[CANCER RESEARCH 46, 4032-4040, August 1986]
Mitomycin-induced Chromatid Breaks in HeLa Cells: A Consequence of Incomplete
DNA Replication1
Marguerite A. Sognier2 and Walter N. Hittelman3
Department of Chemotherapy Research, the university of Texas M. D. Anderson Hospital and Tumor Institute at Houston, and the University of Texas Graduate School
of BiomédicalSciences, Houston Texas 77030
ABSTRACT
The formation of chromosome aberrations induced by alkylating agents
such as mitomycin C has been shown to require the passage of the treated
cell through S phase. However, the exact mechanisms by which mitomycin C-induced DNA lesions are translated into chromosome aberra
tions during S phase are not known. The purpose of these studies was to
better understand the molecular basis of chromosome aberration forma
tion after mitomycin C treatment. The morphology of metaphases of cells
treated in <., phase with mitomycin C resembled that of prematurely
condensed chromosomes of S-phase cells. Consequently we postulated
that chromosome aberrations resulted from cells reaching mitosis without
completing DNA replication. This was tested by treating HeLa cells in
<., phase with mitomycin C and then analyzing these cells at mitosis for
residual DNA damage and DNA content. Utilizing the DNA alkaline
elution assay for DNA damage, we showed that HeLa cells progress
through S phase into mitosis with intact DNA-DNA interstrand cross
links. These cross-links, originally induced into parental DNA, were
associated equally with parental and newly replicated DNA at the time
the cells reached mitosis. This suggests that recombinational events had
taken place during the DNA replication process. Cells that were treated
in G i phase and allowed to proceed to mitosis in the presence of
bromodeoxyuridine to density label newly replicated DNA were analyzed
with cesium chloride density sedimentation. Unreplicated DNA was
present in the mitotic cells of the treated populations but not in the
untreated control cells. Further, flow cytometric measurements, made
under hypotonie conditions in order to reduce chromatin condensation
effects, demonstrated that the mitotic cells from the mitomycin C-treated
populations contained 10-20% less DNA than untreated mitotic controls.
These results indicate that chromosome breaks induced by mitomycin C
are the result of cells reaching mitosis without having fully completed
DNA replication.
INTRODUCTION
Many chemical and physical agents used in the treatment of
malignancy are known to produce DNA lesions that ultimately
result in chromosome aberrations (1). While the existence of
chromosome aberrations has been recognized since Muller's
first description in 1927 (2), little is known about how specific
types of DNA lesions are translated into aberrations at the
chromosomal level (for reviews, see Refs. 3 to 5). The purpose
of the studies reported here was to gain a better understanding
of how DNA damage induced by MMC4 might cause the
formation of one type of chromosome aberration, e.g., the
chromatid break.
Mitomycin C, a therapeutic drug used in the treatment of
Received 2/24/86; revised 4/21/86; accepted 4/22/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by Grants CA-27931 and CA-39534 from the National
Cancer Institute, NIH.
1 Present address: Colorado State University, Department of Radiology and
Radiation Biology. Fort Collins, CO 80523, and the Eleanor Roosevelt Institute
for Cancer Research, 4200 E. 9th Ave.. Denver, CO 80262.
3 To whom requests for reprints should be addressed.
4 The abbreviations used are: MMC, mitomycin C; BrdUrd, bromodeoxyuri
dine: dThd, thymidine; HH, heavy-heavy; HL, heavy-light; LL, light-light; PCC,
prematurely condensed chromosomes.
malignancy, covalently binds to the DNA of cells and produces
both monoadducts and DNA-DNA and DNA-protein cross
links, most likely involving the O6 position of guanine (6).
MMC-induced damage results in a reduction in the rate of
DNA replication and a dose-dependent delay in cell cycle
progression (7, 8). At the chromosome level, MMC is thought
to produce chromosome aberrations by means of an S-phase
dependent mechanism; i.e., chromosome aberrations are not
observed unless the treated cell has undergone replicative DNA
synthesis following the induction of DNA damage (9). When
the treated cells eventually reach mitosis, chromatid-type breaks
and exchanges are observed (10).
The chromatid breaks after MMC treatment are noteworthy
in that the deleted regions can be quite lengthy in appearance,
suggesting that a portion of the genome might be missing.
Similarly, highly damaged metaphase spreads closely resemble
S-phase cells which have been induced into premature chro
mosome condensation. These observations led to the hypothesis
that cells containing MMC-induced damage can eventually
attain mitosis without having fully replicated their DNA. Some
chromosome breaks would therefore be the result of unreplicated chromosome regions.
If this hypothesis holds, the cells that reach mitosis after
MMC treatment should exhibit several distinct characteristics.
(a) The damaged mitotic cells should still contain MMCinduced DNA lesions, (b) These cells should contain a portion
of their DNA in an unreplicated form, (c) The treated cells
should exhibit a diminished DNA content upon reaching mi
tosis. In this paper we have tested and confirmed these predic
tions using a variety of techniques including DNA alkaline
elution (to detect residual DNA-DNA and DNA-protein cross
links), CsCl density sedimentation (to detect unreplicated
DNA), and flow cytometry (to detect mitotic cells with dimin
ished DNA content). Thus, it is likely that MMC-induced
chromatid breaks are a consequence of incomplete DNA repli
cation caused by the presence of residual MMC-induced DNA
lesions.
MATERIALS
AND METHODS
Cell Culture, Cell Synchronization, and Drug Dilution. HeLa cells
were grown as monolayer cultures on Lux 150-mm dishes in Hsu's
modified McCoy's Medium 5A (GIBCO, Grand Island, NY) supple
mented with 10% fetal calf serum (K C Biological, Lenexa, KS), 3 mM
L-glutamine (GIBCO), penicillin (100 ^g/ml) (GIBCO), streptomycin
(100 ^g/ml) (GIBCO), and 0.1 mM CaCl2 (MCB, Norwood, OH).
Cultures were maintained at 37°Cin a humidified 5% CO2 incubator.
The cells were subcultured with 0.125% trypsin (GIBCO). The proce
dures for obtaining HeLa mitotic cells were described previously (11).
Briefly, exponentially growing cells were treated with 2.5 mM dThd for
22-24 h, released from the dThd block, and then accumulated in mitosis
in the presence of 90 psi nitrous oxide. After selective detachment of
mitotic cells, this procedure routinely yielded populations with a mitotic
index of at least 95%. Upon release from nitrous oxide (N2O), the cells
completed mitosis and entered <., phase in a synchronous wave.
