. Wecloned several cell populations from these foci, and threeclones were chosen for further study (Txi , Tx2, and Tx3).

The level of v-Mos in the floating cells is - 0.1 % of totalprotein (Fig. 1A, Lane 3), which is -10-fold higher than inthe Mo-MuSV-infected cells that remain attached at 40 hP.1. (Fig. 1 A, Lane 5) and 60-70 h P.1. (Fig. 1 A, Lane 4). The

level of p39fllO� expression in PTS1 c�mos��u�transformedcells is barely detectable (Refs. 1 5 and 29; Fig. 1 A, Lane 2).As expected, the mobility of c-Mos is faster than v-Mos,which contains 26 extra NH,-terminal amino acids (8).Among the cloned v-mos transformants, Tx3 cells expressthe highest level of v-Mos (Fig. 1A, Lane 6) and show thegreatest morphological alteration (Fig. 1 B, d), while Txlcells express the lowest level of v-Mos (Fig. 1 A, Lane 8) and

are least transformed (Fig. 1 B, b). The Tx2 cells are inter-mediate both in v-Mos expression and transformed pheno-type (Figs. 1A, Lane 7and 18, c). Moreover, the efficiencyof anchorage-independent colony formation in soft agar ofthe v-mos-transformed cell lines also correlates with thelevel of v-Mos expression; the average CFE (the ratio of thenumber of soft agar colonies to the number of viable cellsplated) from two independent experiments was 0.001 forSwiss 3T3 cells, 0.1 3 for Txl cells, 0.24 for Tx2 cells, and0.42 for Tx3 cells. These results confirm earlier studiescorrelating v-Mos overexpression with somatic cell toxicity(30) but also show that the degree of morphological trans-formation correlates directly with the level of Mos expres-sion.

Partial Chromosomal Condensation Induced by HighLevel Expression of v-Mos in the Absence of Histone HiKinase Activity. The majority of the viable floating cells,both cells arrested at 2C and 4C DNA content, contained

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Fig. 2. Inappropriate chromosomal condensation in the floating cells re-suIted troni v-Mos overexpression. The viable floating cells, isolated after

Peroll gradient separation of viable cells from dead or dying cells, wereseeded onto ,� slick’ and examined at X 630. Control cells were grown on

coverslips. After fixation with 1.7/,, formaldehyde, cells were stained withDAPI. A. iriterphase control IT I cell; B. control 3T3 cell in mitosis; Cancl 0,floating ( elk.

Fig. .3. The influence of v-Mos expression on H 1 histone kinase activity andits components, p34’ ‘�‘ 2 and cyclin B. A, cell extracts were precipitated withp1 3” ‘ beads. The beads were subjected to histone Hi kinase analysis as

described in “Materials and Methods.” Control IT.) cells (Lane 1 (, nococla-zole-arrested M-phase control 3T3 cells (Lane 2), floating cells (Lane fl, andTx3 v-mos-transformed cells (Lane 4(. The dead or dying cells were removedfrom the floating cells by Percoll gradient centrifugalion prior to the prepa-ration of lysates. B, p34’’�’ � cell extracts containing �-50 pg of total protein

were sul)jectedl to iO”/,, SDS-PAGE and Iransferrecl onto Imniobilon (Milli-

Pore( sheets. The blots were probedl with anti-p34’ ‘� ‘� COOH-terminalmonoclonal antibody (Santa Cruz(, and anlil)odly-antigen complexes were

dletecled with alkaline phosphatase-conjugateci anti-mouse lgG antibody, as

dlescril)edf in the Fig. 1 legend. The samples in Lanes 1-4 are as in (A(. C,cyclin B: immunoblots, prepared as dlescribedl in (B), were I)rOIR’dI with

anti-cyclin B Polyclonal antibody (Santa Cruz), and antibody-antigen coni-plexes were detected with horseradish peroxidase-conjugateci anti-rabbit

lgG and visualized by chemiluminescent reactions as reconimencleci (Am-

ersham(. The samples in Lanes 1-3 are as in (A). 0, cyclin B in p34’ ‘�

immunoprecipitates: p34’ � � was immunoprecipitated with anti-p34’ “

COOH-terminal monoclonal antibody (Santa Cruz( from control 3T3 cells(Lane 1), nococlazole-arrestecl M-phase 313 cells (Lane 2), andl floating cells

