1975, British Journal of Radiology, 48, 200-208
Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma By Lore v. Szczepanski and K.-R. Trott Abteilung Strahlenbiologie und Biophysik, Institutfur Biologie der Gesellschaftfur Strahlen- und Umweltforschung,8042 Neuherberg, West Germany (Received January, 1974) ABSTRACT
The proliferation kinetics of the transplanted adenocarcinoma 284 of C3H mice from the first to the sixth day after 600 R and from the first to the eighth day after 1,200 R has been studied by repeated labelling of the tumour cells in vivo with 3H-thymidine and measurement of the labelling index and the percentage of labelled mi to tic cells. The time course of the post-irradiation synchronization was followed during the first day. Later, the mean generation times of the tumour cells were usually prolonged and the spread of the generation times increased. Three to fcur days after irradiation, the tumour increased its growth fraction to twice the normal value by triggering resting cells (Go cells) into cycle.
The development of optimal dose-fractionation schedules in radiotherapy requires knowledge of the behaviour of the tumour and normal tissues during the fractionation intervals. In addition to recovery from sublethal radiation damage and reoxygenation, which have already been studied extensively, cell division and recycling of resting cells might be important factors determining the optimal interval between the individual dose fractions. Therefore, we investigated the proliferation pattern of a mouse mammary carcinoma during the first week after single exposures of 600 R and 1,200 R in a study parallel to tumour cure experiments on the optimal spacing of fractions of 600 R and 1,200 R in the same tumour (to be published elsewhere). By that means we hope to find out about the role each of the above-mentioned factors might play in the establishment of optimal fractionation schemes. MATERIAL AND METHODS
The experiments were performed on the murine adenocarcinoma 284 growing in vivo. Female C3H mice were 10-12 weeks old when they entered the experiment. They were fed on Altromin pellets and provided with water ad libitum before and during the experiment. The tumour was serially transplanted by subcutaneous implantation of 2 mm pieces into the right scapular region. It grew to a diameter of 8-10 mm within 12-14 days. The animals were anaesthetized with hexobarbital before irradiation. Fifteen animals were irradiated at a time in a jig with the body shielded by 5-2 mm lead and only the tumour exposed to 300 kV X
rays, filtered by 0-6 mm Cu and 1 -0 mm Al. The exposure rate, at a distance of 51-5 cm was 60 R/minute. The cell-cycle parameters were studied by counting the ratio of labelled to total mitoses at various times after a single injection of 3H-thymidine ( 3 H-TdR). Percent labelled mitoses (PLM) versus time curves were established in unirradiated tumours as well as in tumours which at various times before injection had been irradiated with single exposures of 600 R and 1,200 R. The tumour-bearing animals received 30 /uCi 3H-TdR i.p. (specific activity 2 Ci/mM) at various intervals after irradiation to study the progression of the labelled cells into subsequent mitoses. Every 30 minutes during the first 12 hours and every hour up to 36 hours after labelling, two animals from each group were sacrificed; the tumours were removed and fixed in 4 per cent formalin. They were embedded in paraffin wax and 5ft sections were prepared for autoradiography by dipping in Kodak NTB 2 emulsion. After two weeks' exposure at 8=C the sections were developed and stained with hemalum-eosin. A hundred mitoses were counted in each tumour to define the percentage of labelled mitoses. In some slides, the mitotic index was established by counting 3,000 cells. To establish the fraction of proliferating cells in the tumour at various times after irradiation tumours were repeatedly labelled from three hours to six days after 600 R and from three hours to eight days after 1,200 R. 30 /xCi 3H-TdR was injected i.p. into the animals at the respective times one, twice, three or four times at two-hourly intervals. An hour later two animals from each group were sacrificed and the tumours removed. In the autoradiographs 3,600 cells were analysed in each of the tumours to calculate the labelling index. The experimental points in the figures give the mean value derived from two tumours. RESULTS
The tumour grows rapidly and reaches a diameter of about 1 cm 14 days after transplantation. The volume doubling time of tumours of this size was
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1975
Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma
3-0 days. After irradiation with 600 R and 1,200 R, the tumours regressed rapidly and after 48 hours their volumes were only 15-20 per cent of their preirradiation sizes. These did not change considerably until four to six days after irradiation. Regrowth started six days after 600 R and was not measurable before day 8 after 1,200 R. In earlier passages, the tumour had a very regular structure with chords and lobules surrounded by connective tissue (Fig. 1). Only the marginal zone of one or two cell layers in the lobules were labelled after a single i.p. injection of 3 H-TdR (Hug and
FIG. 1. Adenocarcinoma of C3H-mice. Autoradiograph of the well differentiated part of the untreated tumour after a single i.p. injection of 3 H-TdR. One hour after application the labelled cells are seen only in the marginal zones of the tumour lobules (Szczepanski e£ at. 1971). > 800
Szczepanski, 1969; Szczepanski, Hug and Spangenberg 1971 for detailed description of the tumour histology). The cells in the central part of the lobules were not proliferating and also were morphologically different from the cells of the proliferating zone. With increasing size of the lobules necrotic foci appeared in their centres. This regular structure has not been observed so clearly in more recent passages but its general features are still preserved. If the interval between labelling and killing of the animal is prolonged, the migration of labelled cells through the region of resting cells into necrotic regions takes place. Three days after labelling some dying cells can already be recognized in the centre of the lobules whereas after four days most labelled cells are found in the necrotic region (Fig. 2). The proliferation parameters of the untreated tumour taken from the per cent labelled mitosis (PLM) curve (Fig. 3) are listed in Table I. These
HOURS AFTER TdR
FIG. 3. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into unirradiated animals. ( • experimental points, • A computed curve). ( curve fitted by eye).
TABLE I PROLIFERATION PARAMETERS OF THE UNTREATED C3H ADENOCARCINOMA
Doubling time of tumour volume Labelling index (all histologically "normal" tumour cells included)
f 2. Adenocarcinoma of C3H-mice. Autoradiograph of the well differentiated part of the untreated tumour after a single i.p. injection of 3 H-TdR. Four days later most labelled cells are found in the centralnecrotic regions of the lobules (Szczepanski et al., 1971). x800. FIG.
201
3-0 days 12-5%
Mitotic index
0-37%
Median duration of the Gi phase
4-7 hours
Median duration of the S phase
6-8 hours
Median duration of the G2 phase
1 -9 hours
Median cell cycle time
13-6 hours
Growth fraction
*••> / o
Cell loss factor
0-5
VOL. 48, No. 567
Lore v. Szczepanski and K.-R. Trott were derived from the curve calculated by the computer program of Steel and Hanes (1971). This curve fits the experimental points in the untreated tumour very well. For irradiated tumours, the fit of the computer PLM curve to the experimental data
HOURS AFTER TdR 3 H INJECTION
FIG. 7. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals four days after 600 R. (# experimental points, A- — —A computed curve.) HOURS AFTER TdR- 3 H
INJECTION
4. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals a day after 600 R. (•experimental points, A A computed curve). FIG.
HOURS AFTER TdR- 3 H
INJECTION
8. The percentage of mitoses that times after injection of 3 H-TdR after 1,200 ( • experimental points, A FIG.
HOURS AFTER T d R ^ H
INJECTION
FIG. 5. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals two days after 600 R. ( • experimental points, A A computed curve.)
HOURS AFTER TdR- 3 H
are labelled at various into animals four days R. A computed curve.)
HOURS AFTER TdR
INJECTION
FIG. 6. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals three days after 600 R. ( • experimental points, A -A computed curve.)
FIG. 9. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals six days after 1,200 R. ( • experimental points, AA computed curve.)
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Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma is good in some cases (Figs. 6, 7, 9 and 10). However, in others the fit of the computer curve to the experimental points is poorer (Figs. 4 and 8). In the PLM curve obtained following injection of the label four days after 1,200 R (Fig. 8), the experimental data suggest a double peak 15 hours and 23 hours after labelling rather than the second peak calculated by the computer 17 hours after labelling. In Fig. 4, the experimental points in the trough between the peaks indicate a broad plateau lasting for nearly ten hours. The generation time was prolonged at all the times of observation after 600 R and also eight days after 1,200 R. A slight shortening was seen only four and six days after 1,200 R. In addition to the lengthening of the median generation time, we also found an increased spread in generation times in all cases. In Table II the computed cell cycle parameters at various times after irradiation are listed together with the data taken from the curves fitted by eye.