MMC (Mutamycin, Bristol Laboratories, Syracuse, NY) was dis
solved just before use in sterile distilled water to a 100-¿ig/mlstock
solution and then diluted in McCoy's Medium 5A with 1% fetal calf
serum to a final concentration of 1
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MITOMYCIN-INDUCED
CHROMATID
Experimental Protocol. The general experimental protocol was iden
tical for every type of study described in this paper. HeLa cells were
synchronized to mitosis and selectively detached, and 4 x 106cells were
plated per 150-mm Petri dish. The cells were allowed to divide, attach
to the dishes, and progress into GI phase. Five h after plating, the Gì
cells (labeling index, <1%) were treated with MMC (1 jig/ml) for 1 h,
rinsed 3 times with medium, and reincubated in fresh McCoy's medium
with 10% fetal calf serum. After 18 h, the old medium containing
floating cells was removed, and fresh medium was added. Cells were
then accumulated in mitosis in the presence of N2O, and mitotic cells
were selectively detached for analysis after appropriate time intervals.
For the alkaline elution and CsCI analyses, the cells were radioactively labeled with [I4C]dThd (0.02 MCi/ml) (40-60 Ci/mivi) for two full
cell cycles prior to the first synchronization. For the studies concerning
damage to newly replicated DNA, [MC]dThd was added to the cultures
after MMC treatment. The procedure for alkaline elution has been
previously described (12). For the CsCI assays of unreplicated DNA,
BrdUrd (20 Mg/ml) was added after MMC treatment.
Neutral Cesium Chloride Density Centrifugation. After drug treatment
and cell cycle progression, mitotic cells were accumulated in N2O,
selectively detached, centrifuged, and resuspended in Tris buffer (0.15
M NaCl:0.01 M EDTA:0.01 M Tris, pH 8.1) to a concentration of IO6/
BREAKS
h with MMC, and then allowed to progress to mitosis, chromatid breaks, gaps, and exchanges were observed in metaphase
preparations that were not seen in the untreated controls (Fig.
I, A and B). Ninety % of the total aberrations at this dose were
chromatid breaks, many of which appeared to be the result of
unusually long chromatid deletions, as well as displaced breaks.
At this high dose of MMC (1 ^g/rnl), many cells in the
treated population are not capable of achieving mitosis and
thus remain in interphase for many hours. To visualize the
chromosomes in these drug-delayed interphase populations,
cells were treated in G, phase for 1 h with MMC, reincubated
in drug-free medium for 70 h, and then fused with mitotic cells
to induce premature chromosome condensation. In control
populations, G, cells yield PCC with a single chromatid per
chromosome, while Ga cells yield PCC with two chromatids
per chromosome. The PCC of S phase appear pulverized.
Nevertheless, both single (unreplicated) and double (replicated)
chromosome segments can be observed, with those portions of
the genome actively undergoing replication appearing diffuse
(Fig. 1C) (17, 18). The PCC of the MMC-treated cells resem
bled "normal" S-phase PCC in the sense that both single and
ml. Cells were lysed by addition of Sarkosyl to 0.1 %, incubated for 30
min at 60°Cwith RNase A (80 jig/ml) (Millipore Corporation, Free
hold, NJ), and then incubated for 1 h at 60°Cwith Pronase CB (0.5
mg/ml) (Calbiochem-Behring Company). The cell lysate was mixed
with an equal volume of Sevag [chloroform:isoamyl alcohol (24:1, v/
v)] at 4°C.After centrifugation, the aqueous phase was removed, and
double segments were present. However, the morphology of
these PCC was distinct in that little diffuseness was present,
and condensed single chromatid elements were directly joined
to condensed double chromatid regions (Fig. 1D). Pulse label
ing studies indicated that DNA synthesis was dramatically
reduced in these cells (data not shown).
the Sevag procedure was repeated until the interface between the sample
The fact that the chromosome preparations of the mitotic
and Sevag was clear. The DNA was then precipitated with 2 volumes
of 95% ethanol at 0°Cand collected by a 20-min centrifugation at 4°C cells and the PCC were of similar morphology after MMC
and 10,000 rpm. The DNA was resuspended in 0.15 M NaCl:0.015 M treatment raised the possibility that residual MMC-induced
Na3C6H5O7 (1:100) and used in the preparation of the neutral CsCI
lesions in the DNA blocked the completion of DNA synthesis,
density gradients as previously described (13). Individual gradient frac
and yet the cells continued to prepare for mitosis and eventually
tions were mixed with 1 ml of water and 10 ml of Scintiverse II (Fisher
condensed their chromatin and entered mitosis. For this pos
Scientific, Houston, TX), and radioactivity was measured with a Pack
sibility to hold, one would expect to find residual MMC-induced
ard Model 2650 liquid scintillation spectrometer.
lesions in the mitotic populations of treated cells as well as
Flow Cytometry Analysis. Samples of mitotic cells designated for
indications of incomplete DNA replication. The subsequent
flow cytometric DNA content analysis were washed in physiological
experiments were designed to test these expectations.
saline and then exposed to either hypotonie 0.075 M KCl or physiolog
Residual DNA Damage in MMC-treated Cells at Mitosis. The
ical saline for 25 min in the presence of Colcemid (0.05 Mg/ml).
DNA alkaline elution technique (19) was utilized to measure
Following centrifugation (3 min at 150 g), the cell pellets were resus
pended by mixing on a vortex, fixed in suspension with 70% ethanol
the amount of DNA damage in the HeLa cells after MMC
(4°C),and stored at 4°Cuntil analysis.
treatment. With this technique, radioactively labeled cells are
For DNA fluorochrome staining, 2 x IO6 cells were resuspended in
lysed directly onto filters, and DNA is eluted from the filter in
Dulbecco's phosphate-buffered saline containing 1 mM MgCl2 and
the presence of an alkaline solution, pH 12.1. Under these
centrifuged, 1 ml of 0.5% pepsin (Accurate Chemicals, Hicksville, NY)
conditions, the DNA strands unwind and pass through the filter
was added, and the solution was held for 4 min at room tremperature.