(Lane I). After extensive washing with lysis I)uffer, the immunoprecipitateswere resolved on a 10/ SDS-PAGE and transferred onto Immobilon (Milli-

pore) sheets. The immunoblots were then probe(1 with anti-cyclin B antibodyas described (C) and visualized by chemiluminescent reactions.

Cell Growth A Differentiation 1095

‘ #{163}mpartially condensed chromosomes as revealed with DAPIDNA stairting (Fig. 2, C and D, and data not shown)4 andresemble the state of condensation observed in controlSwiss 3T3 cells only during prophase (Fig. 2B). Interphasecontrol cells stained with DAPI do not show condensation(Fig. 2A).

It has been shown that �34�f�2 kinase induces chromo-somal condensation (31 , 32). Since Mos indirectly activatesand stabilizes MPF (19), we assayed the floating cells forp34((f(2 kinase activity by measuring Hi histone kinaseactivity (Fig. 3A). pi3�’1 bead-precipitated extracts from

nocodazole-treated M-arrested Swiss 3T3 cells displayedhigh levels of histone Hi kinase activity (Fig. 3A, Lane 2).In contrast, extracts from unsynchronized control Mo-MuLV-infected and v-Mos-transformed cell populations, inwhich only a fraction of the cells are in M, displayed theexpected low levels of activity (Fig. 3A, Lanes 1 and 4).Surprisingly, extracts from floating cells lacked detectablehistone Hi kinase activity (Fig. 3A, Lane 3). Although v-Mos-induced chromosome condensation could have re-suIted from transient p34�th2 activation, these results showthat the activation of p34”t’ � is not required for the main-tenance of the chromosome condensation or growth arrest.

States of p34cdc2 and Cyclin B in the Floating Cells. Wefurther examined the floating cell population to determinewhether p’t’ 2 was dephosphorylated and if cyclin B waspresent. We used an antibody directed to the COOH ter-nlinus of � 2 in immunoblot analyses. Control 3T3 cellsin M (Fig. 3B, Lane 2) showed only the expected singleactive form of p34((f(2, while actively growing control cellsand Tx3 cells displayed additional, slower-moving phos-phorylated fornis (Fig. 3B, Lanes 1 and 4, respectively).Only the dephosphorylated form of p34’� � was detected in

the floating cells (Fig. 3B, Lane 3). Even though histone Hikinase activity was not detected (Fig. 3A), cyclin B wasunexpectedly found in the floating cells at levels similar tocontrol cells in M (Fig. 3C, compare Lanes 2 and 3). More-over, cyclin B was not found in anti-p34�’�2 immunopre-cipitates from the floating cells but was coprecipitated withp34((l( 2 from M-arrested control cells (Fig. 3D). We are notcertain why cyclin B is not associated with p34�’2 in thefloating cells, but a similar result was observed previously inMos-transformed NIH3T3 cells (33).

MAPK Activity in Somatic Cells Expressing Mos. Moshas been implicated in MAPK activation during meiosis (25,

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Fig. 4. MAPK activation in cells expressing v-Mos. Control 3T3 cells (Lane 1); v-raf-transformed 3T3 cell line (BxB; Lane 2); floating cells (Lane 3);v-mos-transformed cell lines, Tx3 (Lane 4); Tx2 (Lane 5); Txl (Lane 6). The dead or dying cells were removed from the floating cells prior to the preparation of

lysates. The v-raf-transformed 3T3 cell line with constitulively activated MAPK was included as a positive control (Lane 2). MBP phosphorylation was analyzed

by 18% SDS-PAGE, followed by autoradiography (upperpanel). The kinase activity was also quantitated by scintillation counting, and the data is presented in

bar graphs (lower panel). The lane designations of the auloradiograph and bar graph are the same.

