Download Interference of Daunomycin with Metabolic Events of the Cell Cycle

(CANCER RESEARCH 30, 966-973,
April 19701
Interference of Daunomycin with Metabolic Events of the Cell
Cycle in Synchronized Cultures of Rat Fibroblasts'
R. Silvestrini,2A. Di Marco,and T. Dasdia
Istituto Nazionale per lo Studio e Ia Cura dei Tumori, Sezione di Chemioterapia Sperimentale, Milan, Italy
SUMMARY
The influence of daunomycin on nucleic acid synthesis
during different stages of the cell cycle has been studied in
mammalian cells. Explants from leg muscle of newborn rats
were synchronized by two 24-hr treatments with excess
thymidine, 16 hr apart. The metabolic activity of DNA
and RNA was determined by autoradiographic methods by
measuring the incorporation
of thymidine-3 H, deoxy
cytidine-3H,
and uridine-3H into nuclear structures. In
control
located
@
@
cultures,
DNA synthesis shows two distinct waves
in the early and late S phase, the latter being the
most sensitive to the inhibiting effect of daunomycin. RNA
synthesis takes place in the cells during the entire cell cycle
from the end of mitosis to the late prophase, but two
well-defined peaks can be recognized: the first in the G2
phase just 1 or 2 hr before the mitotic peak and the second
in the middle stages of
. These RNA syntheses involving
both nucleolar and extranucleolar structures are strongly
inhibited by daunomycin treatment. The significance of
these RNA syntheses in control cells and the relationship
between their inhibition and the antimitotic effect of
daunomycin are discussed.
as the inhibition of DNA and RNA synthesis, have also been
demonstrated repeatedly (6, 8, 9, 18, 19, 21).
However, the correlation between the antimetabolic effects
and the antimitotic activity is not clear at present. In this
regard it must be remembered that direct phase-contrast
observations showed that daunomycin can arrest the mitotic
activity of single cells when added up to 20 mm before the
prophase stage (10). Furthermore, on HeLa cell cultures and
on rat methylcholanthrene
sarcoma cells, a remarkable
inhibition of the mitotic index takes place with doses of
daunomycin, which inhibit only slightly the incorporation of
thymidine-3 H into DNA (7).
In view of these observations, the possibility of an anti
mitotic activity of daunomycin unrelated to the effects on
DNA
and
RNA
synthesis
must
also
be considered.
For
further investigation of this problem, a group of experiments
on synchronized cultures with the addition of daunomycin
during different stages of the cell cycle was carried out. The
effects on DNA and RNA synthesis and on the mitotic index
were studied.
MATERIALS AND METHODS
Explants from the leg muscle of newborn rats (24 to 48 hr
old) were cultivated in Porter flasks in a medium consisting
INTRODUCTION
of Hanks' salt solution, human placental serum, and chick
embryo extract (4:4:2). After 30-hr growth the cultures
Daunomycin is an antibiotic of the anthracycline group
were synchronized by two 24-hr treatments with excess
isolated from cultures of Streptomyces peucetius (5 , 12).
thymidine (2 mM), separated by an incubation period of 16
This substance consists of a pigmented aglycone (daunomy
hr(3).
cinone) bound by a glycosidic linkage to an amino sugar
At the end of the treatment with the antibiotic and after
(daunosamine) (1 , 2). In vitro, daunomycin shows a strong
exposure to different precursors, the cultures were fixed in
inhibiting effect on the mitotic activity of different types of
Bouin's solution, washed in 5% trichloracetic acid at 4°and
mammalian cells (10).
in carrier solution, and covered with AR 10 Kodak stripping
The ability of daunomycin to bind to DNA in vitro has
film as described below (15).
been demonstrated by several physicochemical procedures (4,
The rates of DNA and RNA synthesis were evaluated by
13, 14). Some of the antimetabolic effects of this drug, such
counting the number of reduced silver grains in the whole
nucleus and in the nucleolar and extranucleolar structures,
respectively. Each value shown in the graph represents the
average of counts made on at least 100 cells.