in the order of increasing strand length. The presence of single
Digestion was stopped by the addition of 2.5 ml of cold Tris buffer (0.1
or double strand breaks or alkaline labile sites causes more
M Tris base:0.1 M NaCl:0.84 M HCl, pH 7.4). After centrifugation the
cell suspension was resuspended in 1.5 ml of ethidium bromide (25 #<g/ DNA to elute. On the other hand, DNA-DNA or DNA-protein
ml) (Sigma Chemical Co., St. Louis, MO) for IO min, followed by the
cross-linking (making DNA strands effectively larger) masks
addition of 1.5 ml of mithramycin (50 ^g/ml) (Worthington Biochem
strand breakage and retards elution.
ical Corporation, Freehold, NJ) containing 30 mM MgCl2. Immediately
To determine the degree of MMC-induced DNA damage
before analysis each sample received 3 drops of 1% RNase (Worthing
present
immediately after treatment, prelabeled G, HeLa cells
ton) and was filtered through a 65-Mm nylon mesh. The integral DNA
were treated with MMC (l ßg/ml)for 1 h and then immediately
fluorescence emission per cell was measured with an ICP-11 Phywe
analyzed by DNA alkaline elution. As shown in Fig. 2, 90 and
instrument interfaced to a Nuclear Data 256 channel pulse height
analyzer equipped with an HP-26 48A terminal and tape storage device
92.5% of the radioactivity were retained on the filter after
elution for the control and MMC-treated populations, respec
(14, 15).
Cell Fusion. The procedure for cell fusion has previously been de
tively. Thus few strand breaks or alkaline labile sites were
scribed in detail (16). Briefly, mitotic HeLa cells (mitotic index >95%)
apparent immediately after treatment. However, irradiation of
were fused with an equal number of MMC-treated HeLa cells using
the samples on ice just prior to elution demonstrated a de
UV, inactivated Sendai virus. After fusion, the cells were exposed to
creased
rate of elution in the MMC-treated cells compared to
hypotonie, fixed in methanol:glacial acetic acid (3:1, v/v) dropped on
controls. This suggests that DNA-DNA and DNA-protein
clean, wet slides, and stained with Giemsa.
cross-links were present in the MMC-treated cells. To distin
guish between DNA-DNA and DNA-protein cross-links, the
RESULTS
lysed cells were treated with proteinase K prior to elution to
release the DNA-protein cross-links. The fact that proteinase
Visualization of Chromosome Damage after MMC Treatment.
When HeLa cells were synchronized in G, phase, treated for 1 K treatment resulted in only a slight increase in DNA elution
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MITOMYCIN INDUCED CHROMATID BREAKS
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Fig. I. Chromosome morphology of mitomycin C-treated cells. Synchronized HeLa cells were treated in G, phase with or without MMC for 1 h, washed free of
drug, and reincubated in drug-free medium to allow cell cycle progression. A, mitotic figure of untreated control; fi, mitotic figure of MMC-treated cell demonstrating
chromosome damage with many apparent large chromatid deletions (arrows); C, S-phase PCC of an untreated control cell; D, S-phase PCC of MMC-treated cell
blocked in S phase demonstrating continuity of single and double chromatid regions (arrows), x ISOO.
(7 and 10% for the control and MMC treatment, respectively)
suggests that most of the cross-links induced by MMC are
DNA-DNA rather than DNA-protein cross-links.
To determine whether cells treated in d phase could enter
mitosis despite residual DNA damage, prelabeled HeLa cells
were treated in G, phase with MMC and allowed to progress
to mitosis, and then mitotic cells were selectively detached and
analyzed by alkaline elution. As shown in Fig. 3-4, the MMCtreated cells exhibited a slight decrease in DNA retention
compared to the controls (O versus •,respectively). This sug
gested the presence of DNA strand breaks or alkaline-labile
sites in the MMC-treated cells. When the two populations were
irradiated on ice just prior to elution to allow the detection of
residual cross-links, the parental DNA of the MMC-treated
cells still exhibited increased DNA retention on the filters
compared to the controls, suggesting the presence of residual
cross-links in the MMC-treated cells that attained mitosis (Fig.
3A, A versus A, respectively). Proteinase K digestion prior to
elution indicated that the cross-links remaining in the treated
cells at mitosis were DNA-DNA interstrand type.
It is not understood how the treated cell might replicate past
cross-linked DNA, but it is feasible that the DNA synthesized
by the cells after MMC might contain breaks in the regions of
residual cross-links in the parental DNA. To test this, the
parental DNA was prelabeled prior to MMC treatment in GÌ
phase with [14C]dThd, and then the cells were incubated in
[3H]dThd during the S phase after MMC treatment to label the
newly replicated DNA. As before, cells that accumulated at
mitosis were selectively detached and analyzed by DNA alkaline
elution. Surprisingly, the elution rate of 3H-labeled DNA syn
thesized after the MMC treatment was equally retarded as that
of the parental DNA (Fig. 3B). This suggests that DNA syn
thesized after MMC treatment also contained DNA-DNA
cross-links.
That DNA-DNA interstrand cross-links that were present in
equivalent amounts in the parental and newly replicated DNA
after MMC treatment might be a consequence of one of three
mechanisms, (a) The conditions used for liquid scintillation
counting or double radioactive labeling might have produced
an artifact, (b) The formation of MMC-induced cross-links
might occur by a two-step mechanism over a period of hours
similar to that described for ci's-platinum (20), nitrosoureas
(21), and melphalan (22). (c) A recombinational mechanism
might be involved during replication past cross-links (see "Dis
cussion").
The first mechanism was tested by not prelabeling the paren-
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MITOMYCIN-INDUCED
tal DNA but labeling only the newly replicated DNA after
MMC treatment. The elution profiles of these cells after proteinase K digestion were very similar to those seen in the double
label experiments (data not shown). This result indicates that
the double radioisotope labeling procedure was not interfering
with the assay.
The second possible explanation involves a time-dependent
mechanism for cross-link production. If interstrand cross-links
were formed in two discrete steps over a period of time, the
1 00 _
z
LU
z
Q
LU
O
oc
LU
FRACTION
NUMBER
Fig. 2. DNA alkaline elution analysis of control and treated Gt HeLa cells
immediately after MMC treatment. O, untreated control; •.MMC-treated pop
ulation: A, MMC-treated cells irradiated on ice just prior to elution to reveal the
presence of DNA cross-links: Q, MMC-treated cells irradiated on ice and treated
with proteinase K prior to elution to release DNA-protein cross-links; A. untreated
control irradiated on ice just prior to elution: •.untreated control irradiated on
ice and treated with proteinase K just prior to elution.