1096 mos-induced M-phase Phenotypes in Somatic Cells

S T. Choi and G. F. Vande Woude, manuscript in preparation.

26), and the level of MAPK activity is significantly higherduring meiosis than during mitosis in one-cell mouse em-bryos.5 In nontransformed somatic cells, MAPK is activatedonly after mitogenic stimulation in G1 (34-38) and is re-ported to increase slightly at G2-M (39). Here, we examinedthe relationship between the level of MAPK activity, thelevel of v-Mos, and the degree of morphological transfor-mation. MAPK activity was determined in vitro using MBPas a substrate (Fig. 4). MBP phosphorylation was not de-tected in MAPK precipitates from control Swiss 3T3 cellsmaintained for 36 h in 0.5% serum (Fig. 4, Lane 1), whilethe floating cells and the v-mos-transformed cell lines(Txi-3) displayed high levels of MBP phosphorylation ac-tivity (Fig. 4, Lanes 4-6). The level in Tx3 cells is similar tothe level detected in raf-i -transformed cells (Fig. 4, Lane 2).These analyses show that MAPK activity correlates with thelevel of Mos expression as well as with the extent of mor-phological transformation (compare with Fig. 1 ). Moreover,MAPK activation in the floating cells and transformed cellsis constitutive in 0.5�/� serum and occurs in the absence ofmitogenic stimulation.

Similarities of Phenotypes between the Eloating Cellsand Oocytes during Meiotic lnterphase. Low histone Hiki nase activity, chromosome condensation, dephosphory-lated p34cdc2, the presence of cyclin B, and the activationof MAPK in the floating cells are reminiscent of the prop-erties of amphibian and mammalian oocytes during meioticinterphase, the period between meiosis I and meiosis II. InXenopus oocytes during meiotic interphase, MPF is low

(Ref. 40; Fig. 5, A and D), while cyclin B is present (4i , 42)and p34cdc2 remains dephosphorylated (Fig. SB). By mo-bility shift, MAPK remains activated throughout meiosisincluding meiotic interphase (Fig. 5, C and 0). Activated

MAPK is also detected during meiotic interphase by in vitrokinase assays using anti-MAPK monoclonal antibody and

MBP as a substrate (Fig.SD).The Role of Mos and MAPK in Mos-transformed Cells.

The role ofMAPK in gene regulation during G1 (reviewed inRefs. 43-46) is quite different from its proposed role inmicrotubule reorganization during meiosis (23, 24). Wehave proposed that Mos induces, during meiosis, microtu-bule reorganization and that this activity is responsible forthe transformed phenotype (47). Therefore, we examined

the microtubule organization in the floating cells andv-mos-transformed cells by immunostaining with anti-a-tubulin antibody (DMiA; Fig. 6A). Compared with controlSwiss 3T3 cells, the microtubule organization in the floating

cells and in the v-mos-transformed cells was significantlydisrupted (Fig. 6A, b and c). We also examined the floatingcells and v-mos-transformed cells with the monoclonal an-

tibody ZSK2 (Zymed) that was originally prepared againstrat brain tubulin. Surprisingly, the ZSK2 antibody only

stained the spindle of Swiss 3T3 control cells in M, whilecells in interphase were not efficiently stained (Fig. 6B, a-c).However, ZSK2 stained the floating cells and v-mos-trans-formed cells intensely and, more importantly, independentof the cell cycle stage (Fig. 6B, g-j). These results showedthat the ZSK2 antibody preferentially reacted with M-spe-cific epitopes in nontransformed cells and provided evi-dence that these epitopes were constitutively present incells expressing v-Mos. The antigens detected in immuno-

