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Proc. Natl. Acad. Sci. USAVol. 75, No. 10, pp. 5043-5047,
October 1978Cell Biology
Inducers of DNA synthesis present during mitosis of
mammaliancells lacking G1 and G2 phases
(cell cycle/cell fusion/prematurely condensed chromosomes)
POTU N. RAO, BARBARA A. WILSON, AND PRASAD S. SUNKARADepartment
of Developmental Therapeutics, The University of Texas System
Cancer Center, M.D. Anderson Hospital and Tumor Institute,Houston,
Texas 77030
Communicated by David M. Prescott, July 27, 1978
ABSTRACT The cell cycle analysis of Chinese hamster
lungfibroblast V79-8 line by the premature chromosome condensa-tion
method has confirmed the absence of measurable GI andG2 periods.
Sendai virus-mediated fusion of mitotic V79-8 cellswith GI phase
HeLa cells resulted in the induction of both DNAsynthesis and
premature chromosome condensation in the latter,indicating the
presence of the inducers of DNA synthesis abovethe critical level
not only throughout S phase, as it is in HeLa,but also during
mitosis of V79-8 cells. No initiation of DNAsynthesis was observed
whe-n GI phase HeLa cells were fusedwith mitotic CHO cells. These
results indicate that the presenceor absence of a GI period in the
cell cycle depends on the levelsof the inducers of DNA synthesis
present in the cell during mi-tosis.
Studies involving nuclear transplantation (1, 2) and cell
fusionbetween cells in various phases of the cell cycle (3, 4)
haveshown that the cytoplasm of the cells undergoing DNA
repli-cation contains certain factors that can induce DNA
synthesisprematurely in GI nuclei when they are brought in contact
witheach other. Preparations for the initiation of DNA synthesis
inmammalian cells, including the synthesis of these inducers,
arepresumed to take place during the GI phase, which immedi-ately
precedes the period of DNA synthesis. However, untilrecently it was
not clear whether the inducers of DNA synthesisreach a critical
level abruptly at the Gl/S transition or accu-mulate gradually
during the GC period. On the basis of cellfusion studies involving
HeLa cells synchronized at variouspoints in the GC period, Rao et
al. (5) have proposed a modelregarding the availability of the
inducers of DNA synthesisduring the HeLa cell cycle. According to
this model, the in-ducers of DNA synthesis accumulate gradually
during the GCperiod, reaching a critical level by the end of this
period, whenDNA synthesis is initiated. As DNA synthesis is
completed, thelevel of these inducers decreases below the critical
level.Therefore it is of interest to see how such a model would fit
acell line (Chinese hamster V79-8) that has neither GC or G2phase
in its cell cycle (6). In the Chinese hamster V79-8
cells,successive periods of DNA synthesis are interrupted only by
ashort period of mitosis. Hence, the objective of the present
studywas to determine whether the inducers of DNA synthesisremain
above the critical concentration throughout the cellcycle or
decrease during mitosis. The results of these experi-ments indicate
that the factors for the initiation of both DNAsynthesis and
mitosis can be present simultaneously in mitoticcells.
MATERIALS AND METHODSCells and Cell Synchrony. The Chinese
hamster cell line
(V79-8), which lacks both the GI and G2 phases in its cell
cycle,was kindly supplied by R. Michael Liskay, University of
Col-orado, Boulder, CO. V79-8 cells were grown as monolayers
onFalcon plastic culture dishes in McCoy's 5A modified
mediumsupplemented with 15% heat-inactivated fetal calf
serum(GIBCO) in a humidified CO2 (5%) incubator at 37°. Underthese
conditions, this cell line had a generation time of about10 hr (6).