Labeled Substances. These were thymidine-3 H (specific
activity, 160 mCi/mmole), 2 iCi/ml culture medium; deoxy
1These investigations were supported in part by the “Consiglio
cytidine-3 H (specific activity, 500 mCi/mmole), 5 pCi/mi
Nazionale delle Ricerche,―Italia.
culture medium; and uridine-3H (specific activity, 2.4
2ConsiglioNazionale delle Ricerche.
Received July 8, 1969; accepted September 22, 1969.
Ci/mmole), 5 pCi/mi culture medium.
966
CANCER RESEARCH
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Action of Daunomycin
RESULTS
S
MITOTIC
on Synchronized
Cells
INDEX
0
Control Cultures
“a
-a
Mitotic Activity. The mitotic index in untreated cultures is
about 3%; after synchronization the cell population shows a
1st mitotic peak (14 to 17%) within 6 to 10 hr. The 2nd
mitotic peak is not always sharply outlined and the mitotic
index is lower than that of the 1st. There is a period of ca.
23 hr between the 2 mitotic peaks, indicating that the length
of the cell cycle of rat fibroblasts is the same before and
after synchronization (Chart l).@
Incorporation of Deoxycytidine-3 H. After treatment with
excess thymidine, the population emerges in the S phase.
The use of deoxycytidine-3H clearly shows that at the end
of synchronization 90 to 95% of the nuclei are labeled,
showing that almost the entire cell population is in the S
phase.
The percentage of labeled nuclei remains high until the 4th
hr; then it progressively decreases as the cells enter the G2
phase (Chart 2).
The estimation of the nuclear grains demonstrates that the
pattern of deoxycytidine-3H incorporation undergoes marked
changes
during
the S phase (Chart
end of synchronization,
observed.
This
value
3). Immediately
U
a
L.a
“a
..a
a
z
x
0
a
I-
I
U
0
hr
Chart 2. Mitotic activity and percentage of nuclei labeled with
deoxycytidine-3H in synchronized control cultures. Batches of
cultures were fixed after 1-hr contact with the labeled precursor.
after the
a high degree of nuclear labeling is
decreases
rapidly
and
then
reaches
another maximum after 4 to 5 hr. i.e., 3 to 4 hr before the
mitotic peak.
This 2nd period of DNA synthesis occurs during the last 2
hr of the S phase.
3The length of the cell cycle and the single phases in an asynchro
nous population of rat fibroblasts, estimated by a pulse labeling
experiment described by Post et al. (16), is as follows: cell cycle, 21
to 23 hr; G2, 2 hr; M 1 hr; 5, 8 hr; and G1, 10 to 12 hr.
@
—1--@-—
I
I
I
I
I
Incorporation of Uridine-3 H. Cells kept in contact with
uridine-3 H for 1 hr after the end of synchronization show a
progressive increase in labeling from 2 to S hr when a
maximum is observed in both nucleolar and extranucleolar
structures. The nuclear labeling then falls to the basic value
observed at the beginning of the S phase. In this experiment,
the maximum value of the mitotic index (16%) is noted
after 6 hr (Chart 4). Maximum RNA synthesis occurs exactly
1 hr before the mitotic peak but drops to much lower values
by the time the mitotic activity is at its highest point. In the
period of maximum uridine-3 H incorporation, the labelings
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Chart 1. Mitotic activity of rat fibroblast cultures synchronized by two 24-hr treatments with excess thymidine 16 hr apart.
APRIL
1970
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967
R. Silvestrini, A. Di Marco, and T. Dasdia
The RNA synthesis has also been followed in the G1
phase. The results of the labeling in the nucleolar and
extranucleolar regions on incubation of the cultures in the
presence of uridine-3 H for 2-hr periods are reported in Chart
5. Uridine-3
H incorporation
occurring
between
the 2 mitotic
waves undergoes quite marked variations. In the extra
nucleolar area of the nucleus an increase in the number of
grains is observed from the 15th hr until the 19th hr. This
increase is particularly evident at the 15th hr; then the
extranucleolar labeling slowly decreases and at the 2 1st hr
the value is similar to that found at the beginning of the G1
phase. More evident changes are observed in the nucleoli,
where the nucleolar RNA synthesis is rather low in the 1st
hr of the G, phase, then after 14 hr increases rapidly and
reaches a value 3 times higher than at the outset. This rate
of production lasts until the 17th hr, then decreases, but
does not reach the low values of the early G1 phase. These
results have also been confirmed in several experiments.
