de- tected with alkaline phospha- tase-conjugated anti-rabbit. IgG antibody, followed by ...... V-H., Cibert, C. Santa Maria, A., and Maio, B. .... T. A. J., Vincent,.
Vol.
5,
1093-1103,
October
1994
Cell
Growth
& Differentiation
1093
Similarities between Somatic Cells Overexpressing the mos Oncogene and Oocytes during Meiotic lnterphase1
Kenji Eukasawa, Monica S. Murakami, Donald C. Blair, Ryoko Kuriyama, Tim Hunt, Peter Fischinger, and Ceorge E. Vande Woude2 ABL-Basic of Molecular
Research Program Oncology ID.
1K. F., M. S. M., G. F. V. W.l and Laboratory G. B.l, NCI-Frederick Cancer Research and
Development Center, Frederick, Maryland 21 702-1 201 ; Department of Cell 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 Carolina 29425-1020 lP. F.l
Abstract The mos protooncogene encodes a serine/threonine kinase and is a key regulator of oocyte meiotic maturation. After acute infedion of Swiss 3T3 cells with virus containing the v-mos oncogene, cells expressing high levels of v-Mos round up and detach from the monolayer (floating cells), while cells that remain attached express 1 0-fold lower levels of v-Mos and are transformed. The floating cells are growth arrested with their chromosomes partially condensed in the absence of histone Hi kinase adivity, while mitogen-adivated protein kinase adivity is very high. Collectively, these properties are similar to properties observed in maturing oocytes between meiosis I and II. In v-mos-transformed cell populations, mitogen-adivated protein kinase adivity is also elevated, correlating with the degree of morphological transformation and the level of Mos expression. Moreover, phosphoprotein modifications specific for M are found in both the floating cells and in v-mos-transformed cells, regardless of their cell cycle stage. One explanation for both morphological transformation and the phenotypes of the floating cells is that Mos imposes a meiotic program on different stages of the somatic cell cycle. The extent of this meiotic phenotype is proportional to the level of v-Mos expression. These results suggest that both morphological transformation and the phenotypes of the floating cells induced by Mos in Swiss 3T3 cells are related to its normal adivities during oocyte maturation.
viral long terminal repeat promoter, shown to transform cells efficiently
assays (5-7). Similarly, the murine cellular c-mos’, under long terminal repeat control transform cells efficiently (4, 5), demonstrating
The
(1 9), an activity
to
v-Mos
associated
with
the arrest
of oocyte
matu-
to of
meiosis and the production of unfertilized eggs. Mos has been shown to associate with tubulin in vitro and the spindIe and the spindle poles in vivo, suggesting that Mos contributes to meiotic microtubule reorganization (21 , 22).
MAPK has also been implicated in meiotic microtubule reorganization (23) and has been shown to localize to MTOCs during meiosis (24). Recently, Mos has been shown to activate MAPK in vivo and in vitro in Xenopus oocytes or cell-free extracts (25, 26). Acute infection of Swiss 3T3 cells with Mo-MuSV that contains the v-mos protooncogene causes a large percentage of the cells to round-up and detach from the monolayer (floating cells; Refs. 27 and 28), and only the cells that remain attached grow as morphologically transformed cells. We have shown that approximately 70% of the floating cells are growth arrested with 2C or 4C DNA content.4 The cells with 4C DNA content are binucleated and have undergone nuclear division without cytokinesis.4 In this study, we further ing or transformed
characterized by v-Mos
Swiss 3T3 cells overexpressand show, in the floating cells,
partial chromosomal condensation in the absence of Hi kinase activity. Furthermore, phosphoprotein modifications that are specific to M in nontransformed cells are detected floating
cells
and
in v-mos-transformed
of the cell cycle
cells
inde-
stage.