V79-8 cells were synchronized in mitosis by selectivedetachment
after a brief (2-hr) exposure to Colcemid (0.05gg/ml).HeLa cells,
grown in suspension culture, were routinely
maintained in exponential growth by daily diluting with
freshEagle's minimal essential medium supplemented with
heat-inactivated fetal calf serum (10%, vol/vol), nonessential
aminoacids, penicillin/streptomycin mixture, sodium pyruvate,
andglutamine (7). For cell fusion studies, HeLa cells in Gl
phasewere obtained by synchronizing cells in mitosis by the
nitrousoxide block method (8) and then allowing them to divide
overa period of 3 hr after the reversal of the mitotic block. A
3S-minpulse labeling of the Gl population with [3H]thymidine
indi-cated a labeling index of zero. The mitotic index was less
than5%.
Cell Fusion and Cell Cycle Analysis. The cell cycle analysisof
V79-8 cells was performed by the use of the prematurechromosome
condensation method (9). Mitotic and randompopulations of V79-8
cells were fused by the use of UV-inacti-vated Sendai virus to
induce premature chromosome conden-sation. The detailed procedures
for cell cycle analysis by cellfusion have been described (9). This
method of cell cycleanalysis is based on the fact that the
morphology of the pre-maturely condensed chromosomes (PCC) of an
interphase cellindicates its position in the cell cycle at the time
of fusion.
Kinetics of Induction of DNA Synthesis in GI Phase HeLaCells.
Mitotic V79-8 cells, prelabeled with [3H]thymidine, werefused with
HeLa cells in GI phase. Colcemid (0.5 ,g/ml) wasadded to the fusion
mixture along with the virus and kept in themedium throughout the
experiment in order to prevent theV79-8 cells from completing
mitosis. The new mitotic inhibitorMaytansine (10) appeared to be
more effective than Colcemidin holding the V79-8 cells in mitosis
for prolonged periods.Hence, Colcemid was replaced by Maytansine in
later experi-ments.
After fusion, a small sample of the cells was deposited
directlyon a clean slide by the use of a cytocentrifuge, fixed in a
3:1(vol/vol) absolute methanol/glacial acetic acid mixture,
andprocessed forautoradiography to determine the extent of
fusion.The remaining cells were diluted with fresh medium
containing
Abbreviation: PCC, prematurely condensed chromosomes.5043
The publication costs of this article were defrayed in part by
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"ad-vertisement" in accordance with 18 U. S. C. §1734 solely to
indicatethis fact.
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Proc. Natl. Acad. Sci. USA 75 (1978)
[3H]thymidine (1.0 ,gCi/ml, 6.7 Ci/mmol) and Colcemid
(0.5,gg/ml) and then plated in a number of 35-mm Falcon
plasticculture dishes. At hourly intervals, one of the dishes
wastrypsinized and the cell samples were deposited on slides,
fixed,and processed for autoradiography as described above. The
cellswere stained with Giemsa and scored for the presence of
labelon either the nuclei or PCC of HeLa cells residing along
withthe mitotic chromosomes of V79-8 cells in the same
cyto-plasm.
Chinese hamster ovary (CHO) cells, which have a GL periodof 2 to
2.5 hr, were used as a control. Mitotic CHO cells, col-lected by
selective detachment after a 2-hr exposure to Col-cemid (0.5
,gg/ml), were incubated for another 3 hr in the samemedium
containing Colcemid and then fused with GC phaseHeLa cells. After
fusion, cells were incubated with [3H]thy-midine and samples were
taken at hourly intervals, processed,and scored as described above.
About 200 cells were scored foreach sample point.
Cell Cycle Progression of V79-8 Cells after the Reversalof a
Mitotic Block. To determine how rapidly the cells enterS phase
after cell division, we synchronized V79-8 cells in mi-tosis by
selective detachment after a 2-hr Colcemid (0.05,ug/ml) block. The
Colcemid was removed by washing andplating the mitotic cells in
regular medium in a number ofdishes. At 30-min intervals, one of
the dishes was trypsinized.A small fraction of the cells was
deposited on a slide by the useof cytocentrifuge, fixed, stained
with aceto-orcein, and scoredfor mitotic index. The remaining cells
in the sample were fusedwith mitotic V79-8 cells for cell cycle
analysis by the PCCmethod.
