close

Вход

Забыли?

вход по аккаунту

?

DNA synthesis by cultured myocardial cells.

код для вставкиСкачать
DNA Synthesis by Cultured Myocardial Cells '
RUTH E. RUMERY AND WILLIAM 0. RIEKE
Department of Biological Structure, University of Washington,
Seattle, Washington
Myocardial cells cultured from ventricles of 4-day embryonic chick
hearts were labeled with H3-thymidine for the radioautographic study of DNA synthesis
and nuclear cycling. After the myoblasts had been in culture for 48 hours and had
begun to grow out and contract, the cultures were exposed to H3-thymidine, 1 pc/ml of
Hank's balanced salt solution, for one hour. Pairs of cultures were fixed in methyl
alcohol for analysis every two hours over a subsequent period of 60 hours. The total
percentage of labeled cells, together with labeled mitotic figures which developed after
labeling, determined the average values for generation time and nuclear cycle phases
as follows: generation time, 45 hours; DNA synthesis, nine hours; Gz interval, 2-6
hours; Gr interval, 27-33% hours; and mitosis, one-half to one hour. Autonomous
control of DNA synthesis was observed occasionally jn a binucleate cell having only
one labeled nucleus, although both nuclei were i n a similar phase of mitosis. It is
evident from these data that cultured myocardial cells synthesize DNA similarly to
other cells.
ABSTRACT
The mechanism by which striated muscle cells increase in number during growth,
and in the course of regeneration after
injury, has been a subject of considerable
interest and controversy. With the use of
radioautography, a number of recent studies have provided evidence that proliferation of skeletal muscle cells occurs by
synthesis of deoxyribonucleic acid (DNA)
and mitotic division. Both synthesis of
DNA and division have been noted first
in mononucleated myoblasts which later
fused with one another to form multinucleated myotubes. The myotube nuclei,
however, neither synthesized DNA nor did
they divide. These observations have been
reported in the rat (Zhinkin and Andreeva,
'63), in the chick (Stockdale and Holtzer,
'61) and in regenerating muscle of the
mouse (Bintliff and Walker, '60; Walker,
'62).
Cardiac muscle has not been investigated
as extensively with labeled materials.
When Messier and Leblond ('60) injected
H3-thymidine into rats and mice, they
noted that only a rare cardiac muscle nucleus was labeled. I n a more extensive
study of nucleic acid and protein synthesis
in cardiac muscle of mice from birth to
adulthood, Petersen and Baserga ('64)
concluded that the mouse heart grows by
the production of new myocardial cells
mainly during the first 4-6 weeks after
birth, followed thereafter by a n increase
ANAT. REC, 158: 501-508.
in size of the existing muscle cells. They
reported also that the uptake of H3-thymidine by cardiac muscle reached a maximum on the fourth day after birth and
then decreased slowly.
There h a s been some disagreement
among investigators whether or not myocardial cells grown in culture divide mitotically. I n the cultures of cardiac muscle
from the embryonic chick, Cavanaugh and
Cavanaugh ('57) observed mitotic figures
mainly during the first three days after
explanation. Chick cardiac myoblasts in
various stages of mitoses were seen also
by Holtzer, Abbott and Cavanaugh ('59).
In our studies of chick inyoblasts differentiating i n culture over a 4-day period,
mitotic figures were seen only rarely (Rumery and Blandau, '64). Therefore, we were
interested in investigating the mitotic
events of embryonic chick myocardial cells
and in characterizing the nuclear cycle in
relation to DNA synthesis, using the high
resolution provided by autoradiographic
techniques with Hs-thymidine.
MATERIALS AND METHODS
Myocardial cells from the ventricles of
4-day embryonic chick hearts were used
for this study. The method for preparing
the cardiac tissue for culture was the one
described by Rumery and Blandau ('64)
1 Research supported by USPHS grants HE-02083-11
and AI-06150 from the National Institutes of Health.
501
502
RUTH E. RUMERY AND WILLIAM 0. RTEKE
in which the ventricles were minced in
Ca++-free, Mg++-free Tyrode’s solution,
dissociated in 0.03% trypsin for one hour,
and then washed in Hanks balanced salt
solution, before being resuspended in dilute chick embryo extract in preparation
for culturing. The myocardial cells were
cultured on Maximow slides by the double
coverslip method. The small coverslip (344”
diameter) on which the cells were to be
seeded was first coated with a thin layer
of embryo extract-cockerel plasma clot
mixture and then was attached to the large
coverslip (43 X 50 mm). One drop of cell
suspension and one drop of modified
Eagle’s medium with added 10% bovine
amniotic fluid were placed on the coated
coverslip and gently mixed. A Maximow
slide was sealed over each culture which
then was incubated at 37°C in a sittingdrop position.