Chart 3. Mitotic activity and deoxycytidine-3H incorporation/
nucleus in synchronized control cultures. Batches of cultures were
fixed after 1-hr contact with the labeled precursor.
—
—
flucteolar
RP4A
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Because of
the
binding
many
hours
after
its removal
from
the medium.
was
added
to
the
medium
for
3
I
S
hr,
together
concentrations
with
VLB4
(0.5
In this way the total number of cells arrested in
Interference
widely
in 2 different
1
with
DNA
index) could be evaluated.
Synthesis.
with
drug
Interference
caused
concentration
with
deoxy
by daunomycin
and with
time
varies
of administra
tion. Treatment with 0.5 pg/ml in the early S phase does not
affect the DNA metabolic pattern (Chart 8). Treatment with
lower doses (e.g. , 0. 1 pg/mI) over the corresponding period
induces a clear and significant increase in nuclear labeling. In
6
7
S@9.
hours
LP-3H
Chart 4. Mitotic activity and uridine-3H (Ur-3H) incorporation in
synchronized control cultures after 1-hr contact with the labeled
precursor.
of the nucleolus and of the extranucleolar zone are, respec
tively, 3 and 2 times higher than those found before and
after the peak. These results have been confirmed in several
experiments.
968
for
cytidine-3 H incorporation
@
@
absent.
metaphase (stathmokinetic
2
@
completely
in Chart 7, where daunomycin
@.
pg/mi).
@
or
That daunomycin effectively prevents the mitotic division
of cells is shown from the results of the experiment reported
8.
U
As can be observed in Chart 6, in the
capacity of daunomycin to DNA the drug cannot be
removed from the nuclear structures simply by washing the
cell cultures. It must be expected, therefore, that its activity
I
@
L.a reduced
I
/
were treated with daunomycin (0.1 pg/mI) at
of 1 hr from the end of synchronization until the
mitotic activity. After treatment, the antibiotic was
and normal culture medium was added. The
were fixed after 6, 7, 8, or 9 hr and the mitotic
cultures treated with daunomycin, independent of when
treatment
is given, the mitotic activity is considerably
_s0
@
@
@
Cultures
Interference with Mitotic Activity. Batches of synchronized
cultures
intervals
onset of
removed
cultures
HI
— . —-—
Daunomycin-treated
other
words,
the
nuclear
labeling
does
not
decrease
as in
the
controls, but maintains a high value for 2 hr. The treatment
during the last part of the S phase causes a reduction in
deoxycytidine-3 H incorporation which is proportional to the
concentration of the drug.
Interference
with RNA Synthesis. The influence of
4The abbreviation used is: VLB, vinblastine.
CANCER RESEARCH
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Action of Daunomycin
EXT@ANUCtE0LAR
1 @_
on Synchronized
Cells
RPM
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Ur-@H
Chart 5. Mitotic activity and uridine-3H incorporation in synchronized control cultures after 2-hr contact with the labeled precursor.
—
CONTROL
16
0 VLB O.5,ug/ml
1@
. VLB•OaO.lpg/m(
g
U VLB•DaO.5pg/m(
L.a
0
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n
0
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2
Dl
3
0.
5
0.
Da
6
7
8
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Chart 6. Interference of daunomycin (Da) with mitotic activity in
synchronized cultures. Groups of cultures were treated for successive
1-hr periods and washed and incubated in normal medium.
Batches of cultures from each group were fixed during the 6th, 7th,
8th, and 9th hr. Thick lines, time of antibiotic treatments.
@
In
daunomycin on RNA synthesis was followed during all
phases of the cell cycle.