RESULTSCell Cycle Analysis. The cell cycle analysis of V79-8
cells
in exponential growth. by the PCC method revealed that 95.5%of
the cells were in S phase, as indicated by the
"pulverized"appearance of the PCC. The cells in GI phase
constituted lessthan 1%. About 4% of the cells that exhibited PCC
with G2-likemorphology have completed DNA replication except for
oneor two short regions and hence by definition should still
beconsidered as in S phase (see Fig. 1). These data suggest that
theV79-8 cells have practically no G1 and G2 in their cell
cycle.
Rapidity of Mitotic to S Phase Transition in V79-8 Cells.After
the reversal of the Colcemid (0.05 ,ug/ml) block, thesynchronized
mitotic cells completed cell division very rapidly;i.e., within 30
min the mitotic index decreased from 98% to 48%and by 60 min it was
below 5%. The cycle'analysis of this pop-ulation by the PCC method
at 30 min after the reversal of theColcemid block indicated that
90% of the interphase cells ex-hibited PCC with early S phase
morphology (Fig. 1A), whilethe remaining were of theG1 type. These
data suggest that thetransition from mitosis to S phase occurs very
rapidly in V79-8cells.
Induction of Both DNA Synthesis and Premature Chro-mosome
Condensation in GI Phase HeLa Cells by Fusionwith Mitotic V79-8
Cells. Because successive rounds of DNAsynthesis in V79-8 cells are
interrupted by only a brief periodof cell division, we decided to
test whether the inducers of DNAsynthesis are present during
mitosis. The fusion of mitotic V79-8cells with HeLa cells inGI
period resulted in both the inductionof DNA synthesis and premature
chromosome condensationin the HeLa nuclei (Fig. 2). During the
first hour of fusion, theinduction of DNA synthesis in HeLa nuclei
was much morerapid than PCC induction (Fig. 3). Subsequently, both
theseparameters increased linearly as a function of time. The
labelingindex in the mononucleate (U) and binucleate (UU) HeLa
cellsremained essentially zero (U = unlabeled nucleus). In some
ofthe fused cells, DNA synthesis was induced but not premature
.."W
4S'%: I
w*(v
.I JI-
A
GI ,
FIG. 1. PCC of V79-8 cells in exponential growth. (X1600.)
(A)Product of a fusion between mitotic and S phase V79-8 cells. The
PCCare of early S phase morphology, exhibiting "pulverized"
appearance.The darkly stained chromosomes are of the mitotic cell.
(B) Productof fusion between a mitotic and two interphase cells. M
= mitoticchromosomes, well condensed and darkly stained; G1 = G1
PCC withone chromatid each; G2 = PCC of G2 morphology, consisting
of twochromatids except for one or two short segments where DNA
repli-cation is still in progress as indicated by a gap
(arrow).
chromosome condensation (Fig. 2 A and B). These resultsclearly
demonstrate the presence of the inducers of both DNAsynthesis and
premature chromosome condensation in themitotic cells of V79-8
line. In contrast, no DNA synthesis wasinitiated in HeLa nuclei
residing in the binucleate cells formedby the fusion of mitotic CHO
and HeLa GC.