80
CHROMATID
BREAKS
maximum cross-link levels produced would not be observed
immediately after MMC treatment but at some later time (i.e.,
6-17 h after drug treatment). Thus if the first step of cross-link
formation occurred in G, phase while the second step occurred
after some DNA replication had occurred, it would be possible
for newly replicated DNA to be involved in cross-links. To
determine whether MMC forms cross-links by a similar mech
anism, the parental DNA was prelabeled with [l4C]dThd, and
the cells were treated with MMC as before in G, phase. Treated
and control populations were harvested for alkaline elution
analysis at 0, 6, and 15 h after MMC treatment. To allow
detection of cross-links, the cells were irradiated on ice with
400 rads of 7-rays just prior to elution. As shown in Fig. 44,
fewer cross-links were present in the MMC-treated cells 6 h
after treatment compared to that observed immediately after
treatment as evidenced by a decreased DNA retention (D versus
A, respectively). The decrease in retention for the MMC-treated
cells was not due to the initiation of DNA synthesis because
increased elution kinetics was not observed in the unirradiated
control samples (data not shown), and the number of nicks
produced by the irradiation is large compared to the number of
nicks associated with DNA replication. Thus the drop in filter
retention reflected repair of DNA-DNA cross-links. Similarly,
the degree of cross-linking evident in the parental DNA was
also decreased at 15 h posttreatment when compared to that
observed immediately after treatment (Fig. 4Ä).Since the max
imum cross-link levels were observed immediately after MMC
treatment, these results suggest that a delayed two-step mech
anism for cross-linked formation could not explain the finding
that cross-links become associated with DNA synthesized after
MMC treatment.
Demonstration of Unreplicated DNA in Mitotic Cells after
MMC Treatment. The cytological observations described earlier
suggested that some MMC-treated cells might enter mitosis
without completing DNA replication. If so, the cells reaching
mitosis would be expected to contain some unreplicated DNA.
This was tested by prelabeling cells with [3H]dThd for two cell
generations, synchronizing to GÃŒphase, and treating with
MMC as before. After drug treatment, the cells were reincubated in medium containing BrdUrd to density-label the newly
replicated DNA. After appropriate periods of time, the cells
attaining mitosis were accumulated in N2O, selectively detached
(mitotic index, >95%), and the DNA was isolated and subjected
to neutral CsCl density gradient analysis. In this assay, BrdUrd-
_
Z
O
80
Fig. 3. DNA alkaline elution analysis of
cells treated in G, with or without MMC and
selectively detached at mitosis. A, parental
DNA labeled prior to MMC treatment: B.
daughter DNA labeled after MMC treatment
and prior to selection of cells at mitosis. •,
untreated control O, MMC-treated cells; A,
MMC-trealed cells irradiated on ice just prior
to DNA elution: A, untreated control cells
irradiated on ice prior to DNA elution.
468
246
FRACTION
NUMBER
FRACTION
NUMBER
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MITOMYCIN-INDUCED
100
1
1
1
CHROMATID
1
1
1
BREAKS
T
Fig. 4. Alkaline elution analysis of cell pop
ulations al various times after MMC treatment
in Gìphase compared to that found immedi
ately after MMC treatment. A, 0 and 6 h postMMC treatment; B, 0 and 15 h post-MMC
treatment. •.untreated controls immediately
after sham MMC treatment: O, untreated con
trols at 6 or 15 h after sham treatment; A.
MMC-treated cells immediately after treat
ment; D, MMC-lreated cells at 6 or 15 h after
treatment. All populations were irradiated on
ice just prior to DNA elution.
20 _
2
4
6
FRACTION
I
2461
NUMBER
containing replicated DNA can be separated from unreplicated
DNA on the basis of density.
As shown in Fig. 5A, the untreated control mitotic prepara
tions yielded one major DNA peak present at a density of 1.75
g/cm\ typical of DNA replicated once in the presence of
BrdUrd. By weight, the LL region of the control comprised
3.8% of the gradient compared to 85.8% in the HL region. In
contrast, DNA from the MMC-treated populations selected at
mitosis contained two distinct peaks, with 18.5% of the gradient
in the LL peak (1.71 g/cm3) and 67.8% in the HL peak (1.75
g/cm1) (Fig. 5B). In a repeat experiment, approximately 15%
of the gradient of the MMC-treated sample was found in the
LL region, typical of unreplicated DNA. Since the mitotic
indices of the analyzed populations were all greater than 95%,
these results suggest that the MMC-treated populations are
reaching mitosis with some unreplicated DNA.
Detection of Decreased DNA Content in Mitotic Cells after
MMC Treatment. The preceding experiments showed that cells
that enter mitosis after MMC treatment still contain detectable
DNA-DNA interstrand cross-links and some unreplicated
DNA. If chromosome breaks were a consequence of the cell's
inability to complete DNA replication past the residual DNA
damage, one would also expect these drug-treated cells to have
a deficiency in DNA content at mitosis. To test this, the DNA
contents of control and MMC-treated populations selected at
mitosis were determined by flow cytometry using ethidium
bromide and mithramycin as the fluorescent dyes (23, 24).
Since some drugs have been shown to interfere with mithra
mycin staining (25), and since the presence of interstrand cross
links could conceivably diminish the binding of the intercalating
dye ethidium bromide, it was first necessary to ensure that
MMC treatment alone would not affect the DNA content
measurements. To test this, HeLa mitotic cells were treated
with up to 100 fig of MMC per ml for l h in the presence of
Colcemid, and then the cells were analyzed for DNA content.
No difference in the measured DNA content due to MMC
treatment was observed (data not shown). Similar results were
also obtained in MMC-treated mitotic cells exposed to a hy
potonie solution (0.075 M KC1) for 25 min prior to fixation.
Thus, the presence of even high levels of DNA-DNA cross
links does not interfere with the accurate determination of DNA
content.
FRACTION
NUMBER
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20
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40
40
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Fig. 5. Cesium chloride analysis of mitotic populations of cells treated in G,
with or without MMC. washed free of drug, reincubated in medium containing
BrdUrd to density label newly replicated daughter DNA, and selectively detached
at mitosis. A, untreated controls: B, MMC-treated population. Note the presence
of a significant light-light peak in the MMC-treated population demonstrating
the presence of unreplicated and/or recombined DNA.