For the purpose of analyzing to what extent daunomycin
treatment
during the G1 phase interferes with RNA
synthesis, a group of cultures was treated every 2 hr with
uridine-3 H and daunomycin from the 9th to the 2 1st hr
after the end of synchronization,
and then immediately
fixed. A nearly complete inhibition of incorporation of
uridine-3H into the nucleolus was observed (Chart 9). The
degree of inhibition is highest halfway through the
phase
when a peak of nucleolar RNA synthesis occurs in control
cells (see Chart 5). At the same time extranucleolar RNA
_5-@
4_@
J—o
0—/
/—G
Chart 7. Interference of daunomycin (Da) with the stathmokinetic
index in synchronized
cultures.
Daunomycin
at 2 different
con
centrations was introduced for successive1-hr periods (from the 3rd
to the 9th hr after the end of the synchronization) together with
VLB. The cultures were successively washed and incubated until the
9th hr in the medium containing the same concentration of VLB.
synthesis is also inhibited but not as markedly as the
nucleolar synthesis.
Another group of cultures was treated at corresponding
periods and exposed to thymidine-3H from the 21st to the
29th hr. The inhibition of DNA synthesis is first noticed in
the cultures treated from the 11th to the I 3th hr and
becomes pronounced in the cells treated from the 13th hr to
the 17th hr, i.e., the same period as that showing strong
inhibition of nucleolar and extranucleolar RNA synthesis.
APRIL 1970
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969
R. Silvestnni, A. Di Marco, and T. Dasdia
DNA
:i
16
treated at 1-hr intervals (from the end of synchronization
until mitosis) and then immediately fixed (Chart 10). The
treatment with the antibiotic (0.1 pg/ml) induces a strong
inhibition of RNA synthesis, particularly in the nucleolus
(see Chart 5).
Now the question arises of a possible relationship between
the inhibition of RNA synthesis in the G2 phase and the
subsequent inhibition of mitotic activity. For clarification of
this point, cultures were treated with different daunomycin
D CONTROL
0. 0.1pg/mI
@
•[email protected]/mI
1@
x
Lu
0 10
U 8
I..
0
I-. 6
concentrations
4
2
for
1-hr periods
from the 3rd to the 6th hr
T==zI@T@3E.
L
0
2
4
5
6hr
b 702O3O@O5O
GRAINS MJMBER/MJCLEUS
Chart 8. Interference of daunomycin (Da) at different concentra
tions with deoxycytidine-3 H incorporation.
Batches of cultures were
put into contact with the antibiotic and the labeled precursor for
1-hr periods and then immediately fixed. Left, mitotic behavior of
control cultures; thick lines, time of antibiotic treatments.
Thereafter, the inhibiting effect of thymidine-3 H on DNA
synthesis becomes less pronounced.
In order to determine the action of daunomycin on RNA
synthesis during the S and G2 phases, the cultures were
after the synchronization treatment. The effect on both the
uridine-3 H incorporation
in nuclear structures and the
mitotic index was observed. In this experiment (Chart I 1),
the G2 phase of RNA synthesis precedes by 2 hr the mitotic
peak. There is notable reduction in uridine-3H uptake during
the peak period of RNA synthesis at daunomycin con
centrations higher than 0.025 pg/ml. The mitotic activity,
however, is lowered by a concentration of 0.0125 pg/mI
when the antibiotic is added to the medium from the 6th to
the 7th hr, i.e., in the period of maximum mitotic activity.
Hence, the antimitotic effect does not necessarily seem to be
related to the inhibiting effect on the RNA synthesis that
occurs in G2 phase.
DISCUSSION
The
rate
of RNA
synthesis
1$
=
1S
DNA
in rat
fibrobl@ist cultures
CONTROL
o. as pgimL
EXTRANUCLEOLAR
@RHA
NUCLEOLAR
-
14
r@1
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5'
0
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SO
GRAINS
CONTENT
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0
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0
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CONTENT
2lhr
OaUrjsUr
TNYM.'N
Chart 9. Action of daunomycin (Da) on the incorporation of thymidine-3H (Thym-31-f)and uridine-3H (Ur) by synchronized cultures of rat
fibroblasts. After the mitotic wave, the cells were treated with daunomycin (0.5 @sg/ml)
over 2-hr intervals. The cultures were put into contact
with uridine-3H during daunomycin treatment and immediately fixed. Another batch of cultures similarly treated was put into contact with
thymidine-3H from the 21st to the 29th hr and then fixed. Left, mitotic behavior of control cultures; thick lines, time of antibiotic
treatments.