DISCUSSIONA reevaluation of the cell cycle parameters of the
V79-8 cell lineby the PCC method has essentially confirmed the
report ofLiskay (6), who observed that this cell line has no
measurableperiods of GC and G2.A comparison of this cell line
(V79-8) that lacksGC and G2
phases with another, for example, HeLa, that has four
distinctphases (i.e., G1, S, G2, and M) in its cell cycle may help
us tounderstand the basis for cell cycle regulation. The
parameters
5044 Cell Biology: Rao et al.
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Proc. Natl. Acad. Sci. USA 75 (1978) 5045
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FIG. 2. Heterophasic cells resulting from the fusion between
mitotic V79-8 cells prelabeled with [3H]thymidine and the unlabeled
HeLa cellsin G1 period. (X1600.) (A) Cell containing the nucleus of
HeLa on one side and a bunch of darkly stained mitotic chromosomes
of V79-8 (arrow)on the other. In this cell, the HeLa nucleus did
not undergo premature chromosome condensation. (B) Autoradiograph
of the same cell shownin A. Silver grains (black spots) can be seen
on both the mitotic chromosomes and the interphase nucleus. (C)
Cell containing highly condensedand darkly stained mitotic
chromosomes of V79-8 and the lightly stained PCC of the HeLa cell
(arrow). (D) Autoradiograph of the same cellshown in C. Label can
be seen on both the mitotic and the prematurely condensed
chromosomes.
to be compared are: (i) the chromosome condensation, (ii)
thelevels of inducers of DNA synthesis during the cell cycle,
and(iii) the levels of the chromosome condensation
factors.Chromosome Condensation Cycle. The suggestion for the
existence of chromosome condensation cycle in mammaliancells
made by Mazia (11) was subsequently supported by ex-perimental
evidence derived by the use of a variety of tech-niques (9, 12-16).
These studies revealed that the chromosomesachieve the maximum
degree of condensation during meta-phase, gradually decondense
during GI, and become least
condensed at the time of DNA replication in early S phase.
Soonafter the completion of DNA synthesis, the chromosomes
re-condense progressively through the duration of the G2 period(14)
and once again reach the height of their condensationduring mitosis
(Fig. 4A). It is presumed that the degree ofchromosome condensation
that occurs during the G2 period isof a different order of
magnitude than that taking place duringthe initiation of mitosis.
In contrast, the chromosomes of V79-8cells decondense rather
rapidly. Our data indicate that thehighly condensed metaphase
chromosomes of V79-8 cells reach
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Proc. Natl. Acad. Sci. USA 75 (1978)
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._S
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20
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Max
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FIG. 3. Frequency of induction ofDNA synthesis and
prematurechromosome condensation in G1 phase HeLa cells after
fusion withmitotic V79-8 cells. 0, Frequency of PCC induction among
the bi-nucleate cells, consisting of a set of mitotic chromosomes
of V79-8 andan interphase nucleus of HeLa; 0, labeling index on
either nuclei orPCC of HeLa among the mitotic-interphase fused
cells; A, labelingindex among the mono- or the binucleate HeLa
cells.
an extended state of the early S phase within 30 min (Fig.
1A).The condensation process following DNA replication also
ap-pears to be quite rapid, because we did not find many cells
withG2-PCC. The chromosome condensation cycle of V79-8 iscontrasted
with that of HeLa in Fig. 4.
Levels of Inducers of DNA Synthesis during the Cell Cycle.The
levels of inducers of DNA synthesis are known to fluctuateduring
the HeLa cell cycle (Fig. 4C). These inducers remainabove the
critical level only during the S phase, when they caninduce DNA
synthesis in a G1 nucleus upon fusion between Sand G1 cells (5).
Mitotic cells of V79-8, which contain the in-ducers of DNA
synthesis, are capable of inducing both DNAsynthesis and premature
chromosome condensation in G1 phaseHeLa cells within an hour after
fusion (Fig. 3). However, CHOcells blocked in mitosis over a period
of 7-8 hr were unable toinduce DNA synthesis in the nuclei of G1
phase HeLa cellsfollowing fusion. This observation rules out the
notion that,during the extended Colcemid block, the cytoplasm of
CHOor V79-8 cells advances into S phase while the chromosomes
arelocked in metaphase. Thus it is clear that inducers of
DNAsynthesis are present and remain above the critical level in
themitotic cells of V79-8 but not in those of CHO. In spite of
thepresence of the inducers, the metaphase chromosomes of
V79-8cells did not incorporate label during a 2-3 hr incubation
ofColcemid-arrested mitotic cells with [3H]thymidine. In
earlierstudies Rao and Johnson (3) have shown that the chromatin
ofG2 cells (and probably of metaphase chromosomes) is notavailable
for replication until the chromatids are separated.