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MITOMYCIN-INDUCED
CHROMATID BREAKS
II II III
io|-B
10 -A
Control
Fig. 6. Flow cytophotometrie analysis of
mitotic HeLa cell populations treated in d
with or without MMC, allowed to progress to
mitosis, and selectively detached. I. untreated
controls and MMC-treated populations with
out hypotonie pretreatment of populations; B,
untreated controls and MMC-treated popula
tions with hypotonie pretreatment to eliminate
dye-uptake artifacts due to differences in chromatin structure. Internal control lymphocyte
populations were placed at channel 17, and 510 X IO3cells were counted for each measure
ment.
Control
8
8
6
6
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o
4
2
0
10
MMC
=
8
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O
E
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O
10
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MMC
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U
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6
ILA
A ! ! I ! .!. I. I I I I I I I I
16 32 48 64 80 96 112
Channel
To determine whether cells treated in G¡could come to
mitosis with a deficiency in DNA content, HeLa cells were
treated in G! with MMC, selectively detached at mitosis as
described above. The cells were then analyzed by flow cytometry
using human peripheral blood lymphocytes as an internal DNA
content marker. The DNA content measurements for the con
trol (mitotic index, 95%) and MMC-treated (mitotic index,
96%) cell populations are shown in Fig. 6A. While it was
expected that the drug-treated cells would exhibit less fluores
cence than the controls due to a diminished DNA content, just
the opposite was observed (modal peak channel numbers of 52
and 50 for the MMC-treated and control populations, respec
tively).
These results suggested that either the MMC-treated cells
reached mitosis with an increased DNA content or that the
chromatin of MMC-treated cells was more open, allowing
increased dye binding. The latter notion is possible because it
has previously been shown that mitotic cells blocked with
Colcemid for prolonged time periods have their peak channels
diminished by as much as 20% (26). Also, PCC studies have
shown that MMC treatment induces chromatin elongation
during the repair process (27). To minimize the effects of
chromatin condensation differences on DNA content measure
ments, the MMC-treated and control populations were exposed
to hypotonie solution (0.075 M KC1) prior to fixation and
staining with mithramycin and ethidium bromide. Preliminary
studies showed that the greatest dye binding was observed in
cells swollen with hypotonie solution for 25 min (data not
shown). As shown in Fig. 6Ä, when the MMC-treated and
control populations were analyzed by flow cytometry after
hypotonie exposure, the control cells now showed increased
DNA-bound dye when compared to the MMC-treated cells
(modal peak channel numbers of 57 and 52, respectively). Thus
the MMC-treated cells appear to reach mitosis with a decreased
DNA content.
Interestingly, with hypotonie treatment, the control popula
tion increased in peak channel number from 50 to 57 (a net
14% increase), while the MMC-treated cells remained at chan
nel 52. The result with the control population is consistent with
the observation of others that hypotonie treatment increases
Number
16 32 48 64 80 96 112
Channel Number
dye binding (28, 29). The contrasting result with the MMCtreated cells suggests that the chromatin of these cells is already
in a conformation that allows maximum dye binding. Assuming
a HeLa cell in d phase contains 17 pg of DNA while a normal
human lymphocyte contains 6.5 pg of DNA, the MMC-treated
cells appear to be lacking 9-10% of their expected DNA when
they reach mitosis. In a repeat experiment, a 20% difference in
DNA content was observed between the MMC and control
mitotic populations. These figures are on the same order of
magnitude as those obtained by cesium chloride measurements
of the amount of unreplicated DNA in the MMC-treated pop
ulations.
DISCUSSION
The purpose of this study was to determine how MMCinduced DNA lesions present during S phase induce chromo
some aberrations. The preceding experiments demonstrate that
MMC-treated cells reach mitosis without completing either
DNA damage repair or DNA replication and with a decreased
DNA content. This suggests that chromosome breaks result
from incomplete DNA replication prior to mitosis. A similar
idea has been previously postulated to explain the formation of
breaks after fluorodeoxyuridine treatment (30).
DNA alkaline elution analysis of the cells reaching mitosis
demonstrated measurable residual DNA-DNA cross-links in
the mitotic populations of cells that had been treated in d with
MMC. The fact that cross-links can persist in cells for extended
periods of time is consistent with results from several other
studies. Palitti et al. (31) showed that some MMC-induced
damage remains in Chinese hamster ovary cells until 62 phase,
since their repair could be inhibited with hydroxyurea, aphidicolin, or caffeine. Murnane and Byfield (32) reported that a
portion of the cross-links induced in C3H10T'/2 cells by nitro
gen mustard, phosphoramide mustard, or melphalan was not
repairable even though the cells were cross-link repair proficient
if challenged with a second drug dose. Similarly, Sognier and
Hittelman (33) reported that, although some initial repair was
observed during the first 48 h after MMC treatment of densityinhibited normal human fibroblasts, measurable cross-linking
remained in the cells for at least 96 h after a 1-h treatment with
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MITOMYCIN-INDUCED
G,
PHASE
MITOSIS
S PHASE
CHROMATID BREAKS
were ever detected in these reported studies (42, 43), and it was
believed that recombination probably did not occur in mam
malian cells. On the other hand, recent evidence has suggested
that recombination can occur in mammalian cells after dam
aging treatment. Using UV endonuclease to probe the location
of thymidine dimers in 3T3 cellular DNA after UV treatment,
Meneghini et al. (44) showed that dimers originally induced
into parental DNA were transferred to newly synthesized DNA.
Earlier studies had failed to show this effect, since it mainly
occurs soon after damage induction and declines thereafter.
Subsequently, Fournace (45) confirmed Meneghini's results
I
Fig. 7. Diagrammatic representation of a postulated mechanism for MMCinduced chromatid break formation involving inability to complete DNA repli
cation past cross-links and recombinational events occurring at the sites of blocks
to replication.
MMC (3 ¿¿g/ml).
Slow repair kinetics of MMC-induced cross
links was also reported by Dorr et al. (34). These studies all
suggest that a fraction on the cross-links induced in cellular
DNA might be irrepairable. Indeed, some evidence indicates
that there might be regions of the genome that are resistant to
repair functions (35).
Since DNA damage in these studies was measured with
alkaline elution, only the presence or absence of breaks and
cross-links was measured. However, MMC induces monoadducts at 5 times the frequency of cross-links (36). It is therefore
not possible to exclude a contributory role for monoadducts on
the effects observed. However, it is unlikely that monoadducts
are primarily responsible for these results for several reasons.
