THE RELATION BETWEEN DIVISION RATE
AND THE RADIOSENSITIVITY OF CELLS
CHARLES PACKARD
(From Columbia University, Imtitute oj Cancer Research, F. C . IVood, Director)
One of the many unsolved problems of radiology is the significance of susceptibility of cells and tissues. Why the germinal
epithelium of the testis should be readily injured by a dose which
leaves the connective tissue unchanged, or why the lymphocytes
are relatively susceptible, while the red cells with their high iron
content are not, remain unanswered questions. Still more difficult to understand is the fact that a cell may change within the
space of a few minutes from a condition of relative resistance to
one of high sensitivity. At present there is no generally accepted hypothesis to account for these phenomena; so little is
known about them that we can only describe the physical and
physiological conditions common to cells which are susceptible
or resistant. Such descriptions are often used as explanations,
as for instance, when the statement is made that certain cells
are sensitive because they are immature. Clearly this does not
throw much light on the subject because we do not know the
pertinent characters of immature cells. A thorough-going
analysis of the conditions common to susceptible cells must be
made before we can form an hypothesis which will correlate
the scattered information now at hand.
One such condition was noticed very early in the study of radiation effects; actively dividing cells were found to be more sensitive than those which are quiescent. The growing root tips of
young seedlings are injured by doses that leave unharmed the
ungerminated seeds or the adult plants. It is not true, however,
that any rapidly growing tissues are more sensitive than any in
which growth is slow. A squamous cell epithelioma grows
rapidly but is resistant, while a slowly growing basal cell tumor is
sensitive. But in general, rapid cell division and high sensitivity
are closely associated.
359
360
CHARLES PACKARD
One method of studying this relation is to test the susceptibility in cells whose rate of cell division can be controlled by temperature. The effect of temperature on the rate of development has
been studied extensively. Ascaris eggs kept at 37" C. divide
about forty times as rapidly as they do at 0" C. These extremes
are not injurious. Drosophila eggs cannot survive so wide a
range; 13" is about the minimum, while 28" is close to the mnximum. At the higher temperature development is more than
five times as fast as at the lower. In the early embryo, mitosis
occurs every fifteen minutes a t 2g0, while a t 13' more than an
hour is required for each division. These differences are sufficiently large for experimental purposes.
That temperature per se does not affect susceptibility to any
marked extent is shown by the experiments of Redfield and
Bright (I) who exposed the unfertilized eggs of the marine worm
Nereis to the beta rays of radium, a t 0 and 24" C. The effect
which they measured was almost the same a t both temperatures,
the temperature coefficient for a rise of 10" C. being 1.14. This
result was to be expected in view of the fact that the radiations
produce a photochemical effect. Since the eggs in this expesiment were unfertilized they show very little metabolic activity;
this factor, which complicates all the tests in which dividing
cells are used, was practically absent.
Some observations on the effect of X-radiations on' cells dividing at different temperatures, that is, at different rates, have already been described. Holthusen (2) exposed Ascaris eggs, in
the one cell stage, at temperatures ranging from 0 to 23" C. and
determined the ratio between the injured and uninjured embryos
which developed. Between 0 and 10" the injuries increased
about 60 per cent; the same proportion held also between 10" and
23" C. Dognon (3) found no change in the sensitivity of these
eggs radiated a t these temperatures, but stated that between 20"
and 33" C. it practically doubles. At still higher temperatures
it increased more and more, but this was undoubtedly due to the
injurious effect of the heat added to that of the radiations.
Both Holthusen and Dognon believe that sensitivity is due to
at least two factors, one which is independent of temperature,
RADIOSENSITIVITY
OF CELLS
361
and one which is dependent on it. The first is a photochemical
effect; the second is in some way connected with the activity of
the cell. Holthusen thinks that it is associated with the condition of the nucleus at the time of division, while Dognon holds
that it is related to cell metabolism.
Politzer (4) has made a study of the number of mitoses occurring in the salamander cornea after radiation at 26" and 10" C.
At the time of exposure the number found in the warm tissue was
about two and one half times as great as that found in the other.
One hour after radiation the number of cells in division in the
first had dropped to less than 50 per cent of the initial number;
in the tissue radiated at 10"C. the decrease was considerably less.
This difference continued through the succeeding hours. In the
first, all division ceased after seven hours; in the second, after
eighteen hours.