970
CANCER RESEARCH
VOL.30
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Action
0 CONTROL D DA@J@VCIN
O.lpg/ml
/I\\
RNA
1@
@
@
-
)@%
12
Lu
0
10
RNA
on Synchronized
EXTRAM/CLEXXARNUCLEOLAR
14
MXLE@AR
EXTRAMXLE@m
of Daunomycin
RNA
.
I
ld
--------“@
MITOTIC
RNA
INOEX
-4
1
iI1IIII1II1IIi1IH
Cells
____________
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2
n
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2
4
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8
1U1―@
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0702030
GRAINSCONTENT GRAINSCONTENT
Chart 10. Interference of daunomycin with the incorporation of
uridine-3 H into nucleolar and extranucleolar
structures. Batches of
cultures were put into contact with the antibiotic and the labeled
U control
S O.O@25
Mg/mi
@I
U
2t@
I
£0 b
10
Ô
GRAINS CONTENT GRAIS C@WTENT
ID 0.0250 @@g/mI
. 0.05 @g,mI
‘a
0.1
@‘mI
Chart 11. Interference of different doses of daunomycin with RNA
synthesis and with' mitotic activity. Left, mitotic behavior of control
cultures; thick lines, time of antibiotic treatments.
precursor for 1-hr periods and then immediately fixed. Left, mitotic
behavior in control cultures; thick lines, time of antibiotic treatments.
undergoes considerable variations during the different stages
of the cell cycle. The uridine-3H incorporation begins to
increase 2 hr before the onset of the mitotic wave both in
the nucleolus and in the extranucleolar area of the nucleus
and reaches a maximum 1 or 2 hr before the peak of the
mitotic wave. Considering the length of the G2 phase (2 hr),
is is reasonable to conclude that RNA synthesis occurs in the
nuclei when the DNA synthesis is already complete and the
mitotic
process
has not
yet
begun.
(Nucleolar
RNA
con
tributes mainly to this increase, while extranucleolar RNA
plays a minor role.) Towards the end of G2 , RNA synthesis
progressively diminishes and remains at a very low level
during mitosis and the first hours of the G1 phase. Then the
RNA synthesis again begins to increase, and halfway through
G1 a well-defined
increase in incorporation
of labeled uridine
is observed. Once again, this is due mainly to the presence of
nucleolar RNA. Taylor (23) and Robbins and Sharff (17) did
not observe this periodical RNA synthesis in synchronized
cultures. However, both these authors measured the rate of
synthesis on samples of cells withdrawn from the cultures.
Another point which deserves consideration is the use, in
our experiments, of diploid cells after the first in vitro
transplant.
How far could the antimitotic effect of daunomycin be
related to the inhibiting effect of RNA synthesis? One of the
points of highest sensitivity to daunomycin, as far as the
antimitotic activity is concerned, occurs in the G2 phase.
Therefore, the inhibitory effect on the mitotic process
obtained by treating the cells during this phase must be
related to the interference with those metabolic events which
occur when the DNA synthesis is already complete and
before the mitotic process begins. The experiments carried
out to determine the influence exerted by daunomycin on
RNA synthesis during the G2 phase clearly demonstrate an
inhibition of uridine-3 H incorporation
during the hours
immediately preceding mitosis. This inhibition, involving
mainly the nucleolar structures and to a lesser extent the
extranucleolar
ones, is greatest when the rate of RNA
synthesis reaches a maximum in the control cultures. It
would therefore be reasonable to assume that the nuclear
RNA synthesized during G2 plays an important role in the
occurrence of mitosis. A more detailed analysis of the results
shows that the relationship between the inhibiting effect on
RNA synthesis during G2 and on mitosis is not a strict one.
In fact, the occurrence of mitosis is inhibited by con
centrations of daunomycin which have no effect on the
synthesis of RNA; furthermore, mitosis can be inhibited by
treating the cells after the period of maximum RNA
synthesis has passed.