(a) Monofunctional alkylating agents are less efficient inducers
of chromsosome aberrations than polyfunctional alkylating
agents (37); (b) O6-alkyl guanine adducts are poor inducers of
chromosome aberrations, and some cell lines can successfully
replicate past these lesions (38, 39); (c) the repair rates for O6alkyl guanine are relatively short in human cells due to the
presence of alkyltransferase, so many of the lesions would be
repaired before DNA replication was initiated (40); and (d)
damage induced by bifunctional MMC is potentiated by hydroxyurea in G? while damage induced by monofunctional
MMC is not (31).
It is not known how cells replicate past lesions such as intact
DNA-DNA cross-links. Cleaver has suggested that the presence
of two inhibitory DNA lesions in a replicón might prevent
synthesis of the DNA between the lesions (41). Interestingly,
however, our DNA alkaline elution data suggest that the DNADNA cross-links become associated with DNA synthesized
after drug treatment. Since control experiments showed that
this result was not a consequence of a delayed two-step mech
anism of cross-link formation nor an artifact of the radioisotope
labeling and counting conditions, this finding suggests that
some form of recombinogenic process takes place when the cell
replicates past these DNA lesions. One possible mechanism for
this process is illustrated in Fig. 7 where two cross-links within
a replication unit prevent the completion of DNA synthesis
between the cross-links. If a recombinogenic process occurred
at or near the cross-links, the resultant structure would satisfy
our observations; i.e. (a) cross-links would be present and would
be distributed in both parental and daughter DNA; (b) both
parental and daughter DNA would also have single strand
breaks; and (c) a portion of the genome would remain unreplicated.
A number of investigators have attempted to demonstrate
the existence of recombination in mammalian cells by looking
for the presence of HH DNA in CsCl density gradients. How
ever, only very small amounts of HH DNA (i.e., 0.1-0.2%)
using DNA alkaline elution. Interestingly, a mammalian en
zyme similar to the RecA protein in E. coli, known to be
involved in recombinational events, has recently been isolated
(46).
We have shown in this paper that cells reaching mitosis after
treatment in GI phase with MMC contain unreplicated DNA
and exhibit a decreased DNA content when compared to un
treated control populations. In previous reports, other investi
gators reached a different conclusion when they measured DNA
content after either UV irradiation and caffeine (47) or X-ray
treatment (48). Although these studies involved different dam
aging agents, it is possible that diminished DNA content was
actually present in the treated populations but was not detect
able using standard flow cytophotometry methods. The results
presented here suggest that an altered chromatin configuration
might be present in the damaged cells that allows increased
fluorescent dye uptake compared to the control cells. Indeed, it
has recently been demonstrated that agents such as UV irradia
tion and alkylating agents induce a chromatin decondensation
process that can be visualized in the interphase PCC (27, 4951) as well as in mitotic figures (52). However, hypotonie
treatment of the cell populations prior to flow cytophotometric
analysis decreases the influence of chromatin condensation
differences between the two populations and allows a more
accurate estimation of DNA content.
The results of this study raise the question of how a cell can
proceed to mitosis with unrepaired and unreplicated DNA. The
transition to mitosis in treated cells can be thought of as a
product of two counterbalancing processes. First, as the cell
proceeds through the cell cycle, chromosome condensation/
mitotic factors gradually accumulate to prepare the cell for
mitosis (53, 54). On the other hand, cell fusion studies have
shown that both S-phase cells and damaged quiescent cells can
block G2 cells from progressing to mitosis (55), and extracts
from damaged cells have been shown to contain factors that
bind to and inactivate mitotic factor activity (56, 57). Thus,
when a cell has unrepaired or extensive damage, it often be
comes blocked in S or G2 phase, and only the least damaged
cells reach mitosis (58, 59). Eventually, however, the deconden
sation activity may subside, and the accumulated mitotic con
densation factors finally force the cell to enter mitosis even
though neither DNA repair nor DNA replication is complete.
A similar situation has been observed in cells that exhibit a
temperature-sensitive
mutation for S-phase functions (60).
After periods of time at nonpermissive temperature, these cells
still undergo chromosome condensation without completion of
DNA synthesis and exhibit an S-phase PCC morphology. Fur
ther, these cells can induce PCC in other cells, suggesting that
even though they cannot complete DNA replication, they still
continue to produce chromosome condensation factors (61).
Taken together, the results of this study suggest the following
model for chromosome break production. Due to the presence
of residual DNA lesions, the treated cells are unable to complete
DNA replication. Nevertheless, some cells continue to prepare
for and eventually enter mitosis. The unreplicated portion of
4038
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MITOMYCIN-INDUCED
CHROMATID
the chromosome would then appear as a chromatid deletion at
mitosis.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Todd Johnson for help in the flow
cytophotometric measurements; Josephine Neicheril for assistance in
preparation of the manuscript; James L. Cooper for computer program
ming and use of equipment; and Dr. Ramesh C. Adlakha, Dr. Frances
M. Davis, and Dr. Potu N. Rao for encouragement and helpful discus
sions.
REFERENCES
1. Kihlman, B. A. Actions of Chemicals on Dividing Cells. Englewood Cliffs,
NJ: Prentice-Hall, 1966.
2. Muller, H. J. Artificial transmutation of the gene. Science (Wash. DC), 66:
84-87, 1927.
3. Bender, M. A. Relationship of DNA lesions and their repair to chromosomal
aberration production. Basic Life Sci., 15: 245-265, 1980.
4. Evans, H. J. Molecular mechanisms in the induction of chromosome aber
rations. In: D. Scott, B. A. Bridges, and F. H. Sobéis(eds.). Progress in
Genetic Toxicology, pp. 57-74. Amsterdam: North-HollandrElsevier
Biomedicai Press, 1977.
5. Kihlman, B. A. Molecular mechanisms of chromosome breakage and rejoin
ing. Adv. Cell Mol. Biol., /: 59-107, 1971.
6. Tomasz, M., Mercado, C. M., Olson, J., and Chatterjee, N. The mode of
interaction of mitomycin C with deoxyribonucleic acid and other polypeptides
in vitro. Biochemistry, 13: 4878-4887, 1974.
7. Barlogie, B., and Drewinko, B. Lethal and cytokinetic effects of mitomycin
C on cultured human colon cancer cells. Cancer Res.. 40: 1973-1980, 1980.
8. Shatkin. A. J.. Reich. E., Franklin. R. M., and Tatum, E. L. Effect of
mitomycin C on mammalian cells in culture. Biochim. Biophys. Acta, 55:
277-289, 1962.