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Cell Growth & Differentialion 1097

Fig. 5. Analysis of Hi histone ki-nase and MAPK activities during

the meiotic interphase of Xenopus

OOcytes. Stage VI oocytes, preparedas described) in “Materials andMethodls,” were induced for matu-ration by incul)ation in 5 pg/mI pro-

gesterone ( Prog. (. In ordler to exam-

ne the meiotic interphase (Mint,),sve achievedl the synchronization of

maturing oo ytes by setting thetime of germinal vesicle breakdown

(GVBD(, determined by the ap-pearance of a white 5001 in the an-

imal heniisphere, as “0” time, andlysates were 1)r(’Paredl relative tothat time schedule. A, analysis ofHi histone kinase activity diuring

meiotic interphase. The oocyte ly-

sates PreI)ar(’dI at each time pointwere measured for MPF activity in

vitro using histone H 1 as a substrate

as described previously (79). The in

vitro kinase reaction mixtures were

analyzedi by 1 2/ SDS-PAGE. B,immunoblot analysis of immuno-

precipitatedl p34’ “ 2 using anti-phosphotyrosi n(’ (anti-P-Tyr) mono-

clonal antibody. The lysates were

immunoprecipitated with anti-

� ‘� 2 COcwi-terniinal peptideantil)ody (Santa Cruz(. The immu-

noprecipitates were resolved by1 2/ SOS-PAGE and transferredonto Immohilon (Millipore) sheets.

The blots were then probed with

anli-P-Tyr monoclonal antibody(PY-20(. Antibody-antigen complexwas delectedl by chemillumines-

cent reactions as described in the

Fig. 3 legend. C immunohlol anal-ysis of MAPK during meiotic inter-

phase. The lysates resolvedi in iO#{176}k

SDS-PAGE were transferred ontoImmohilon )Millipore) sheets andprobed with anti-MAPK monoclo-nal antibody (Zymed). Antibody-

antigen complex was dietected by

chemilluminescent reactions as de-scribed in the Fig. 3 legendl. MAPK’indicates the phosphorylated, ac-

five form of MAPK. 13, MPF (histone

Hi phosphorylalion( activity andMAPK, in vitro MBP phosphoryla-tion activity of anti-MAPK mono-

clonal antibody immunoprecipi-tales, were also dluantitated byscintillation counting and summa-rized in (0).

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blot analyses by ZSK2 were not tubulin molecules but werereminiscent of the antigens detected with the M-specificmonoclonal antibodies, MPM-2 (48) and CHO3 (Ref. 49;see below).

M-specific Phosphorylation during Interphase. Themonoclonal antibody MPM-2 reacts with phosphorylatedprotein epitopes that are specifically modified during mito-sis or meiosis but not during interphase (Refs. 48, 50, and

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.�;Fig. 7. Immunoblot analysis of v-Mos-expressing cells with monoclonal antibodies directed against M-specific phosphoprotein epilopes. Cell extracts, prepared

as in the Fig. 1 legend and containing -50 pg of total protein, were run on a 6-i 2% gradient SOS-PAGE andl then transferredl onto Immobilon )Millipore) sheets.The immunoblots were then probed with the monoclonal antibodlies: (A) MPM-2; (B) CHO3; and (C) ZSK2. Antibody-antigen (omplexes were detected withhorseradish peroxidase-conjugaledi anti-mouse lgG antil)odly (Boehringer Mannheim; for MPM2 and ZSK2( or anti-mouse 1gM antibodly )Boehringer Mannheim;for CHO3(, followed I)y chemilluminescent reactions. Lane 1, control 3T3 cells; Lane 2. nocodlazole-arrested M-phase control 313 cells; Lane 3, floating cells;

Lane 4, Tx2 dells; Lane 5, Tx.) cells. The detection of M-phase antigens in cells expressing v.Mos (Lanes 3-5) was not due to a higher percentage of transformed

cells being in M-phase, since by flow cytometry analysis, v-rnos-transformedl cells show cell cycle phase distribution similar to contrd)l Swiss 3T3 cells (data notshown(. More so than with the MPM-2 and CHO3 antibodies (Fig. 7 and data not shown), the ZSK2 antibodly is more reactive with antigens in cells expressingv-Mos (Figs. 6 aridl 7) than M-phase control Swiss 3T3 cells.