mos
cellular virus,
Received I
that
ration at metaphase of meiosis II, the state corresponding an unfertilized egg (20). Thus, Mos is a key regulator
pendent
gene was originally identified as an acquired sequence carried by the acute transforming retroMo-MuSV3 (i-4). Recombinant v-mos, under a pro-
homologue,
was shown
and c-Mos have essentially the same biological activities and that inappropriate expression of the mos protooncogene in fibroblast cells is sufficient to cause morphological transformation (3, 4, 8, 9). The c-mos protooncogene product is normally expressed in germ cells (iO) and is required throughout oocyte maturation in Xenopus to activate MPF (11-18). Mos has also been identified as an active component of cytostatic factor
in the
Introduction
was the first oncogene in DNA transfection
Research
5/27/94; sponsored
revised 7/29/94; in part
accepted
by the National
8/9/94. Cancer
Institute,
DHHS,
under
contract No. N01-CO-74101 with ABL. Research funding for K. F. was provided 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-diamidino-2-phenylindole; MBP, myelin basic protein; PBS, phosphate-buff-
Results Morphological Transformation and v-Mos Expression. To obtain high levels of Mos expression in somatic cells, we infected Swiss 3T3 cells with Mo-MuSV containing the v-mos oncogene. Control cells were either mock-infected or infected with helper Mo-MuLV. As previously reported, 60-70
90%)
h P.1., the majority
round
ered saline; Tris-buffered ride. K. Fukasawa
4
of Mo-MuSV-infected
up and detach
SDS-PAGE, sodium dodecyl saline; DTT, dilhiothreitol; and
G.
F. Vande
cells
from the monolayer
Woude,
sulfate-gel electrophoresis; PMSF, phenylmethylsulfonyl submitted
for
(70-
as floating
publication.
TBS, fluo-
1094
rnos-incluced
M-phase
Phenotypes
in Somatic
Cells
A kDa
1
46
2
3
5
4
-
7
8
I
,
,
6
v-mos c-mos
..-
...
.-
1!,
30-
Fig. 1. The immunoblot of v-Mos expression fected with Mo-MuSV
0’
C ..
c-rnos” transformed cell line; Lane 3, floating cells; Lane 4, MoMuSV-infectecl cells that remain attached to the dish 40 h P.1.; Lane 5, Mo-MuSV-infected cells that remain attached to the dish 70 h P.1.; Lane 6, Tx3 line; Lane 7, Tx2 line; Lane 8, Txi line. B, cells grown in culture dish were directly photographed using an inverted niicro-
sVa>
.-
:
analysis cells inand pho-
tomicrographs of cloned v-mos transformed cell lines. A, cell cxtracts containing 1 00 pg of total proteins were run on a i0”/ SOSPAGE and transferred onto Immobilon (Millipore( sheets. The blots were then prc)bed with anti-Mos” polyclonal antibody ZC-75. Antibody-antigen complex was detected with alkaline phosphatase-conjugated anti-rabbit IgG antibody, followed by reaction with bromochloroinclolyl phosphate-nitroblue letrazolium (BioRad(. Lane 1, control Mo-MuLVinfected cells; Lane 2, PTS1
B
-
in
..
% -
.
.
.;
(.
.
.
-
-
..
-
:
.
“i:,’’
,
:,
,
.-
..
.
,.
.7’
,
..-,
.
.
:-‘
.
scope. a, control Txi cells; c, Tx2 x 100.
-
Swiss cells;
3T3 cells; b, d, Tx3 cells.
4r:’,.:d4
.
c-.
;
..
-:_
-:.
‘
J
.,
#{163}
.
.(
.
“-I
.
,---#{149}---.-.--
.
-
. ..
,
-
.
-:
..
‘,
1
cells.4 Approximately 70% of floating cells exclude trypan blue but do not proliferate, while the remaining cells are dead or dying (27). Only the cells that remain attached grow as foci of morphologically transformed cells. We cloned several cell populations from these foci, and three clones were chosen for further study (Txi , Tx2, and Tx3). The level of v-Mos in the floating cells is - 0.1 % of total protein (Fig. 1A, Lane 3), which is -10-fold higher than in the Mo-MuSV-infected cells that remain attached at 40 h P.1. (Fig. 1 A, Lane 5) and 60-70 h P.1. (Fig. 1 A, Lane 4). The level of p39fllO expression in PTS1 cmosutransformed cells 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 express the highest level of v-Mos (Fig. 1A, Lane 6) and show the greatest morphological alteration (Fig. 1 B, d), while Txl cells express the lowest level of v-Mos (Fig. 1 A, Lane 8) and
-
.