Levels of the Mitotic Factors. Our earlier studies
involvingfusion of early, middle, and late G2 cells revealed that
thefactors for the initiation of mitosis in HeLa cells
accumulateduring the G2 period (17). The closer the cells are to
mitosis,the greater is the level of the mitotic factors they
contain. The
TimeFIG. 4. An idealized diagram to show the differences
between
HeLa and V79-8 cells regarding some cell cycle parameters.
Chro-mosome condensation cycle of (A) HeLa and (B) V79-8; the
processof decondensation is gradual in HeLa, whereas it is very
abrupt in thecase of V79-8 cells. Levels of inducers of DNA
synthesis during thecell cycle of (C) HeLa and (D) V79-8 cells; the
broken line indicatesthe critical level. Levels of the mitotic
factors during the cell.cycle of(E) HeLa and (F) V79-8 cells; the
broken line indicates the criticallevel.
cell enters mitosis when the mitotic factors reach a critical
level(Fig. 4E). However, in the case of V79-8 cells, the
accumulationof mitotic factors must take place during the S phase
becauseit has no significant G2 period (Fig. 4F).On the basis of
these data, we suggest that the presence or
absence of a Gl period in the cell cycle depends on the levelsof
the inducers of DNA synthesis in a cell at the time of mitosis.If
the inducers are present above a critical level during mitosis,the
cell enters S phase immediately after cell division. If not,the
cell requires a GI period to synthesize the inducers. Thus,the
duration of GI period is directly related to the time requiredby
the cell to achieve a critical concentration of the inducers.The
slower the synthesis of these inducers, the longer the
GIperiod.
This investigation was supported by U.S. Public Health
ServiceGrants CA-16480, CA-11520, CA-19856, and CA-14528 from
theNational Cancer Institute and GM-23252 from the Institute of
GeneralMedical Sciences.
1. Gurdon, J. B. (1967) Proc. Natl. Acad. Sci. USA 58,545-552.2.
de Terra, N. (1967) Proc. Natl. Acad. Sci. USA 57,607-614.3. Rao,
P. N. & Johnson, R. T. (1970) Nature (London) 225,
159-164.4. Graves, J. A. M. (1972) Exp. Cell Res. 72,393-403.5.
Rao, P. N., Sunkara, P. S. & Wilson, B. A. (1977) Proc. Natl.
Acad.
Sci. USA 74, 2869-2873.
. *I.- IA Chromosome condensation cycle B
M +G1 S G2 4M M+--S 1MI
I I I IM-
,,'.40
, S o~~~~~~~~t00
I/o,~~~~~~dI AIII
1~~~~~~~~~
____AA
.... . ..- C Inducers of DNA synthesis D
M G1---- S -4----2- -M------- 1
M G1 IS G2-EM LMS S im
5046 Cell Biology: Rao et al.
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1
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Cell Biology: Rao et al.
6. Liskay, R. M. (1977) Proc. Natl. Acad. Sci. USA 74,
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7. Rao, P. N. & Engelberg, J. (1965) Science 148,
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D. G. (1975) Biochem. Pharmacol. 24,751-754.11. Mazia, D. (1963)
J. Cell Comp. Physiol. 62, Suppl. 1, 123-
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Proc. NatI. Acad. Sci. USA 75 (1978) 5047
12. Pederson, T. (1972) Proc. Natl. Acad. Sci. USA 69, 2
2224-2228.
13. Pederson, T. & Robbins, E. (1972) J. Cell. Biol. 55,
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(1975) J. Cell Sci.
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Physiol. 95,333-
342.17. Rao, P. N., Hittelman, W. N. & Wilson, B. A. (1975)
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