Si ; Fig. 7A, compare Lanes 1 and 2). Like MPM-2, the ZSK2

and CHO3 antibodies also recognize epitopes that are sen-sitive to alkaline phosphatase treatment (data not shown),and all three antibodies react predominantly with M-spe-cific phosphorylated epitopes (compare Fig. 7A, B, and C,Lanes 1 and 2). Strikingly, with each of the monoclonalantibodies, we found that antigens detected in the extractsof the floating cells (Fig. 7A, B, and C, Lane 3) or v-mos-transformed cell lines (Fig. 7A, B, and �, Lanes 4 and 5),were very similar to those found in the M-arrested controlcells. However, low molecular weight antigens detected byeach antibody are more prevalent in the v-Mos-expressingcells. We conclude that Mos expression induces M-specificphosphoprotein modifications at all stages of the somaticcell cycle.

Discussion

Our results suggest a model for transformation by the Mos

oncogene, whereby the program that is normally restricted

to the process of meiosis runs concurrently with the pro-

gram of the highly ordered somatic cell cycle, and the

degree of morphological transformation correlates with the

level of Mos and/or MAPK activation. At very high levels,

Mos expression in somatic cells induces MAPK activation,

growth arrest, binucleated cells,4 and inappropriate chro-

mosome condensation. Our studies also show that Mos

expression in somatic cells induces M phosphorylation

events at nonmitotic phases of the cell cycle. Davis et a!.

(52) have shown that MPM-2 antibody does not affect entryinto mitosis but blocks exit from mitosis, perhaps by pre-

Fig. 6. Indlirect immunofluorescence staining of v-Mos-expressing cells with monoclonal antibodies directed against o-tubulin andi M-phase phosphorylation-

dlependlent epit(ipes. A, cells either growing d)fl a coverslip (control IT 3 cells andl Tx3 v-rnos-transformed cells) or seededl on a coverslip by low speedcentrifugation (floating cells) were fixed andl immunostained with anti-c tubulin monoclonal antibody )DM1A(, followedi by a fluorescein isothiocyanate-conjugatedl anti-mouse lgG antibodly (Amershani). a. control Swiss 3T3 cells; b, floating cells; c, Tx3 v-mos-transformed cells. x 630. B, cells were stained withDAPI (d-t andl treatedl with monoclonal ZSK2 antibody )Zymed; a-cand g-j). The ZSK2 antibody-antigen complexes were detectedi using a Texas redl-conjugated

anti-mouse lgC antibody (Amersham(. a and d. the ZSK2 and DAPI staining of control 3T3 cells at interphase; b and e, c andl 1, control 3T3 cells during mitosis.White arrosss, the mitotic cells dletectedi by condensed chromosomes (DAPI). ZSK2 dloes not efficiently stain the interphase cells. ZSK2 stained Mo-MuSV-infected

cells at 4)) h P.1. (g(, floating cells (h(, Tx3 v-rnos-transformed cells (i), andl Tx2 v-rnos-transformed cells )j( indlependent of their cell cycle stages. x 250.

1100 mos-induced M-phase Phenotypes in Somatic Cells

U, K. Fukasawa and N. G. Ahn, unpublished observations.

venting the dephosphorylation of the M-specific antigens.The high levels of v-Mos could induce growth arrest bypreventing sufficient dephosphorylation of certain M-spe-cific antigens to allow cell cycle progression.