are least transformed (Fig. 1 B, b). The Tx2 cells are intermediate both in v-Mos expression and transformed phenotype (Figs. 1A, Lane 7and 18, c). Moreover, the efficiency of anchorage-independent colony formation in soft agar of the v-mos-transformed cell lines also correlates with the level of v-Mos expression; the average CFE (the ratio of the number of soft agar colonies to the number of viable cells plated) from two independent experiments was 0.001 for Swiss 3T3 cells, 0.1 3 for Txl cells, 0.24 for Tx2 cells, and 0.42 for Tx3 cells. These results confirm earlier studies correlating v-Mos overexpression with somatic cell toxicity (30) but also show that the degree of morphological transformation correlates directly with the level of Mos expression.
Partial
Chromosomal
Level Expression Kinase Activity. both
cells
arrested
of v-Mos
Condensation
The majority at 2C and
by High of Histone Hi
Induced
in the Absence
of the viable floating cells, 4C DNA content, contained
Cell
.
A
.
histone
C
..
II 34cdc2
23
D
formaldehyde, cells were stained with B. control 3T3 cell in mitosis; Cancl 0,
partially condensed chromosomes as revealed with DAPI DNA stairting (Fig. 2, C and D, and data not shown)4 and resemble the state of condensation observed in control Swiss 3T3 cells only during prophase (Fig. 2B). Interphase control cells stained with DAPI do not show condensation (Fig. 2A). It has been shown that 34f2 kinase induces chromosomal condensation (31 , 32). Since Mos indirectly activates and stabilizes MPF (19), we assayed the floating cells for p34((f(2 kinase activity by measuring Hi histone kinase activity (Fig. 3A). pi3’1 bead-precipitated extracts from nocodazole-treated M-arrested Swiss 3T3 cells displayed high levels of histone Hi kinase activity (Fig. 3A, Lane 2). In contrast, extracts from unsynchronized control MoMuLV-infected and v-Mos-transformed cell populations, in which only a fraction of the cells are in M, displayed the expected low levels of activity (Fig. 3A, Lanes 1 and 4). Surprisingly, extracts from floating cells lacked detectable histone Hi kinase activity (Fig. 3A, Lane 3). Although vMos-induced chromosome condensation could have resuIted from transient p34th2 activation, these results show that the activation of p34”t’ is not required for the maintenance of the chromosome condensation or growth arrest. States of p34cdc2 and Cyclin B in the Floating Cells. We further examined the floating cell population to determine whether p’t’ 2 was dephosphorylated and if cyclin B was present. We used an antibody directed to the COOH ternlinus of 2 in immunoblot analyses. Control 3T3 cells in M (Fig. 3B, Lane 2) showed only the expected single active form of p34((f(2, while actively growing control cells and Tx3 cells displayed additional, slower-moving phosphorylated fornis (Fig. 3B, Lanes 1 and 4, respectively). Only the dephosphorylated form of p34’ was detected in
I
-
Fig. 2. Inappropriate chromosomal condensation in the floating cells resuIted troni v-Mos overexpression. The viable floating cells, isolated after Peroll gradient separation of viable cells from dead or dying cells, were seeded onto , slick’ and examined at X 630. Control cells were grown on
Hi
1234
lRI1IIpp
@
A Differentiation
234
-
B
coverslips. After fixation with 1.7/,, DAPI. A. iriterphase control IT I cell; floating ( elk.
I
Growth
cyclin
B
cyclin
B
23 “4,
‘ #{163}m Fig. .3. The influence of v-Mos expression on H 1 histone kinase activity and its components, p34’ ‘‘ 2 and cyclin B. A, cell extracts were precipitated with p1 3” ‘ beads. The beads were subjected to histone Hi kinase analysis as described in “Materials and Methods.” Control IT.) cells (Lane 1 (, nococlazole-arrested M-phase control 3T3 cells (Lane 2), floating cells (Lane fl, and Tx3 v-mos-transformed cells (Lane 4(. The dead or dying cells were removed from the floating cells by Percoll gradient centrifugalion prior to the preparation 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-terminal monoclonal 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 coniplexes were detected with horseradish peroxidase-conjugateci anti-rabbit lgG and visualized by chemiluminescent reactions as reconimencleci (Amersham(. 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 immunoprecipitates were resolved on a 10/ SDS-PAGE and transferred onto Immobilon (Millipore) sheets. The immunoblots were then probe(1 with anti-cyclin B antibody as described (C) and visualized by chemiluminescent reactions.