Summarizing the data in Table II, minor changes in the cell-cycle times could be found within the first week after irradiation. The length of the DNA synthetic period is remarkably constant but there is a tendency for this to become shorter after irradiation. Also, the G2-period is very constant, but was observed to double 24 hours after 600 R. The Giphase showed the greatest variations, partly due to scatter in the later experimental points. Therefore, these values have to be taken with reservation. There were considerable fluctuations in the mitotic index after irradiation (Fig. 11). Twelve hours after irradiation it was essentially zero after 1,200 R and 0-1 per cent after 600 R. It then increased for three days after 600 R when it had reached the control level again. The following day, the mitotic index was about twice the control value, to which it decreased again six to eight days after irradiation. The post-irradiation overshoot in mitotic index after 1,200 R was observed one to three days later than after 600 R.
8
sts
8d after 1200 R Oill
I --y 1 v
60
s -" 40
;
HOURS AFTER TdR^H
INJECTION
FIG. 10. The percentage of mitoses that are labelled at various 3 times after injection of H-TdR into animals eight days after 1,200 R. ( • experimental points, A.- - -A. computed curve.)
TIME (DAYS)
11. Mitotic index at various times after radiation doses of 600 R and 1,200 R. FIG.
TABLE II THE CELL PROLIFERATION PARAMETERS (IN HOURS), AFTER IRRADIATION
Ts
TGI
Time after irradiation Control 1 dav after 600 R 2 davs after 600 R 3 days after 600 R 4 days after 600 R 4 days after 1,200 R 6 days after 1,200 R 8 davs after 1,200 R
PLM curve fig3 4 5 6 7 8 9 10
Tc
TG2
Computer
Fitted by eye
Computer
Fitted by eye
Computer
Fitted by eye
Computer
Fitted by eye
4-7 6-8 6-4 6-5 8-2
4-3 9-2 7-5 6-5 8-1
6-8 6-5 6-5 64 6-3
7-0 6-7 6-8 6-4 6-2
1-9 3-5 2-4 1-9 1-7
2-0 3-4 2-2 2-0 1-8
13-6 17-6 160 15-2 16-9
13-3 19-3 16-5 151 161
20 19 23 31 36
2-1 3-7 10-2
3-7 4-3 10-0
6-3 51 6-3
6-5 5-5 6-8
2-1 2-4 2-3
2-0 2-2 2-2
121 11-8 19-8
12-2 12-0 190
28 26 38
C.V.* (%)
*Coefficient of variation in the distribution of the generation times calculated by the computer program of Steel and Hanes (1971). 203
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1200 R
60
TdR^H
INJECTIONS
TdR-^
12. Labelling index after one, two, three, or four injections of tritiated thymidine (interval two hours) at various times after radiation doses of 600 R. FIG.
INJECTIONS
13. Labelling index after one, two, three, or four injections of tritiated thymidine (interval two hours) at various times after radiation doses of 1,200 R. FIG.
TABLE III
TABLE IV
CELLS IN S PHASE AND FLUXES INTO AND OUT OF S PHASE AT VARIOUS TIMES AFTER IRRADIATION WITH 600 R
CELLS IN S PHASE AND FLUXES INTO S PHASE AT VARIOUS TIMES AFTER IRRADIATION WITH 1,200 R
Time after 600 R (h)
Control 3 6 12 24 48 72 96 144
CellsO ' in S o
12 11 2-5 1-5 8 13 23 23 17
Inflow (% Per h) 5-0 0-3 0-5 0-8 2-8 3-2 4-2 4-2 3-7
Time after 1,200 R
Outflow* (% Per h)
(h)
5-0 3-0 0-2
Control 3 6 12 24 48 72 96 144 192
Cells in S phase °o
12 2 1 1 6 10 16 23 25 16
Inflow into S (° o per h) 5-0 0-2 0-4 0-8 2-3 2-3 3-7 3-7 3-2 2-5
*As explained in the text, outflow could be calculated during the first six hours only.