In the floating cells, in spite of the presence of dephos-phorylated p34cdc2 and cyclin B, histone Hi kinase activityis not detectable, although the floating cells display chro-mosome condensation. This phenotype may be similar tooocytes during meiotic interphase, when MPF is low, butthe chromosomes remain condensed (53), and the levels ofMos and MAPK are high. These kinases and the cascade ofkinases and phosphorylation events they regulate may besufficient to maintain growth arrest and chromosome con-densation. MAPK is activated by Mos (25, 26) and is local-ized to MTOCs present at the poles and kinetochores of themeiotic spindles. Recently, Mos has been localized to kin-etochores (54). Moreover, both MPM-2 and CHO3 mono-clonal antibodies that constitutively recognize M-phase an-tigens in cells expressing Mos also stain MTOCs at mitoticspindle poles and kinetochores (55-58), and at least someMPM-2 and CHO3 epitopes are generated by MAPK duringmeiosis (59). Furthermore, using partially purified microtu-bules from Swiss 3T3 cells as a substrate for MAPK in vitro,at leastone high molecular weight protein (Mr 230,000) isphosphorylated and becomes an epitope for MPM-2 andCHO3 monoclonal antibodies.6 It is possible, therefore,that Mos expression in somatic cells either directly or, morelikely, through activating a cascade of specific kinases thatare normally restricted to meiotic maturation such as MAPKand possibly Raf(60, 61), promotes the inappropriate phos-phorylation of M-phase epitopes at all stages of the cellcycle.

Boveri (62) first implicated defective spindle poles as acausative factor in malignant tumors, and binucleated tu-mor cells were one of the earliest unexplained histopatho-logical observations found in Mo-MuSV-induced mousesarcomas (63). Cells transformed by v-mos have beenshown to be genetically unstable (64), and genetic instabil-ity is a component of tumor cell progression. The sisterchromatid exchange and chromosome segregation thatuniquely occur during meiosis is likely to use differentcheckpoint functions for fidelity than somatic cells (65).Superimposing a meiotic program on a mitotic cell cyclecould possibly influence and compromise somatic cellcheckpoint function. Other oncogenes such as ras (16,66-68) and raf(60, 61) induce meiotic maturation, and allactivate MAPK (69-72). Constitutive activation of MAPK isa property common to many oncoproteins; therefore, theMAPK family of proteins (73-75) would make ideal down-stream targets for clinical intervention.

Materials and Methods

Cells. Swiss 3T3 cells used in this study were maintained incomplete medium (Dulbecco’s modified Eagle’s medium-10% fetal bovine serum with 100 units/mI penicillin and100 pg/mI streptomycin) and grown in an atmosphere con-taming iO% CO2. Cultures were maintained at a subcon-fluent density by passing 7.5 x i05 cells per 100-mm dishtwice weekly.

Virus and Virus Infedion. Mo-MuSV 1 24 is a clonalisolate from the original Mo-MuSV stock (76). Twenty-four

h before infection,i x i06 Swiss 3T3 cells were plated per100-mm dish with complete medium containing 4 pg/mIPolybrene; followed by a 1-h incubation period with fil-tered medium from either Mo-MuSV i 24-transformed pro-ducer cells (TB cell line) or Mo-MuLV producer cells, alsocontaining 4 pg/mI Polybrene. The multiplicities of infec-tion were typically 5-iO focus-forming units per cell forboth Mo-MuSV 1 24 and Mo-MuLV. Mock-infected cellswere treated identically, except that no virus was added.After infection, the cells were cultured in complete me-dium.