There are some observations, however, which seem to show
that susceptibility and the rate of division are not related.
Ancel and Vintemberger (5) radiated frog and hen eggs at low
and high temperatures and then allowed them to develop under
optimum conditions. The degree of injury appeared to be the
same, regardless of the temperature, and hence of the division
rate a t the time of exposure. Similarly, Ancel (6) exposed seedlings a t high and low temperatures and then measured their
growth rate. She was not able to find any constant difference
between the two lots.
It is of interest to test this relation on the eggs of Drosophila
for their rate of development can be varied at will by changing
the temperature at which they are incubated, while their semitivity can be measured quantitatively with considerable accuracy.
EXPERIMENTAL
The eggs of Drosophila are in the early cleavage stage when
used for experiment, and are in active mitosis even at room temperatures. All of the cells in an egg divide at the same moment.
This cannot be seen in the living material because of the thick
shell, but it is obvious in stained sections. Unlike h c a r i s eggs,
the members of a sample of Drosophila eggs are not all in the
362
CHARLES PACKARD
same stage of division a t the same moment. For this reason no
inferences can be drawn as to the most susceptible stage of mitosis. The results obtained represent the average susceptibility
of many eggs dividing at equal rates but not simultaneously.
The freshly laid eggs were radiated at 13", 23O, and 28" C.,
these temperatures being about the minimum, optimum, and
maximum for normal development. I n order to have them a t
the desired temperature during radiation I kept them in a constant temperature chamber for thirty minutes or more previous
to exposure. If the preliminary treatment is shorter than this
the results are irregular.
The chamber consists of a large Thermos bottle partly filled
with water of the desired temperature. I t is covered with a
celluloid cap which transmits the X-rays without appreciable
loss in intensity. The eggs, on small strips of filter paper, wet
with banana juice, are placed in a celluloid float resting directly
on the water. Readings taken before and after the experiment
showed that the temperature remained constant within 0.5 C.
In each experiment the dose was 150 KV. 5 Ma. at 40 cm.
distance. The filtration was 0.25 mm. copper and 1.0 mm.
aluminum. Measurements showed that the intensity of the
beam falling on the eggs was 17.0 r/min, including the scatter
from the water and the walls of the chamber. After 5, 10, 15,
and 20 minutes of radiation, samples of eggs were removed and
incubated in a moist chamber at room temperature. The percentage of eggs hatching is an index of the effect.
The results of these experiments, in which more than 8000 eggs
were radiated, are shown in Table 1. The data of the many
TABLE 1
Temperature during r d i t i o n
Expmure
13 C.
Alive Dead
.
6 min.. . . . . . . .
10 min.. . . . . . . . .
16 min.. . . . . . . .
20 min.. . . . . . . . .
.
23 C.
Con. Alive Dead
28 C.
Con. Alive Dead
Con.
------------
87 85.5 88.1
669 45 93.7 96.6 469 79 85.6 88.2 612
661 288 66.1 68.1 242 209 63.8 55.4 476 626 43.2 44.5
429 664 43.2 44.5 171 380 32.2 33.2 334 1096 23.4 24.1
238 695 25.6 26.3
RADIOSENSITIVITY
OF CELLS
363
separate tests have been condensed, only the totals appearing.
In the figure, the duration of exposure is given on the abscissa,
and the percentage of eggs hatching, on the ordinate. The
differences in the effect of the rays a t these three temperatures
show best after exposures of 10 and 15 minutes. There can be
no doubt that the eggs are less semitive at 13" than at 28".
After a 10 minute exposure in the former temperature, 68 per
cent hatch; in the latter, 47 per cent. This difference is far
greater than any normal variation. The results of radiation at
23" lie between the other two, and in a few crtses, overlap them,
but there is a real difference bet ween them.
The differences in sensitivity can be calculated in this way;
the length of exposure needed to kill 50 per cent of the eggs at 23"
is 10.5 minutes, as shown in the figure. At 13"it is 13.2 minutes,
and a t 28" it is 9 minutes. Taking the sensitivity at 23" as 100
(since the measurements in all previous experiments have been
364
CHARLES PACKARD
made at about this temperature) the susceptibility a t 18" is 80
per cent of the standard, and a t 28" it is 117 per cent.
A comparison between the sensitivity at these temperatures
and the division rate shows that the two are by no means parallel.