The labelling index (LI) after repeated injections of 3 H-TdR for six hours with the first injection given at various times after 600 R is shown in Fig. 12. In the unirradiated tumour, the labelling index rapidly increased from 12-5 per cent to 42 per cent. When the first injection was given three hours after 600 R, the curve was essentially horizontal at 11 per cent. When the first injection was given six and twelve hours after 600 R, a slight rise in the labelling index was seen, but the values were all at or below 5 per cent. When the repeated labelling was started 24 hours after 600 R the labelling index rose from 8 per cent to 25 per cent and 48 hours after 600 R
from 14 per cent to 33 per cent. Seventy-two hours and ninety-six hours after 600 R the starting point at 23 per cent was well above the control value, rising to nearly 50 per cent within six hours. Six days after 600 R the curve was similar to the control curve rising from 16 to 38 per cent. From the slope of these curves the inflow of cells into S-phase can be calculated. At early times, when the individual curves overlap, the flow of cells out of S-phase can be calculated from the difference between the inflow of cells into S-phase and the simultaneous change in the labelling index (LI) after pulse labelling. These data are presented in Table III.
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Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma The analogous curves and data after 1,200 R are shown in Fig. 13 and Table IV. In contrast to the curves after 600 R, there was a very low level of labelling index already three hours after 1,200 R which did not change after repeated injections. Twenty-four plus four hours after 1,200 R there seems to be a rather sudden increase in the labelling index from 7 to 20 per cent within two hours. The other curves are similar to those after 600 R with the only exception that they are delayed by about one day. Taking all data presented in Figs. 3-13 and Tables II-IV together, the proliferation behaviour of the cells of the tumour after irradiation during the following six days can be described as follows: The exposure of 600 R induced a block at the end of the G2-phase preventing the proliferating cells from entering mitosis and the Gi-phase. Accordingly the mitotic index fell very rapidly to very low values. However, as long as there were still cells in the Giphase the inflow into the S-phase and the outflow into the G2-phase took place as before. About three hours after irradiation, the Gi-pool was exhausted and no more cells entered the S-phase whereas the progression of cells from S-phase into the G2-phase proceeded at nearly normal speed until the S-pool also was nearly empty. Twelve hours after 600 R over 80 per cent of all proliferating cells accumulated in the G2-phase. The release from this block occurred between 12 and 24 hours after 600 R. During this time of blocked proliferation, the volume of the tumour decreased rapidly. During the mitotic delay period a great deal of cell death was observed in the marginal zones of the tumour lobules. The first wave of DNA synthesis occurred about 24 hours after 600 R. Forty-eight hours after irradiation, the proliferation seemed to be nearly normal again—only the cell cycle time was prolonged by three hours. Three days after irradiation, a sudden increase in the growth fraction occurred, which after 600 R amounted to nearly a doubling of the pre-irradiation growth fraction and which lasted for two days. Six days after 600 R the pre-irradiation proliferation pattern was attained again, whereas the tumour was still half of its pre-irradiation size. Essentially similar interpretation can be given to the data after 1,200 R. Differences concern mainly the appearance of a block of DNA-synthesis in cells in S-phase and a block at the Gi/S border which lead to a rapid decrease of the labelling index within three hours to 2 per cent. The increase in growth fraction is delayed about 1 day and lasts about three days. Eight days after 1,200 R the growth fraction
is nearly normal again, but the median generation time is still six hours longer than before irradiation.