Immunoblotting. Cells were washed twice in PBS andthen lysed in modified sample buffer [2% SDS, 10% glyc-erol, and 60 mM Tris (pH 6.8)1. Cell extracts were cleared bya 30-mm centrifugation at 1 00,000 X g. The lysate dena-tured at 95#{176}Cfor 5 mm in sample buffer [2% SDS, iO%glycerol, 60 mt�i Tris (pH 6.8), 5% f3-mercaptoethanol, and0.Oi % bromophenol blue]. Samples were resolved by SDS-PAGE. Fractionated proteins were then transferred to Im-mobilon (Millipore) sheets, and the blots were incubated inblocking buffer [5% (w/v) nonfat dry milk-0.2% Tween 20in TBS)] for 2 h at room temperature and then with primaryantibodies for 2 h at room temperature. The blots wererinsed in TBS-T (0.2% Tween 20 in TBS) and incubated witheither alkali ne phosphatase-conj ugated or horserad ish per-oxidase-conjugated secondary antibodies for 1 h at roomtemperature. The blots were then rinsed in TBS-T, and theantibody-antigen complex was visualized with either bro-mochloroindolyl phosphate (Bio-Rad) and nitroblue tetra-zolium (Bio-Rad) as substrates in 100 m�i NaCI-5 ms’tMgCl2-iOO mM Tris (pH 9.5) for the alkaline phosphatasereaction or visualized by using the ECL chemiluminescencemethod (Amersham), according to the manufacturer’s in-structions for horseradish peroxidase reaction.

Indired Immunofluorescence. Cells, either seeded onslides by low speed centrifugation or grown on coverslips,were washed twice with PBS and then fixed with 3.7%formaldehyde for 20 mm at room temperature. After fixa-tion, the cells were washed with PBS and permeabilizedwith 1% Nonidet P-40 in PBS for 5 mm at room tempera-ture. Coverslips were incubated with blocking solution(1 0% normal goat serum in PBS) for i h and then incubatedwith primary antibodies, followed by incubation with eitherfluorescein isothiocyanate or Texas red-conjugated second-ary antibodies for 1 h at room temperature. Coverslips werewashed three times with PBS after each incubation. Cover-slips were then embedded in aqueous nonfluorescingmounting medium.

Ceneration and Preparation of Floating Cells. Over theperiod of 72 h after infection of Swiss 3T3 cells with Mo-MuSV, �90% ofthe cells round up and float into the media(floating cells). The floating cells were directly collectedfrom the cell culture media. The examination of their via-bilities by trypan blue exclusion assay showed that -70%ofthe floating cells were viable (excluding trypan blue), and-30% were dead or dying. The ratio of viable to dead ordying cell populations are constant in repeated experimentswith the SE of less than 5%. When replated into fresh media,the floating cells remain viable for at least 1 week.4

Histone Hi Kinase Analysis. Cell extracts containing-300 pg total protein were incubated with 25 p1 pi3sU1�beads (Oncogene Science) for 3 h at 4#{176}C.The beads werecollected, washed five times with lysis buffer [1 50 m�NaCI, 50 mM Tris (pH 7.5), i mtvi EDTA, i % Nonidet P-40,1 mM DTT, 50 mM NaF, 1 misi Na3VO4, 2.5 mivi PMSF, 2

Cell Growth & Differentiation 1101

pg/mI leupeptin, 2 pg/mI aprotinin)l, and resuspended in 20

p1 histone Hi kinase buffer [80 ms’i sodium �-glycerophos-phate, 20 mM EGTA, 50 mtvt MgCI2, 20 msi N-2-hydroxy-ethylpiperazine-N’-2-ethanesulfonic acid (pH 7.2), 1 mivtDTT, 2.5 mM PMSF, 2 pg/mI leupeptin, 2 pg/mI aprotinin,

and 10 �M protein kinase A inhibitor (Sigma Chemical Co.)].Histone Hi kinase assay was performed by adding i 2 p1 ofa mixture containing i mg/mI histone Hi, i mr’�iATP, andi pCi [y-32P]ATP. The reaction was incubated at roomtemperature for i 5 mm and stopped by the addition of 30 p12X sample buffer 1100 mist Tris-CI (pH 6.8), 4% SDS, 20%glycerol, 10% �-mercaptoethanol, and 0.2% bromophenolblue)].