the floating cells (Fig. 3B, Lane 3). Even though histone Hi kinase activity was not detected (Fig. 3A), cyclin B was unexpectedly found in the floating cells at levels similar to control cells in M (Fig. 3C, compare Lanes 2 and 3). Moreover, cyclin B was not found in anti-p34’2 immunoprecipitates from the floating cells but was coprecipitated with p34((l( 2 from M-arrested control cells (Fig. 3D). We are not certain why cyclin B is not associated with p34’2 in the floating cells, but a similar result was observed previously in Mos-transformed NIH3T3 cells (33). MAPK Activity in Somatic Cells Expressing Mos. Mos has been implicated in MAPK activation during meiosis (25,
1095
1096
mos-induced
M-phase
Phenotypes
in Somatic
Cells
10000’
1
2
3
8000’
0. 0
4
5
6 MBP
C
0 6000’ >1
0 0. Cl)
4000
0
0.
20003
I
2
Fig. 4. MAPK activation in v-mos-transformed cell lines, lysates. The v-raf-transformed by 18% SDS-PAGE, followed bar graphs (lower panel). The
3
4
the floating cells (Txi-3) displayed
and the v-mos-transformed cell lines high levels of MBP phosphorylation ac(Fig. 4, Lanes 4-6). The level in Tx3 cells is similar to
tivity the level detected in raf-i -transformed cells (Fig. 4, Lane 2). These analyses show that MAPK activity correlates with the level of Mos expression as well as with the extent of morphological transformation (compare with Fig. 1 ). Moreover, MAPK activation in the floating cells and transformed cells is constitutive in 0.5/ serum and occurs in the absence of mitogenic stimulation.
of Phenotypes during Meiotic
between the Eloating Cells lnterphase. Low histone Hi
ki nase activity, chromosome condensation, dephosphorylated p34cdc2, the presence of cyclin B, and the activation of MAPK in the floating cells are reminiscent of the properties of amphibian and mammalian oocytes during meiotic interphase, the period between meiosis I and meiosis II. In Xenopus oocytes during meiotic interphase, MPF is low
S
T. Choi
and
G. F. Vande
6
cells expressing v-Mos. Control 3T3 cells (Lane 1); v-raf-transformed 3T3 cell line (BxB; Lane 2); floating cells (Lane 3); 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 3T3 cell line with constitulively activated MAPK was included as a positive control (Lane 2). MBP phosphorylation was analyzed by autoradiography (upperpanel). The kinase activity was also quantitated by scintillation counting, and the data is presented in lane designations of the auloradiograph and bar graph are the same.
26), and the level of MAPK activity is significantly higher during meiosis than during mitosis in one-cell mouse embryos.5 In nontransformed somatic cells, MAPK is activated only after mitogenic stimulation in G1 (34-38) and is reported to increase slightly at G2-M (39). Here, we examined the relationship between the level of MAPK activity, the level of v-Mos, and the degree of morphological transformation. MAPK activity was determined in vitro using MBP as a substrate (Fig. 4). MBP phosphorylation was not detected in MAPK precipitates from control Swiss 3T3 cells maintained for 36 h in 0.5% serum (Fig. 4, Lane 1), while
Similarities and Oocytes
5
Woude,
manuscript
in preparation.