9. Crooke, S. T. Mitomycin C—an overview. In: S. T. Crooke and A. W.
Prestayko (eds.). Cancer and Chemotherapy. Vol. 3. pp. 49-60. New York:
Academic Press, 1980.
10. Doi, O., Takai, S., Aoki, Y., Higashi. H.. and Kosaki, G. Effects of mitomycin
C on HeLa cells at various stages of the division cycle. Gann, 58: 125-137,
1967.
11. Rao, P. N. Mitotic synchrony in mammalian cells treated with nitrous oxide
at high pressure. Science (Wash. DC), 160: 774-776, 1968.
12. Sognier, M. A., and Hittelman, W. N. The repair of bleomycin induced DNA
damage and its relationship to chromosome aberration repair. Mutât.Res..
02:517-527, 1979.
13. Kondoleon, S. K., and Stambrook, P. J. Replication of mammalian DNA in
BrdU: appearance of a component with intermediate density. Mol. Gen.
Genet.. ISO: 523-530, 1980.
14. Dean, P. N., and Jeff, J. H. Mathematical analysis of DNA distribution
derived from flow microfluorometry. J. Cell Biol.. 60: 523-527. 1974.
15. Johnston, D. A., White, R. A., and Barlogie, B. Automatic processing and
interpretation of DNA distributions: comparisons of several techniques.
Comp. Biomed. Res., II: 393-404, 1978.
16. Sognier, M. A., and Hittelman, W. N. Effect of DNA repair inhibition on
induction and repair of bleomycin induced chromosome damage. Mutât.
Res., 60:61-72, 1979.
17. Johnson, R. T., and Rao, P. N. Mammalian cell fusion: induction of pre
mature chromosome condensation in interphase nuclei. Nature (Lond.), 226:
717-722, 1970.
18. Sperling, K., and Rao, P. N. Mammalian cell fusion. V. Replication behavior
of heterochromatin as observed by premature chromosome condensation.
Chromosoma (Beri.), 45: 121-131, 1974.
19. Kohn, K. W., Ewig, R. A., Erickson, L. C., and Zwelling, L. A. Measurement
of strand breaks and cross-links by alkaline elution. In: E. C. Friedberg and
P. C. Hanawalt (eds.), DNA Repair—A Lab Manual of Research Procedures,
Part B, pp. 379-401. New York: Marcel Dekker, Inc., 1981.
20. Zwelling, L. A., Kohn, K. W., Ross, W. E., Ewig, R. A. G., and Anderson.
T. Kinetics of formation and disappearance of a DNA cross-linking effect in
mouse leukemia L1210 cells treated with cis- and fra/is-diamminedichloroplatinum(Il). Cancer Res., 38: 1762-1768, 1978.
21. Ewig, R. A. G., and Kohn, K. W. DNA damage and repair in mouse leukemia
L1210cells treated with nitrogen mustard, l,3-bis(2-chloroethyl)-l-nitrosourea, and other nitrosoureas. Cancer Res., 37: 2114-2122, 1977.
22. Ross, W. E., Ewig, R. A. G., and Kohn, K. W. Differences between melphalan
and nitrogen mustard in the formation and removal of DNA cross-links.
Cancer Res., 38: 1502-1506, 1978.
23. Crissman, H. A., Mullaney, P. F., and Steinkamp, J. A., Methods and
applications of flow systems for analysis and sorting of mammalian cells.
Methods Cell Biol., 9: 179-246, 1975.
24. Kerker, M., Van Dilla, M. A., Brunsting, A., Kratohuil, J. P.. Hsu, P., Wang,
D. S., Gray, J. W., and Langlois, R. G. Is the central dogma of flow cytometry
(i.e., fluorescence intensity is proportional to cellular dye content) true?
Cytometry. 5:71-78, 1982.
25. Alabaster, O., Tannenbaum, E., Habbersett, M. C., McGrath. I., and Her
man, C. Drug induced changes in DNA fluorescence intensity detected by
flow microfluorometry and their implications for analysis of DNA content
distributions. Cancer Res., 38: 1032-1035, 1978.
BREAKS
26. Haag, D., Unlweauxhunfwn zur Proportionalität zwischen DNA-Gehalt und
fluoreszenzintensitat fluorochromierter ein zelzellen. Histochimie, 36: 283291, 1973.
27. Hittelman, W. N. Prematurely condensed chromosomes: a model system for
visualizing effects of DNA damage, repair, and inhibition at the level of
chromosome structure. In: A. Collins, S. Downes, and R. T. Johnson (eds.),
DNA Repair and Its Inhibition, pp. 341-371. Oxford: IRL Press, 1984.
28. Garcia, A. M. Studies on DNA in leukocytes and related cells of mammals.
VI. The Feulgen-DNA content of rabbit leukocytes after hypotonie treatment.
J. Histochem. Cytochem., 17:47-55, 1969.
29. Ringertz, N. R., Gledhill, B. L., and Darzynkiewicz, Z. Changes in deoxyribonucleoprotein during spermiogenesis in the bull. Exp. Cell Res., 62: 204218, 1970.
30. Taylor, J. H., Haut, W. F., and Tung, J. Effects of fluorodeoxyuridine on
DNA replication, chromosome breakage, and reunion. Proc. Nati. Acad. Sci.
USA, 48: 190-198, 1962.
31. Palitti, F., Tanzarella, C., Degrassi, F., De Salvia, R., Fiore, M., and Natarajan, A. T. Formation of chromatid type aberrations in the G2 stage of the
cell cycle. Mutât.Res., 110: 343-350, 1983.
32. Murnane, J. P., and Byfield, J. E. Irrepairable DNA cross-links and mam
malian cell lethality with bifunctional alkylating agents. Chem.-Biol. Inter
act., J«:75-86, IMI.
33. Sognier, M. A., and Hittelman, W. N. Loss of repair of DNA interstrand
crosslinks in Fanconi's anemia cells with culture age. Mutât.Res., 108: 383393, 1983.
34. Dorr, R. T., Bowden, G. T., Alberts, D. S., and Liddil, J. D. Interactions of
mitomycin C with mammalian DNA detected by alkaline elution. Cancer
Res., «.-3510-3516, 1985.
35. Wilkins, R. J.. and Hart. R. W. Preferential DNA repair in human cells.
Nature (Lond.), 247: 35-36, 1974.