At the upper temperature mitosis takes place about five times as
rapidly as at the lower, while the sensitivity is less than 40 per
cent greater. This is somewhat less than the figure given by
Holthusen in his experiments on Ascaris eggs.
The foregoing experiments show that the rate of cell division
at the time of exposure to X-rays is correlated with the sensitivity of the eggs. In the second series of tests it will be shown
that the rate of cell division after exposure is also a factor in
determining susceptibility.
Some observations on this point have already been described.
Ancel and Vintemberger (7) radiated chick embryos in ovo and
then placed some at a low temperature, and others in an incubator at a high temperature. The former did not show any increase in injury after some days, while the latter did. The
authors believe that the actual amount of injury was the same in
both lots, but that in the cold it did not develop; that is, the
latent period was greatly lengthened. At the high temperature
when cell division was stimulated, this period was short.
Strangeways and Fell (8) have thrown additional light on the
problem. They also radiated chick embryos and then kept some
at 0" C. for five hours (this treatment is not injurious), and others
in an incubator for a similar time. They then prepared tissue
cultures from each. Those made from the chilled embryos
grew fairly well; the others grew very little or none a t all.
Their conclusion is that if the division rate of the radiated cells
is kept at a low level after exposure the injury not only does not
develop, as Ancel and Vintemberger believe, but actually tends
to disappear. Strangeways and Fell are of the opinion that the
sensitivity is lowered in the cold because the metabolic rate is
lowered.
Similar experiments with Drosophila eggs were made in this
way. The eggs were radiated at room temperature and immediately afterward divided into two lots, one of which was
RADIOSENSITIVITY
OF CELLS
365
kept at 18" and the other at 28" C. until hatching. I made a
number of tests in which the eggs were kept at 13O, but they were
unsuccessful; the death rate was considerably higher than that
of eggs incubated at 23", probably because this low temperature
is so close to the lower limit of tolerance that some eggs, already
injured by radiation, cannot survive the added effect of the cold.
But a temperature of 18" is definitely beneficial, as may be seen
by the results given in Table 2 and in Fig. 2. After the cold
treatment, half of the eggs are found to be killed by an exposure
of 17.5 minutes, while after the warm treatment, the time is 14.1
minutes. The effect on eggs incubated at 23" is half way between these values. By the method of calculation already mentioned we find that the sensitivity of the chilled eggs is 89 per
cent of that of eggs reared at room temperature; the sensitivity
of the warmed eggs is 110 per cent. While this difference, which
is about 21 per cent, is not large, it far exceeds the limits of
ordinary variation.
CHARLES PACKARD
TABLE
2
Temperature after radiation
18 C.
Expcaure
28
C.
.Ee
,E,
a&
-----------Alive
6 min.. . . . . . . . .
9 min.. . . . . . . . .
l l m i n. . . . . . . . . .
14mi n . . . . . . . . . .
16 i n . .. . . . . . . .
23 C.
Dead
Con. Alive Dead
294 24 92.4 95.3
632 110 85.2 87.8 156 34 82.1
250 63 80.0 82.5 146 46 76.0
1198 041 65.1 67.1 615 436 68.5
733 685 51.7 53.3 208 241 46.3
Corr. Alive Dead
Corr.
250 25 90.9
84.6 927 360 72.0
78.3
60.2 787 678 53.5
47.7 291 417 41.2
93.7
74.2
55.1
42.1
The results of separate tests with the chilled eggs varied more
from the average than was expected, but this was probably due
to the fact that in some cases the moist chamber in which the
eggs were incubated was cooled before they were put into it,
while at other times it was not. With the first method, any cells
which may have been ready to begin mitosis again after exposure
were immediately prevented from so doing, a result which appears to be beneficial, for a larger proportion hatched than when
the other method was followed.
DISCUSSION
One of the hypotheses to account for susceptibility is based on
the fact that cells are especially sensitive at a definite period of
their division cycle. If then they are in active division during
exposure they will pass through this phase more than once and
will therefore be more severely injured than they would be if
their division rate was slow. That there is a decided variation
in susceptibility during mitosis is true, although at present
there is no agreement as to when the critical stage occurs.
Strangeways and Hopwood (9) state that it is just before mitosis
begins; Regaud (10) believes that there are two maxima of sensitivity, one during the prophase and the other during the anaphase. The commonly accepted view has been that the metaphase is the time of least resistance. Finally, Vintemberger (11)
has shown that sensitivity increases steadily from the beginning
of division up to the teIophase, after which it falls abruptly.