Fig. 5, A and D), while cyclin B is present (4i , 42) remains dephosphorylated (Fig. SB). By mobility shift, MAPK remains activated throughout meiosis including meiotic interphase (Fig. 5, C and 0). Activated MAPK is also detected during meiotic interphase by in vitro kinase assays using anti-MAPK monoclonal antibody and MBP as a substrate (Fig.SD). (Ref. 40;
and
p34cdc2
The Role of Mos and MAPK in Mos-transformed Cells. The role ofMAPK in gene regulation during G1 (reviewed in Refs. 43-46) is quite different from its proposed role in microtubule reorganization during meiosis (23, 24). We have proposed that Mos induces, during meiosis, microtubule reorganization and that this activity is responsible for the transformed phenotype (47). Therefore, we examined the microtubule organization in the floating cells and v-mos-transformed cells by immunostaining with anti-atubulin antibody (DMiA; Fig. 6A). Compared with control Swiss 3T3 cells, the microtubule organization in the floating cells and in the v-mos-transformed cells was significantly disrupted (Fig. 6A, b and c). We also examined the floating cells and v-mos-transformed cells with the monoclonal antibody ZSK2 (Zymed) that was originally prepared against rat brain tubulin. Surprisingly, the ZSK2 antibody only stained the spindle of Swiss 3T3 control cells in M, while cells in interphase were not efficiently stained (Fig. 6B, a-c). However, ZSK2 stained the floating cells and v-mos-transformed cells intensely and, more importantly, independent of the cell cycle stage (Fig. 6B, g-j). These results showed that the ZSK2 antibody preferentially reacted with M-specific epitopes in nontransformed cells and provided evidence that these epitopes were constitutively present in cells expressing v-Mos. The antigens detected in immuno-
Cell
Prog. 0
GVBD Q
01
Growth
& Differentialion
1097
M.int. 0
20V21
2345
6
(h)
A S..
Fig. 5. Analysis of Hi histone kinase and MAPK activities during the meiotic interphase of Xenopus OOcytes. Stage VI oocytes, prepared as described) in “Materials and Methodls,” were induced for maturation by incul)ation in 5 pg/mI progesterone ( Prog. (. In ordler to examne the meiotic interphase (Mint,), sve achievedl the synchronization of maturing oo ytes by setting the time of germinal vesicle breakdown determined by the appearance of a white 5001 in the animal heniisphere, as “0” time, and lysates were 1)r(’Paredl relative to that time schedule. A, analysis of Hi histone kinase activity diuring meiotic interphase. The oocyte lysates PreI)ar(’dI at each time point were measured for MPF activity in vitro using histone H 1 as a substrate as described previously (79). The in vitro kinase reaction mixtures were (GVBD(,
analyzedi immunoblot
by
p34’
phosphotyrosi
n(’ (anti-P-Tyr)
clonal antibody. immunoprecipitated
“
The
2
blot analyses reminiscent monoclonal see below).
GVBD
Mint.
0
0
0
1
2
OV2/41/2234
5
6
(h)
B
‘tigH p34CdC2(pTyr)
.
using
antimonolysates were with anti-
‘ 2 COcwi-terniinal peptide antil)ody (Santa Cruz(. The immunoprecipitates were resolved by 1 2/ SOS-PAGE and transferred onto Immohilon (Millipore) sheets. The blots were then probed with anli-P-Tyr monoclonal antibody (PY-20(. Antibody-antigen complex was delectedl by chemilluminescent reactions as described in the Fig. 3 legend. C immunohlol analysis of MAPK during meiotic interphase. The lysates resolvedi in iO#{176}k SDS-PAGE were transferred onto Immohilon )Millipore) sheets and probed with anti-MAPK monoclonal antibody (Zymed). Antibodyantigen complex was dietected by chemilluminescent reactions as described in the Fig. 3 legendl. MAPK’ indicates the phosphorylated, acfive form of MAPK. 13, MPF (histone Hi phosphorylalion( activity and MAPK, in vitro MBP phosphorylation activity of anti-MAPK monoclonal antibody immunoprecipitales, were also dluantitated by scintillation counting and summarized in (0).
Prog.
0
1 2/ SDS-PAGE. B, analysis of immuno-
precipitatedl
-HistoneH1
Prog.
GVBD
M.int.
0
0
0
01
C
234
201/21
56(h)
-
-
D
I
-
-
.-
-MAPK
Melosls
II
MelosIsI
Progesterone GVBD
Melotlc Inlerphase
E a 3000 C
Metaphase arrest
II
1
E
a
0
U
C 0 0
2000
C
a
C
0 C
I
0 C
2000
a
a
U)
a
S
0. C
0
3 .±
by ZSK2 were not tubulin of the antigens detected antibodies, MPM-2 (48)
1000
molecules but were with the M-specific and CHO3 (Ref. 49;
M-specific
Phosphorylation
during
Interphase.