MAP Kinase Analysis. Cells were washed twice in PBSand then lysed in Triton X-1 00 buffer [1 % Triton X-i 00,

0.1% SDS, 50 mM Tris (pH 8.0), 50 ms�i f3-glycerophos-phate, 50 mM sodium fluoride, 5 ms’i MgCI2, 1 mivi Na3VO4,

i mM DTT, i 0 mM EGTA, 2.5 m M PMSF, i 0 pg/mI leupep-tin, and 10 pg/mI aprotinin] by incubation for 30 mm onice. The cell lysates were cleared by centrifugation for 30mm at 100,000 X g at 4#{176}C.The lysate containing -1 00 pgof total protein was immunoprecipitated with anti-MAPkinase antibody i9i3 (77). Antibody-MAPK complex was

collected with protein-A agarose beads and washed fivetimes with Triton X-iOO buffer and once with histone Hikinase buffer. The MAPK activity was measured using MBP

as a substrate. The assay was performed in 30 p1 of histoneHi kinase buffer containing MBP (i mg/mi), 2 pCi [y32-

PIATP, and i 0 pm ATP. After i 5 mm at room temperaturefollowed by incubation at 30#{176}Cfor i 5 mm, the reaction wasstopped by the addition of 30 p1 2X sample buffer. MBP

phosphorylation activity of maturing oocytes were assayed

as described previously (78).Colony Formation in Soft Agar. Analysis of v-mos-trans-

formed cell lines for anchorage-independent growth wasperformed in 60-mm dishes by overlaying i 0� cells inDulbecco’s modified Eagle’s medium containing 10% fetal

bovine serum, 0.3% (w/v) Noble agar (Difco), and 0.1 2%Bactopeptone onto a base of 0.5% (w/v) Noble agar and0.2% Bactopeptone prepared in the same medium. Cob-nies were allowed to grow for 2 weeks at 37#{176}Cin anatmosphere containing i 0% CO2.

Oocyte Preparations. Xenopus !aevis females were pur-chased from Xenopus i (Ann Arbor, Ml). Oocytes weresurgically removed and defolliculated by incubation inmodified Barth solution [88 misi NaCI, i mt�i KCI, 2.5 m�iNaHCO3, 1 0 mM N-2-hydroxyethylpiperazine-N’-2-eth-anesulfonic acid (pH 7.5), 0.82 ms’t MgSO4, 0.33 mtvi

Ca(NO3)2, and 0.4i mM CaCI2] containing collagenase A(i .5 mg/mI; Boehringer Mannheim Biochemicals) for 2 h at20#{176}C. The oocytes were washed extensively in modifiedBarth solution, and stage VI oocytes were selected andallowed to recover i 6 h in 50% Leibovitz’s L-i 5 medium(GIBCO). Oocyte maturation was induced by incubation in5 pg/mI progesterone (Calbiochem) in 50% L-i 5 medium.

Percoll Cradient Separation. The gradient was devel-oped in PBS (90% Percoll solution and 10% PBS) by cen-trifugation for i 5 mm at 23,000 X g in a fixed-angle rotor.The cells collected from the culture dish (--i X i06 cells)were resuspended in 4 ml of complete medium. The cellsuspension was applied onto the Percoll gradient and sub-jected to centrifugation for 1 5 mm at 600 x g, followed bycentrifugation for 30 mm at i 000 X g. More than 95% ofthe cells in the band formed after centrifugation consisted of

viable cells, while cells diffused in the upper layer almostexclusively consisted of dead or dying cells.

AcknowledgmentsWe thank J. A. Cooper for anti-MAPK antibody, P. N. Rao for MPM-2

antibody, and D. K. Morrison for v-raftransformed cells. We also thank S.

Hughes, R. Zhou, M. Strobel, I. Daar, P. Donovan, L. Parada, and D. Kaplanfor critical reading of this manuscript. We are grateful to M. Rosenberg for

information on ZSK-2 (Zymed) antibody. We thank M. Reed for the prepa-ration of this manuscript.

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