DISCUSSION
In the transplantable adeno-carcinoma of the C3H mouse profound changes in the proliferation pattern took place after irradiation. After a period of synchronization lasting for about 24-48 hours and a period of nearly normal proliferation a transient but extensive increase in the growth fraction was observed. Mammary carcinomas of the C3H mouse are a very commonly-used experimental system for the study of the effects of irradiation. The kinetic parameters taken from the PLM curve and tabulated in Table I compare well with those published by others {e.g. Denekamp, 1970; Begg, 1971). The tumour has a rather low growth fraction of only 25 per cent as a result of its well-organized structure. Proliferation takes place in a small rim near the connective tissue which separates the individual lobules. Proliferation ceases at distances where, according to calculations by Thomlinson and Gray (1956) and by Tannock (1972) enough oxygen, to allow normal proliferation, should still be available. There is a continuous migration of cells from the proliferation zone near the blood vessels through the compartment of resting cells with gradually decreasing oxygen concentration into regions of obvious necrosis. The turn-over time of the non-proliferative compartment in the unirradiated tumour seems to be short, since two to three days after pulse labelling, labelled cells can already be found in the necrotic region. A more indirect approach to quantitative evaluation of cell flow can be made by comparing the curves of increasing labelling index after repeated 3H-TdR-injections before irradiation and after one week following 600 R when proliferation may be supposed to be normal again. In the unirradiated tumour the labelling index increases from 13 to 43 per cent within six hours, and in the regrowing tumour from 17 to 38 per cent within the same period of time. This rise in the labelling index is due to: (1) the inflow of unlabelled cells into S-phase; (2) mitotic division of labelled cells; (3) elimination of unlabelled cells from the tumour. The steeper rise of the LI in controls compared to the regrowing irradiated tumours is mostly due to loss of unlabelled cells from the tumour at a rate of more than 1 per cent/hour, whereas it can be assumed that in the regrowing tumour no preferential loss of unlabelled cells occurs.
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The earliest effect of irradiation was synchronization of the proliferating cells of the tumour. After 600 R, the main effect was a G2-block lasting about a generation time which is in good agreement with data from cells in culture (Sinclair, 1968). After higher doses, additional effects on DNA synthesizing cells and cells entering the DNA synthesis phase were observed. Similar effects have been described in vitro, also (e.g. Mak and Till, 1963). During the period of mitotic delay the tumour shrank at a rate considerably greater than that caused by normal cell loss. This fast regression implies that it takes place mainly in the non-proliferating compartment. Typical radiation-induced cell death appears already during the period of mitotic delay but is maximal between 12 and 24 hours. Obviously, death of cells trapped in mitosis is not the only way irradiated tumour cells die, contrary to observations in cell and tissue cultures (Lasnitzki, 1940; Hurwitz and Tolmach, 1969). From the second day after irradiation onwards, the tumour was much smaller than before irradiation but consisted mainly of doomed cells. The growth fraction did not differ appreciably from that in the unirradiated tumour. Therefore, the post-irradiation regression of tumour mass affected the proliferative and non-proliferative compartments at about equal rates. The prolongation of the generation times two to four days after 600 R was mainly due to an extension of the Gi period. (Table II, Figs. 5-7). The very flat trough between the two peaks in Fig. 4 looks to us rather like a synchronization effect where the bulk of the proliferating, partly synchronized cells was still in pre-synthetic stages of the cell cycle at the start of the PLM experiment. In no instance other than four and six days after 1,200 R did we find a shortening of the generation time; in fact, it usually was prolonged by two to six hours. In addition to this, the coefficient of variation of the generation time distribution was increased from 19 per cent to greater than 30 per cent. On the fourth to fifth day after 600 R and the fifth to seventh days after 1,200 R, a marked increase in the growth fraction was recorded. Since the duration of the S-phase in the PLM curves did not change much and the labelling index after continuous labelling increased steadily at normal rates, the labelling index was proportional to the growth fraction. A shortening of the cell cycle (with normal S-phase duration) to such a degree as to account for the increase in the labelling index is ruled out by the PLM curves and the continuous labelling curves which show an inflow from proliferating but not
labelled cells. Disappearance of the Gi-period, which has been suggested by Hermens and Barendsen (1969) to explain their findings, did not occur in our tumour. The data presented above can only be interpreted as an indication of a sudden increase in growth fraction three days after 600 R and four days after 1,200 R up to nearly twice the normal value, while cells in cycle proliferate at normal or slightly slower speed. This effect was transitory and finished about six days after 600 R and eight days after 1,200 R. To explain the mechanisms for this increase in growth fraction there are several theoretical possibilities most of which can be ruled out by our observations : (1) A sudden loss of 60 per cent of cells in the nongrowing fraction three to four days after irradiation is very unlikely and would have been easily detected in our measurements of the tumour weights. (2) The fact remains that the absolute number of proliferating cells doubled within one day. These cells might have come, either from the growth fraction, or from the non-growth fraction. If all cells in the growth fraction divided once and no cell left the proliferative compartment, a doubling of the absolute number of proliferating cells ought to be found. However, such a sudden change in the post-mitotic pathway for one day only at the third post-irradiation division seems very unlikely. (3) On the other hand, we have direct evidence concerning the origin of increase in growth fraction shown by the PLM curve starting four days after 1,200 R (Fig. 8). Whereas the computer tried to fit the data to include a normal second peak, the experimental data suggest two peaks or rather a dip in the second peak. The depression in the percentage of labelled mitoses 16-21 hours after labelling is due to a sudden increase in the absolute number of unlabelled mitoses. The mitotic index, which was 0-33 per cent 14 hours after labelling, increased to 0-66 per cent 18 hours after labelling. Obviously four days after 1,200 R there were at least two populations in the tumours one of which was partially synchronized. The most satisfactory explanation for the dip in the second peak of the PLM curve of Fig. 8 is the assumption that some resting cells had been triggered into cycle in a parasynchronous way which divided first five days after 1,200 R. This is consistent with the overshoot of the mitotic index five days after 1,200 R.