The
monoclonal antibody MPM-2 reacts with phosphorylated protein epitopes that are specifically modified during mitosis or meiosis but not during interphase (Refs. 48, 50, and
1098
max-induced
M-phase
Phenotypes
in Somatic
Cells
A
B
0
0 4-
-
p
0 4.-
4.-
Cell
A
B
MPM2
C
CHO3
12345
12345
Growth
Differentiation
ZSK2 12345
Kd
200#{163}
I
.
I.. 97.4-
a !-
.
69
-
-I.’
46
-
I -
4’
I: 30
-
7.
Immunoblot
analysis
_pI_I
-
-
.;
21.5-
Fig.
.
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 with horseradish 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 not shown(. 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 expressing v-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 sensitive to alkaline phosphatase treatment (data not shown), and all three antibodies react predominantly with M-specific phosphorylated epitopes (compare Fig. 7A, B, and C, Lanes 1 and 2). Strikingly, with each of the monoclonal antibodies, we found that antigens detected in the extracts of the floating cells (Fig. 7A, B, and C, Lane 3) or v-mostransformed cell lines (Fig. 7A, B, and , Lanes 4 and 5), were very similar to those found in the M-arrested control cells. However, low molecular weight antigens detected by each antibody are more prevalent in the v-Mos-expressing cells. We conclude that Mos expression induces M-specific phosphoprotein modifications at all stages of the somatic cell 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 program 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 chromosome 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 entry into 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 phosphorylationdlependlent 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 speed centrifugation (floating cells) were fixed andl immunostained with anti-c tubulin monoclonal antibody )DM1A(, followedi by a fluorescein isothiocyanateconjugatedl 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 with DAPI (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.
1099
1100
mos-induced
M-phase
Phenotypes
in Somatic
Cells
venting the dephosphorylation of the M-specific antigens. The high levels of v-Mos could induce growth arrest by preventing sufficient dephosphorylation of certain M-specific antigens to allow cell cycle progression. In the floating cells, in spite of the presence of dephosphorylated p34cdc2 and cyclin B, histone Hi kinase activity is not detectable, although the floating cells display chromosome condensation. This phenotype may be similar to oocytes during meiotic interphase, when MPF is low, but the chromosomes remain condensed (53), and the levels of Mos and MAPK are high. These kinases and the cascade of kinases and phosphorylation events they regulate may be sufficient to maintain growth arrest and chromosome condensation. MAPK is activated by Mos (25, 26) and is localized to MTOCs present at the poles and kinetochores of the meiotic spindles. Recently, Mos has been localized to kinetochores (54). Moreover, both MPM-2 and CHO3 monoclonal antibodies that constitutively recognize M-phase antigens in cells expressing Mos also stain MTOCs at mitotic spindle poles and kinetochores (55-58), and at least some MPM-2 and CHO3 epitopes are generated by MAPK during meiosis (59). Furthermore, using partially purified microtubules from Swiss 3T3 cells as a substrate for MAPK in vitro, at least one high molecular weight protein (Mr 230,000) is phosphorylated and becomes an epitope for MPM-2 and CHO3 monoclonal antibodies.6 It is possible, therefore, that Mos expression in somatic cells either directly or, more likely, through activating a cascade of specific kinases that are normally restricted to meiotic maturation such as MAPK and possibly Raf(60, 61), promotes the inappropriate phosphorylation of M-phase epitopes at all stages of the cell cycle. Boveri (62) first implicated defective spindle poles as a causative factor in malignant tumors, and binucleated tumor cells were one of the earliest unexplained histopathological observations found in Mo-MuSV-induced mouse sarcomas (63). Cells transformed by v-mos have been shown to be genetically unstable (64), and genetic instability
is a component
of tumor
cell
progression.