At the end of 48 hours, the cultures were
examined and 12-14 of them which had
vigorously contracting myocardial cells
were selected for each labeling series. The
Maximow slides were removed and the
small coverslips with the cultured cells
were placed in sterile Petri dishes. As soon
as the medium was withdrawn, H3-thymidine in Hanks solution (final concentration of 1 uc H3-thymidine per ml of Hanks
solution, a specific activity 6.05 c/mM)
was dropped onto the cells. After an incubation period of one hour at 37”C, the HSthymidine was removed and each culture
was immersed in two changes of Hanks
solution and then re-affixed to a large coverglass. Fresh medium which contained unlabeled thymidine in Hanks solution, in a
concentration 100 times that of the H3thymidine, was added. The cultures were
sealed again to Maximow slides and returned to the incubator. Those cultures
which served as controls were not labeled
but were fixed in absolute methanol at the
time when the labeled cells were given
fresh medium.
At two-hour intervals, from 0 to 72 hours
after labeling, a pair of cultures (experimental and control) was fixed in absolute
methanol. Before fixation, however, each
culture was examined under the microscope to be sure that the cells were still
viable and contracting. When fixation was
complete, the coverslips with the cultured
cells were attached to 25 x 75 mm glass
slides, cell side uppermost. These coverslips with their cells were coated with photographic emulsion by lowering a wire
loop, bearing a film of melted Kodak NTB,
emulsion, over the culture. The preparations were exposed to the einulsion for one
week, then developed and poststained using MacNeil’s tetrachrome staining procedure.= Finished slides were coded and
analyzed without knowledge of the labeling interval. Between 500 and 1000 cells
were scored on each slide in order to determine the percentage of labeled cells. At
least 50-100 figures in various mitotic
phases were observed in order to establish
the percentage of labeled mitoses.
RESULTS
Six groups, of 12-14 cultures, were used
in this investigation. The outgrowth from
the trypsin-dissociated cells of the 4-day
embryonic chick ventricles was composed
almost entirely of myoblasts since the action of the trypsin inhibited the growth of
either fibroblasts or endothelial cells
considerably.
There was active DNA synthesis in dl
cultures which were exposed to the H3-thymidine. An average of 20% of all myocardial cells (range 16-24% ) showed distinct
nuclear labeling at the earliest interval
(one hour) following exposure to H3-thymidine (fig. 1). Cultures serving as controls and not exposed to Hs-thymidine were
consistently devoid of any labeling.
Labeled mitotic figures in all stages were
observed at intervals of two hours or more
after exposure to H3-thymidine (figs. 2,3,4).
This confirmed the previous assumption
that labeled cells seen at the earliest intervals were synthesizing DNA in preparation
for mitotic division. Binucleate cells were
not uncommonly observed. Usually these
were unlabeled, but occasionally both nuclei, and rarely one nucleus, were labeled.
Whether binucleate cells arose by fusion
of adjacent cells or by karyokinesis could
not be determined, but mitotic activity was
often noted in each member of the nuclear
pair (fig. 5). One interesting evidence of
nuclear autonomy and asynchrony in syn2 Conn, H. J.
1953 Biological Stains. W. F.
H u m p h r e y Press, Inc., Geneva, New York. Pages 244249.
DNA SYNTHESIS
IOO-
D
cr,
-
I
0
r,
I
I
I
-8
b
$50-
I
9
I
Q
\
r,
s
s
Q
-
["'a\
I
I
'i
: 1
503
Appearance of labeled mitotic
figures i n cultures of embryonic
chick heart cells labeled for one
hour with H3-thymidine.
\
\
\
\
'L
-p ----
om--
I
thetic events was observed in a binucleate '63), the duration of the DNA synthesis
cell in which both nuclei were in prophase phase S is calculated by measuring the disstage but only one was labeled (fig. 6). tance between the 50% points on the asIt seems clear in this situation that what- cending and descending limbs of the curve
ever controls a common cytoplasm may of labeled figures. This yields an average
exert over nuclear events, the labeled nu- value for S of nine hours.
cleus had a longer DNA synthetic phase
The majority of the myocardial cells
and a shorter postsynthetic rest phase than exhibited a postsynthetic rest phase (Gz)
the unlabeled member of the pair.
of 4-6 hours, the time from the presentaWith the evidence that myocardial cells tion of the H3-thymidine until the appearsynthesize DNA in preparation for mitotic ance of the first labeled mitosis. A few
division, it became possible to analyze the
duration of the phases of the nuclear cycle cells remained, however, in the G, phase
by analyzing the temporal characteristics for a brief two hours while several more
of the wave of labeled mitotic figures cells continued in this phase for nearly
which appeared after the cultures were eight hours. The majority of the cells had
labeled with H3-thymidine. This method of a G, phase of 4-6 hours under the present
estimating the duration of the nuclear culture conditions. Myocardial cells which
cycle and its phases has been employed had been in culture four days (2 days after
widely and its advantages have been sum- being labeled) often showed degenerative
changes and, therefore, it was not possible
marized by Wimber ('63 >.