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Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma Taking all the evidence together, we conclude that, depending on the dose of radiation three or four days after irradiation, resting cells are triggered into cycle to increase the growth fraction for some days. The nature of this trigger remains obscure. It is unlikely that it is the oxygen concentration, since the oxygen tension should be rather high at the border of the non-proliferative compartment, already. If it is the reduction in tumour volume or cell number, one would have to explain the fact that the higher dose which leads to more cell destruction takes longer to trigger resting cells. There probably exists some dose-dependent proliferative delay for resting tumour cells forced to recycle. Such a presynthetic delay has been observed in a number of normal cells where resting cells can be triggered into cycle, as for example in liver (Kelly et al., 1957) or in salivary gland (Winter, 1972). In the C3H adenocarcinoma, however, the irradiation is the trigger as well as the delaying factor. The post-irradiation proliferation kinetics of tumours have been studied by several authors, but usually at one arbitrary time point and by means of the PLM curve only. Denekamp and Thomlinson (1971) discussed the problems encountered in the determination of the cell-cycle parameters shortly after irradiation by the PLM method. Since they concluded that only after all doomed cells had been removed this technique revealed reliable results, they started their kinetic analysis only after regrowth had started {e.g. 14 days after 1,500 rads to a C3H mammary carcinoma). They did not find any conspicuous changes in the generation times nor in the growth fraction. Whereas Denekamp and Thomlinson could be certain of studying surviving cells only, most cells in our studies, especially during the first few days, were doomed to die sooner or later from their radiation damage. However, it has been shown in cell culture that the proliferation kinetics of surviving and inactivated cells after irradiation are not significantly different (Elkind, Sutton and Moses, 1961). Brown and Berry (1968) and Brown (1970) studied the PLM curves of induced squamous cell carcinomas in the cheek pouch of Syrian hamsters 24 hours after 500 R and 72 hours after 1,000 R. They did not observe big changes in the growth fraction in the first five days after 500 R, but a slight prolongation of the generation times which was mostly due to an increase in the duration of the Gi-phase. Frindel, Vassort and Tubiana (1970) demonstrated marked changes in the generation times of the XCTC 2472 tumour, in its ascitic form, 20 hours
and 48 hours after exposure to 250 R, resulting from lengthening of the Gi and S phases of the cells. No such changes were seen in the solid form 48 hours after 600 R (Tubiana, Frindel and Malaise, 1968). In contrast to these papers and our data, which did not demonstrate a speeding up of the cell cycle but rather a slowing down after irradiation, Hermens and Barendsen (1969) and Van Peperzeel (1970) recorded a shortening of the generation times in irradiated tumours. Van Peperzeel studied the labelling index in a very fast growing adenocarcinoma of DBA mice at various times after a single dose of 210 rads. At the peak of the post-irradiation synchronization wave of the labelling index (LI = 75 per cent) she injected 3 H-TdR and analyzed the progression of these cells through the subsequent mitoses by the PLM method. The analysis of the PLM curve revealed a shortening of the cell cycle from 14*5 hours to 11 hours and an increase in the growth fraction from 87 to 100 per cent. Hermens and Barendsen (1969) studied the postirradiation proliferation kinetics in a rat rhabdomyosarcoma four days after exposure to 2,000 R when the tumour had reached its minimal volume and the number of clonogenic cells was just about to rise again. They observed the most dramatic change in generation time which anybody