RADIOSENSITIVITY
OF CELLS
367
The only fact that emerges from this confusion is that cells do
vary in susceptibility to radiations as they pass through their
mitotic cycle.
But the assumption that actively dividing cells are severely
injured because they pass through this critical stage more than
once during a long exposure is certainly incorrect. As a matter
of fact, very few cells begin to divide, once radiation has commenced. This has been shown by Canti and Spear (12) who
exposed chick tissue cultures to gamma rays of radium. Two
and one half minutes of radiation is long enough to check the
onset of division for some time, while thirty minutes greatly
reduces the number of dividing cells. From the latter dose there
is a partial recovery, a few cells beginning to divide soon after
the radium is removed.
Cells actually in mitosis when the exposure begins complete
the process. Regaud, Lacassagne and Juvin (13) examined
chick embryos fixed at frequent intervals, beginning directly
after the end of a heavy dose which was sufficient to kill the
embryos a t the end of three days. At the close of radiation
mitoses were numerous, some being abnormal; ten to fifteen
minutes later, division stages had almost disappeared. There
were no prophases or telophases to be seen, a fact which indicates
that the cells which were diGding fifteen minutes before had
completed their division, while no cells had commenced to divide.
Some intermediate stages were abnormal. Several hours after
the end of the exposure, mitosis began again in some cells, but
was abnormal. Differences in sensitivity among cells dividing
at different rates must therefore be due to their condition at the
moment that radiation begins.
The rapidity with which cell division occurs in normal tissue
at the time of radiation determines the length of time elapsing
between exposure and death. Actively dividing cells quickly
succumb, that is, the latent period is short, while in slowly
growing or quiescent tissues the visible injury develops slowly
if a t all. Politzer observed that cells exposed at the higher
temperature, when mitosis was rapid, died seven hours later,
while the slowly dividing cells lived for eighteen hours.!
368
CHARLES PACKARD
After the end of an exposure many cells which have been
abruptly stopped from beginning to divide, become active again.
When the radiation has been brief, the decrease in the number of
dividing cells is followed by a great increase after which the
normal rhythm is resumed. (12) But even in tissues which are
so severely injured that they finally die, some cells make an
attempt at division. (13) When such activity is encouraged
by high temperature, as in the second series of experiments,
the effect is definitely injurious. On the other hand, if by cold
cells are prevented from dividing, they have a chance to recover.
Apparently the reparative processes are favored by low temperatures, that is, they proceed more readily when cell division is
checked. Loeb and Drake (14) observed the same phenomenon
when they placed on ice some cells which previously had been
heated above their maximum temperature. They recovered
more completely than when kept at room temperature.
This behavior places a difficulty in the way of accepting
Crowther's theory of the action of radiations. (15) He assumes
that each kind of cell must be hit in a particular, sensitive spot
by a definite number of X-ray quanta before death can occur.
If the quanta are large, that is, produced at high voltages, a
smaller number of hits will be required than if they are small.
Thus Condon and Terrill (16) ialculate that if one quantum
produced a t 190 KV. will kill a Drosophila egg, then two or three
will be needed if they are produced at lower voltages. But in
view of the results presented in this paper, it would appear that
the number of quantum hits needed to produce death must vary
with the activity of the egg after the close of radiation. This
situation makes the acceptance of the theory in its present form
very difficult.
The various kinds of cells and tissues which have been mentioned,--Drosophila eggs, amoebocytes of Limulus, salamander
cornea, chick tissue cultures,--all respond to changes in temperature; their division rate can be modified at will. And it is in
these cells that the results of altering the division rate before and
after radiation can most easily be studied. Since these changes
in activity cannot be produced in human patients or in warm
RADIOSENSITIVITYOF CELLS
369
blooded animals generally, we cannot experimentally test on
them the results discussed in this paper. But undoubtedly they
are of general significance. The division rate at the time of
radiation is a real factor, though not a large one, in determining
sensitivity to radiations, and the activity of the cells after exposure is another factor.
SUMMARY
The influence of division rate on the sensitivity of Drosophila
eggs to radiations has been determined quantitatively. Susceptibility rises as the rate of division at the time of exposure increases, but the two are not parallel.
The activity of cells after exposure is a second factor in determining the amount of injury produced. The effect is lessened
when the activity is decreased.
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