Measurements of the phases of the nu- to measure their generation time by followclear cycle are plotted from the curve and ing labeled cells through two consecutive
data shown in text figure 7 and table 1. mitotic waves. The average generation
As is most commonly accepted (Wimber, time was estimated from the expression
% of labeled cells after pulse labeling ( 1 hour)
Duration of DNA synthesis
20%
100%
-9hourH
GT
~.
&=
900
~
20
100%
- ___
Generation time
~
-45 hours
504
RUTH E. RUMERY AND WILLIAM 0. RIEKE
TABLE 3
Percentage labeled mitoses in cultured embryonic chick heart cells after exposure
to H3-thymidine f o r one hour
Hours
arter
Ha-thymidine
exposure
1
2
3
4
6
8
10
12
14
16
22
24
26
30
Number of
cultures
5
4
1
4
4
4
4
5
5
5
4
3
1
4
Percentaee labeled mitoses
Individual
cultures
0
3,4,4,5
4.5
11,16,23,30
80,85,86,97
94,97,97,100
79,85,86,98
44, 76,82,84,89
28,40,45,60,67
lo,%, 29,37,50
10,13, 15,22
2,6,7
15
10, 11, 12,23
It should be noted that this calculation
of generation time is obviously dependent
on the validity of the required assumption
that all the myocardial cells in each culture
were capable of growth and were randomly
distributed among the various phases of
the proliferative cycle at the time they
were labeled with H'-thymidine. The absence of any degenerative changes in the
myocardial cells when the H3-thymidine
was introduced, together with the fact that
the percentage of labeled cells was relatively constant in different cultures (1624 % ) , would seem to offer sufficient evidence to substantiate this assumption.
The generation time as obtained was
multiplied by the mitotic index, which
ranged from 1-2%, in order to obtain the
time for mitosis (M) of 0.45-0.90 hours.
Finally, the presynthetic phase (GI) was
estimated by subtracting the sum of the
other events in the nuclear cycle from the
generation time. This phase was approximately 27-33% hours.
I n addition to providing information
about the duration of the various phases
of the nuclear cycle, the curve of labeled
mitosis (text fig. 7) reached 100% at eight
hours after I-13-thymidine labeling and
thereby established that all cells recognizable a s being in division had passed
through a preceding period of DNA syntkesis.
Mean
Standard
error
0.81
-
0.0
4.0
4.5
20.0
f
87.0
_t
97.0
87.0
75.0
48.0
30.0
15.0
5.0
15.0
14.0
f 2.4
2 8.2
7.1
f 7.9
t 17.9
? 15.6
2 14.9
f 5.1
2 2.6
*
-
6.0
DISCUSSION
This study presents a particular phase,
that of replication, of the embryonic chick
myocardial cells during growth and differentiation in culture. From these data it is
evident that myocardial cells do synthesize
DNA and do divide mitotically. One hour
following exposure to H'-thymidine, 20 %
of the cells showed uptake of H3-thymidine
in their nuclei, and this percentage remained the same for a t least three hours
after labeling. The fact that the percentage of labeled cells did not increase during
the first three hours (table 2 ) shows that
the H3-thymidine was removed effectively
by the washing procedure and by dilution
with the added nonradioactive thymidine.
Therefore, increases in labeled cells, after
three hours, resulted from division of the
labeled cells only and not from delayed
incorporation of H3-thymidine. As the labeled cells divided, the average grain count
decreased, providing further evidence for
the absence of free H'-thymidine.