Thus, the mitotic inhibition achieved under these con
ditions cannot be ascribed to a block in the synthesis of
RNA in the G2 period and other metabolic events must
come into play to explain the antimitotic
effect of
daunomycin. This does not mean that this RNA is not
important for triggering off the mitotic process. In fact,
doses of daunomycin which are able to inhibit this RNA
synthesis are also able to prevent the cells from entering the
mitotic stage. Modification of the structure of daunomycin
which impairs the binding capacity to DNA is also very
effective in reducing the antimitotic activity of this drug.
Both are, in fact, considerably reduced in the N-acetyl
daunomycin derivative (9). There is also the possibility that
the antimitotic effect observed under these conditions may
be due to a modification of the tertiary structure of DNA,
which does not permit its passage from the interphase to the
mitotic chromosomes. Experiments carried out to determine
the importance of the RNA synthesized during G1 on
subsequent synthesis demonstrated that daunomycin causes a
nearly complete inhibition of the nucleolar uridine-3 H
incorporation and a considerable but less evident reduction
of uridine-3 H incorporation into the extranucleolar region of
the nucleus. The maximum inhibition of DNA synthesis
during the following S phase is reached when the cells are
treated in the middle of the G1 phase, i.e., when maximum
RNA synthesis occurs in control cells. These observations are
consistent with the hypothesis that in the middle stages of
APRIL 1970
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971
R. Silvestrini, A. Di Marco, and T. Dasdia
the G1 phase a sequence of biochemical events essential to
DNA replication occurs in the cell (1 1). As these events can
be . influenced by an inhibition of DNA-dependent RNA
synthesis, they could be related to the formation of RNA
molecules bearing the information necessary for the synthesis
of the enzymes essential to DNA replication.
The pattern of DNA synthesis shows marked variations
during the S phase. The autoradiographic determinations of
nuclear labeling show 2 distinct peaks of DNA synthesis
localized, respectively, in the early and late S phases. The
2nd wave of DNA synthesis, according to postulates by
another worker (22), may be reasonably identified as the
replication of heterochromatin.
The inhibitory effect of
daunomycin on DNA synthesis varies according to the
concentration of the drug and the period of the S phase
during which the cells come in contact with daunomycin.
DNA synthesis in the early S phase is not inhibited and even
increases with low doses of daunomycin, as shown by the
increase
in deoxycytidine-3
H incorporation.
This increased
incorporation
must be interpreted
as a lack of DNA
synthesis regulation, rather than a real increase.
On the contrary, inhibition of deoxycytidine-3 H incorpora
tion proportional to the antibiotic concentration is observed
when the cells are treated in the last stages of the S phase. It
is in this period that the maximum binding of daunomycin
to the nuclear structures has been observed (20). Since
synthesis of heterochromatin occurs during the last stages of
the S phase, it is probable, as already noted for actinomycin
(22), that the maximum binding of daunomycin also occurs
during the synthesis of heterochromatin and that this impairs
the synthesis of DNA.
In conclusion,
the uptake of daunomycin
produces
different effects according to the physiological activity of
the cells at the time of treatment. In cells in the G1 phase,
there is inhibition of synthesis of enzymes relevant to DNA
replication. In cells in the S phase, there is inhibition of
DNA replication,
not necessarily followed by mitotic
inhibition; irreversible damage to the chromosomal structures
appears in a high percentage of the cells. In cells in the G2
phase, there is blockage of the synthesis of nuclear and
particularly nucleolar RNA, which could be essential for
triggering off the mitotic process. In cells in mitosis, there is
mitotic blockage when the drug is given just a few minutes
before the prophase.
All these effects are related to the ability of daunomycin
to bind to DNA, and can be obliterated or reduced by
modifying the daunomycin molecule in such a way that its
binding capacity with DNA is reduced. In fact, the N-acetyl
derivative of daunomycin, endowed with a low binding
capacity to DNA (4), shows almost none of these effects.
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Interference of Daunomycin with Metabolic Events of the Cell
Cycle in Synchronized Cultures of Rat Fibroblasts
R. Silvestrini, A. Di Marco and T. Dasdia
Cancer Res 1970;30:966-973.
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