36. Lown, J. W. The molecular mechanism of antitumor action of the mitomycins. In: S. K. Carter and S. T. Crooke (eds.), Mitomycin C—Current Status
and New Developments, pp. 5-26, New York: Academic Press, 1979.
37. Sturelid, S. Chromosome breaking capacity of tepa and analogues in Vicia
faba and Chinese hamster cells. Hereditas, 68: 265-276, 1971.
38. Abanobi, S. E., Columbano, A., Mulivar, R. A., Rajalakshmi, S., and Sarma,
D. S. R. In vivo replication of hepatic deoxyribonucleic acid of rats treated
with dimethylnitrosamine: presence of dimethylnitrosamine-induced
O6methylguanine, A^-methyl guanine, and W'-methyl adenine in the replicated
hybrid deoxyribonucleic acid. Biochemistry, 19: 1382-1387, 1980.
39. Ledda, G. M., Columbano, A., Rao, P. M., Rajalakshmi, S., and Sarma, D.
S. R. In viro replication of carcinogen-modified rat liver DNA: increased
susceptibility of O'-methylguanine compared to W-methylguanine in repli
cated DNA to SI nuclease. Biochem. Biophys. Res. Commun., 95:816-821,
1980.
40. Medcalf, A. S. C., and Lawley, P. D. Time course of O'-methylguanine
removal from DNA of A'-methyl-jV-nitrosourea treated human fibroblasts.
Nature (Lond.), 289: 796-798. 1981.
41. Cleaver, J. E. Regulation of the responses to DNA damage in the hypersensitivity diseases and chromosome-breakage syndromes. In: J. German (ed.).
Chromosome Mutation and Neoplasia, pp. 235-249. New York: Alan R.
Liss, Inc., 1983.
42. Loveday, K. S., and Latt, S. A. Search for DNA interchange corresponding
to SCE in CHO cells. Nucleic Acids Res., 5: 4087-4104, 1978.
43. Romelaere, J.. and Miller-Faures. A. Detection by density equilibrium centrifugation of recombination like DNA molecules in somatic mammalian
cells. J. Mol. Biol., 98: 195-218, 1975.
44. Meneghini, R., Menck, C. F. M., and Schumacher, R. I. Mechanisms of
tolerance to DNA lesions in mammalian cells. Q. Rev. Biophys., 14: 381432, 1981.
45. Fournace, A. J., Jr. Recombination of parental and daughter strand DNA
after UV irradiation in mammalian cells. Nature (Lond.), 304: 532-534,
1983.
46. Kenne, K., and Ljungquist, S. A DNA-recombinogenic activity in human
cells. Nucleic Acids Res.. 12: 3057-3068, 1984.
47. Cremer, C., Cremer. J., and Gray, J. W. Induction of chromosome damage
by UV light and caffeine: correlation of cytogenetic evaluation and flow
karyotype. Cytometry, 2: 287-290, 1982.
48. Griffiths. T. D., and Tolmach, L. J. Age dependence of X-ray induced
deficiency in DNA synthesis in HeLa S3 cells during Generation 1. Radiât.
Res., 63: 501-520, 1975.
49. Hittelman, W. N., and Pollard, M. Induction and repair of DNA and
chromosome damage by neocarzinostatin in quiescent normal human fibro
blasts. Cancer Res., 42: 4584-4590. 1982.
50. Schor, S. L.. Johnson. R. T., and Waldren, C. A. Changes in the organization
of chromosomes during the cell cycle: response to ultraviolet light. J. Cell
Sci., 17: 539-565, 1975.
51. Waldren, C. A., and Johnson. R. T. Analysis of interphase chromosome
damage by means of premature chromosome condensation after X- and
ultraviolet-irradiation. Proc. Nati. Acad. Sci. USA, 71: 1137-1141, 1974.
52. Mullinger, A. M., and Johnson, R. T. Manipulating chromosome structure
and metaphase status with ultraviolet light and repair synthesis inhibitors. J.
Cell Sci., 73: 159-186. 1985.
53. Rao. P. N., and Adlakha. R. C. Chromosome condensation and decondensation factors in the life cycle of eukaryotic cells. In: A. L. Maizel and R.
Ford (eds.). Mediators in Cell Growth and Differentiation, pp. 45-69. New
York: Raven Press. 1985.
54. Sunkara, P. S., Wright. D. A., and Rao, P. N. Mitotic factors from mam
malian cells induce germinal vesicle breakdown and chromosome condensa
tion in amphibian oocytes. Proc. Nati. Acad. Sci. USA, 76: 2799-2802,
1979.
4039
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1986 American Association for Cancer Research.
MITOMYCIN-INDUCED
CHROMATID
55. Rao, P. N., and Smith, M. L. Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis. J. Cell Biol., 88:649653, 1981.
56. Adlakha, R. C.. Sahasrabuddhe, C. G., Wright, D. A., and Rao, P. N.
Evidence of the presence of inhibitors of mitotic factors during G, period in
..
n i /•Tin- r n-r n/n mi
inoi
mammalian cells^J. Cell¡B.ol.,97: 1707-1713. 1983
57. Adlakha, R. C.. Wang. Y. C.. Wright, D. A., Sahasrabuddhe, C. G., Bigo,
H., and Rao, P. N. Inactivation of mitotic factors by U. V. irradiation of
HeLa cells in mitosis. J. Cell Sci., 65: 279-295, 1984.
58. Hittelman. W. N., and Rao. P. N. Premature chromosome condensation. I.
BREAKS
Visualization of X-ray induced chromosome damage in interphase cells,
Mutât.Res., 23: 251-258, 1974.
59. Hittelman, W. N.. and Rao, P. N. Bleomycin induced damage in prematurely
condensed chromosomes and its relationship to cell cycle progression in
n c"° cells- CanE." Res-<34: ^l33"?43'•' 9D74'
60. Nishimoto, T., Eilen, E., and Basilico, C. Premature chromosome condensation in a ts DNA~ mutant of BHK cells. Cell, 75: 475-483, 1978.
6, Hayashi A Yamamoto. s.. Nishimoto. T., and Takahashi, T. Chromosome
condensing factor(s) induced in tsBN2 cells at a nonpermissive temperature:
evidence for transferable material by cell fusion. Cell Struct. Fund., 7: 291294, 1982.
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Mitomycin-induced Chromatid Breaks in HeLa Cells: A
Consequence of Incomplete DNA Replication
Marguerite A. Sognier and Walter N. Hittelman
Cancer Res 1986;46:4032-4040.
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