The number of cells which became labeled was somewhat lower than usual for
actively proliferating mammalian cells
which, in culture, devote approximately
one- to two-thirds of their life cycle to DNA
synthesis (Defendi and Manson, '63). Some
cells may have ceased to develop and therefore were not labeled. Marchok ('65) reported that the number of embryonic chick
505
DNA SYNTHESIS
TABLE 2
Percentage total labeled cells in cultured embryonic chick heart cells after exposure
to H3-thymidine f o r one hour
Hours
after
dine
Hs-thymi-
1
2
3
5
4
4
4
4
6
Percentage total labeled cells
Individual
M~~~
Standard
cultures
error
Number of
cultures
1
16, 18, 19,23,24
15, 17, 19, 25
20
18,19,25,26
24,27,27,30
skeletal muscle cells which were synthe-
sizing DNA decreased gradually as the
incubation period progressed. Myocardia1
cells cultured from hearts of &day embryonic chicks were observed by Cavanaugh
and Cavanaugh ('57) to divide most fiequently between l2 and 70 hours in
but rarely thereafter. If such were the case
in these cultures, more mvocardial cells
might have been labeled if kxposed to H3thymidine earlier than 48 hours after explantation. If more cells could be labeled
at an earlier interval, it is clear that the
present estimate of generation time would
decrease in relation to the increased number of cells which were synthesizing DNA.
No striated myofibrils were seen in the
cells which were in mitosis. This is in accord with the common observation that
cells do not differentiate and divide concurrently but, rather, differentiate after division is complete. Stockdale and Holtzer
('61) stated that skeletal myoblasts and
myotubes with myofibrils did not incorporate thymidine because they were unable
to synthesize DNA and contractile proteins
simultaneously. Similarly, Zhinkin and
Andreeva ('63) suggested that as differentiation proceeds some nuclei synthesize
special proteins while others undergo reproduction. The myocardial cells in the
present study appear to have similar metabolic patterns.
LITERATURE CITED
Bintliff, S., and B. E. Walker 1960 Radioautographic study of skeletal muscle regeneration.
Am. J. Anat., 106: 233-246.
20.0
19.0
20.0
22.0
27.0
"3.3
14.3
-
2 4.0
2 2.4
Cavanaugh, M. W., and D. J. Cavanaugh 1957
Studies on the pharmacology of tissue cultures.
I. The action of quinidine on cultures of dissociated chick embryo heart cells. Arch. Int.
Pharm. et Ther., 110: 43-56.
Defendi, F., and L. A. Manson 1963 Analysis
Of the life-cycle in mammalian cells. Nature,
198: 359-361.
Holtzer, H., J. Abbott and M. W. Cavanaugh
1959 Some
of embryonic
myoblasts. Exp. Cell Res., 16: 595-601.
Marchok, A. 1965 Cell division and nucleic acid
synthesis in cells of the developing chick muscle. Abstracts Fifth Annual Meeting of the
American Society for Cell Biology, No, 122,
p. 63A.
Messier, B., and C . P. Leblond 1960 Cell proliferation and migration as revealed by radioautography after injection of thymidine H3 in
male rats and mice. Am. J. Anat., 106: 247286.
Petersen, R. O., and R. Baserga 1964 Nucleic
acid and protein synthesis in cardiac muscle of
growing and adult mice. Abstracts Fourth Annual Meeting of the American Society for Cell
Biology. No. 145, p. 72A.
Rumery, R. E., and R. J. Blandau 1964 The
cytodifferentiation of myocardial cells from 4day embryonic chick hearts grown in culture.
Acta Anat., 58: 116-130.
Stockdale, F. E., and H. Holtzer 1961 DNA
synthesis and myogenesis. Exp. Cell Res., 24:
508-520.
Walker, B. E. 1962 A radioautographic study
of muscle regeneration in dystrophic mice. Am.
J. Path., 41: 41-54.
Wimber, D. E. 1963 Methods of studying cell
proliferation with emphasis on DNA labels.
Cell Proliferation. Eds. L. F. Lamberton and
R. J. M. Fry, Blackwell Scientific Publications,
Oxford.
Zhinkin, L. N., and L. F. Andreeva 1963 DNA
synthesis and nuclear reproduction during embryonic development and regeneration of muscle tissue. J. Embryol. Exp. Morph., 11: 353367.
PLATE 1
EXPLANATION OF FIGURES
1
Radioautograph of 4-day embryonic chick myocardial cells one hour
after exposure to H3-thymidine in culture. Note labeled interphase
cells and unlabeled mitotic figures.
2 4 Radioautographs of 4-day embryonic chick mycardial cells a t intervals of 2-10 hours after exposure to H?-thymidine in culture. Note
labeled mitotic figures in various stages.
506
5
Radioautograph showing a binucleate myocardial cell in culture with
both nuclei in prophase but unlabeled. Compare with figure 6 .
6
Radioautograph showing a binucleate myocardial cell in culture with
both nuclei in prophase but only one labeled with H3-thymidine.
DNA SYNTHESIS
Ruth E . Ruinery and William 0. Rieke
PLATE 1
507
Документ
Категория
Без категории
Просмотров
0
Размер файла
465 Кб
Теги
synthesis, myocardial, dna, culture, cells
1/--страниц
Пожаловаться на содержимое документа