The
sister
chromatid exchange and chromosome segregation that uniquely occur during meiosis is likely to use different checkpoint functions for fidelity than somatic cells (65). Superimposing a meiotic program on a mitotic cell cycle could possibly influence and compromise somatic cell checkpoint function. Other oncogenes such as ras (16, 66-68) and raf(60, 61) induce meiotic maturation, and all activate MAPK (69-72). Constitutive activation of MAPK is a property common to many oncoproteins; therefore, the MAPK family of proteins (73-75) would make ideal downstream targets for clinical intervention. Materials
and Methods
Cells. Swiss 3T3 cells used in this study were maintained in complete medium (Dulbecco’s modified Eagle’s medium10% fetal bovine serum with 100 units/mI penicillin and 1 00 pg/mI streptomycin) and grown in an atmosphere contaming iO% CO2. Cultures were maintained at a subconfluent density by passing 7.5 x i05 cells per 100-mm dish twice weekly. Virus and Virus Infedion. Mo-MuSV 1 24 is a clonal isolate from the original Mo-MuSV stock (76). Twenty-four
U,
K. Fukasawa
and
N. G. Ahn,
unpublished
observations.
h before infection, i x i 06 Swiss 3T3 cells were plated per 100-mm dish with complete medium containing 4 pg/mI Polybrene; followed by a 1-h incubation period with filtered medium from either Mo-MuSV i 24-transformed producer cells (TB cell line) or Mo-MuLV producer cells, also containing 4 pg/mI Polybrene. The multiplicities of infection were typically 5-iO focus-forming units per cell for both Mo-MuSV 1 24 and Mo-MuLV. Mock-infected cells were treated identically, except that no virus was added. After infection, the cells were cultured in complete medium. Immunoblotting. Cells were washed twice in PBS and then lysed in modified sample buffer [2% SDS, 10% glycerol, and 60 mM Tris (pH 6.8)1. Cell extracts were cleared by a 30-mm centrifugation at 1 00,000 X g. The lysate denatured at 95#{176}C for 5 mm in sample buffer [2% SDS, iO% glycerol, 60 mti Tris (pH 6.8), 5% f3-mercaptoethanol, and 0.Oi % bromophenol blue]. Samples were resolved by SDSPAGE. Fractionated proteins were then transferred to Immobilon (Millipore) sheets, and the blots were incubated in blocking buffer [5% (w/v) nonfat dry milk-0.2% Tween 20 in TBS)] for 2 h at room temperature and then with primary antibodies for 2 h at room temperature. The blots were rinsed in TBS-T (0.2% Tween 20 in TBS) and incubated with either alkali ne phosphatase-conj ugated or horserad ish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The blots were then rinsed in TBS-T, and the antibody-antigen complex was visualized with either bromochloroindolyl phosphate (Bio-Rad) and nitroblue tetrazolium (Bio-Rad) as substrates in 100 mi NaCI-5 ms’t MgCl2-iOO mM Tris (pH 9.5) for the alkaline phosphatase reaction or visualized by using the ECL chemiluminescence method (Amersham), according to the manufacturer’s instructions for horseradish peroxidase reaction. Indired Immunofluorescence. Cells, either seeded on slides 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 fixation, the cells were washed with PBS and permeabilized with 1% Nonidet P-40 in PBS for 5 mm at room temperature. Coverslips were incubated with blocking solution (1 0% normal goat serum in PBS) for i h and then incubated with primary antibodies, followed by incubation with either fluorescein isothiocyanate or Texas red-conjugated secondary antibodies for 1 h at room temperature. Coverslips were washed
three
times
with
PBS after
each
slips were then embedded mounting medium. Ceneration and Preparation
in
period
of Swiss
of 72 h after
infection
incubation.
aqueous
of Floating 3T3
Cover-
nonfluorescing Cells. cells
Over the with Mo-
MuSV, 90% ofthe cells round up and float into the media (floating cells). The floating cells were directly collected from the cell culture media. The examination of their viabilities 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 or dying cell populations are constant in repeated experiments with 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 were collected, 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
leupeptin, 2 pg/mI aprotinin)l, and resuspended in 20 p1 histone Hi kinase buffer [80 ms’i sodium -glycerophosphate, 20 mM EGTA, 50 mtvt MgCI2, 20 msi N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (pH 7.2), 1 mivt DTT, 2.5 mM PMSF, 2 pg/mI leupeptin, 2 pg/mI aprotinin, pg/mI
and 10
protein
M
Histone
Hi
kinase A inhibitor
kinase
assay
was
(Sigma Chemical
performed
by adding
Co.)]. i 2 p1 of
a mixture containing i mg/mI histone Hi, i mr’i ATP, and i pCi [y-32P]ATP. The reaction was incubated at room temperature 2X sample
for i 5 mm and stopped buffer 1100 mist Tris-CI
glycerol, 10% -mercaptoethanol, blue)]. MAP Kinase Analysis. Cells
by the addition of 30 p1 (pH 6.8), 4% SDS, 20%
and 0.2% were
bromophenol
washed
twice
then lysed in Triton X-1 00 buffer [1 % Triton X-i 00, SDS, 50 mM Tris (pH 8.0), 50 msi f3-glycerophosphate, 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 leupeptin, and 10 pg/mI aprotinin] by incubation for 30 mm on ice. The cell lysates were cleared by centrifugation for 30 mm at 1 00,000 X g at 4#{176}C. The lysate containing -1 00 pg
with anti-MAP
kinase antibody i9i3 (77). Antibody-MAPK complex was collected with protein-A agarose beads and washed five times with Triton X-iOO buffer and once with histone Hi kinase buffer. The MAPK activity was measured using MBP as a substrate. The assay was performed in 30 p1 of histone
Hi
kinase buffer containing MBP (i mg/mi), 2 pCi [y32PIATP, and i 0 pm ATP. After i 5 mm at room temperature followed by incubation at 30#{176}C for i 5 mm, the reaction was stopped by the addition phosphorylation activity as described previously
of 30 p1 2X sample buffer. MBP of maturing oocytes were assayed (78). in Soft Agar. Analysis of v-mos-trans-
Colony Formation formed cell lines for anchorage-independent performed in 60-mm dishes by overlaying Dulbecco’s modified bovine serum, 0.3%
Bactopeptone
onto
growth was i 0 cells in
Eagle’s medium containing (w/v) Noble agar (Difco),
a base of 0.5%
(w/v)
10% fetal and 0.1 2%
Noble
agar and
0.2% Bactopeptone prepared in the same medium. Cobnies were allowed to grow for 2 weeks at 37#{176}Cin an atmosphere containing i 0% CO2. Oocyte Preparations. Xenopus !aevis females were pur-
chased
from
surgically modified NaHCO3,
Xenopus
i (Ann
Arbor,
Ml).
Oocytes
were
removed and defolliculated by incubation Barth solution [88 misi NaCI, i mti KCI, 2.5 1 0 mM N-2-hydroxyethylpiperazine-N’-2-eth-
anesulfonic
acid
Ca(NO3)2, (i .5 mg/mI; 20#{176}C. The
(pH
7.5),
0.82
and 0.4i mM CaCI2] Boehringer Mannheim oocytes were washed
ms’t MgSO4, 0.33 containing collagenase Biochemicals) extensively
in mi mtvi
A
for 2 h at in modified
Barth solution, and stage VI oocytes were selected and allowed to recover i 6 h in 50% Leibovitz’s L-i 5 medium (GIBCO). Oocyte maturation was induced by incubation in 5 pg/mI
progesterone
Percoll Cradient oped in PBS (90%
(Calbiochem)
in 50%
L-i 5 medium.
Separation. The gradient was develPercoll solution and 10% PBS) by cen-
trifugation for i 5 mm at The cells collected from were resuspended in 4 suspension was applied
23,000 X g in a fixed-angle rotor. the culture dish (--i X i06 cells) ml of complete medium. The cell onto the Percoll gradient and sub-
jected to centrifugation for 1 5 mm at 600 x g, followed centrifugation for 30 mm at i 000 X g. More than 95%
by of
the cells
of
in the band
formed
after centrifugation
consisted
while
cells
consisted
diffused
of dead
in the upper or dying
layer
1101
almost
cells.
Acknowledgments We 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. Kaplan for critical reading of this manuscript. We are grateful to M. Rosenberg for information on ZSK-2 (Zymed) ration of this manuscript.
.
in PBS
0.1%
immunoprecipitated
cells,
exclusively
& Differentiation
antibody.
We
thank
M.
Reed
for the
prepa-
of mice.
NaIl.
Cancer
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