has measured so far. While the growth fraction decreased from 30 to 24 per cent, the mean cycle time was shortened from 28 hours to 11 hours in the tumour periphery. This was due to complete disappearance of the Gi-phase of the cycle. Also, during the period of regrowth the growth fraction was usually smaller than in unirradiated tumours. Only eight days after irradiation Hermens (1973) found a rise in the "fraction of cycling cells" above control levels in the tumour centre. During the regrowth period, irradiated human lung metastases tend to grow appreciably faster for some time than before irradiation (Van Peperzeel, 1972; Malaise et al., 1972). In animal experiments it has been shown that this can be due to changes either in the growth fraction or the generation times or in the cell loss factor. In the C3H mouse adenocarcinoma, irradiation leads to an increase in the growth fraction by triggering resting cells, which might be called Go cells, into cycle. Not much is known about the radiosensitivity of Go cells in vitro or in vivo. Several authors have supposed that they might be considerably more radioresistant than cells in cycle (Hug and Szczepanski, 1969; Tubiana, 1971; Reinhold, 1973). This being so, the triggering of Go cells into the cycle by
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radiation may be one mechanism rendering fractionated radiotherapy more effective than single large doses of irradiation. For the tailoring of optimal fractionation schedules for various human tumours, besides knowledge of the time courses of cell synchronization, reoxygeneration and cell multiplication after each dose, the time when triggering of resting cells into cycle occurs should also be taken into account. ACKNOWLEDGMENTS
IAEA symposium on Radiation Induced Carcinogenesis, Athens, pp. 85-95 (IAEA, Vienna). HURWITZ, C , and TOLMACH, L. J., 1969. Time-lapse cine-
micrographic studies of X-irradiated HeLa S-3 cells. I. Cell progression and cell disintegration. Biophysical Journal, 9,607-633. KELLY, L. S., HIRSCH, J. D., BEACH, G., and PALMER, W.,
1957, The time function of 32P incorporation into DNA of regenerating liver; the effect of irradiation. Cancer Research, 17, 117-121. LASNITZKI, I., 1940. The effect of X-rays on cells cultivated in vitro. British Journal of Radiology, 13, 279-283. MAK, S., and TILL, J. E., 1963. The effects of X-rays on the progression of L-cells through the cell cycle. Radiation Research, 20, 600-618.
We are grateful to Professor O. Hug for his advice, MALAISE, E. P., CHARBIT, A., CHAVAUDRA, N., COMBES, interest, and stimulation throughout this project. We are P. F., DOUCHEZ, J., and TUISIANA, M., 1972. Change in greatly indebted to Dr. G. G. Steel, Sutton, for the comvolume of irradiated human metastases. Investigation of puter analysis of all PLM data. We thank Miss I. Fettinger repair of sublethal damage and tumour repopulation. and Mrs. R. Gessner for their technical assistance. British Journal of Cancer, 26, 43-52. REINHOLD, H., 1973. Radiosensitivity of capillary endothelium. XIII Congreso Internacional de Radiologia, Madrid. REFERENCES BEGG, A. C , 1971. Kinetic and histological changes of a SINCLAIR, W. K., 1968. Cyclic X-ray responses in mammalian cells in vitro. Radiation Research, 33, 620—643. serially transplanted mouse tumour. Cell and Tissue Kinetics, 4, A0X-4W. STEEL, G. G., and HANES, S., 1971. The technique of labelled mitoses: Analysis by automatic curve-fitting. Cell and BROWN, J. M., 1970. The effect of acute X-irradiation on Tissue Kinetics, 4, 93-105. the cell proliferation kinetics of induced carcinomas and their normal counterpart. Radiation Research, 43, 627- SZCZEPANSKI, L. V., HUG, O., and SPANGENBERG, G., 1971. 653. Fraktionierungsstudien an transplantablen Tiertumoren unter zellkinetischen Aspekten. In: Prdoperative TumorBROWN, J. M., and BERRY, R. J., 1968. Effects of X-irradiabestrahlung, ed. O. Hug (Munich: Urban and Schwartion on cell proliferation in normal epithelium and in zenberg). tumours of the hamster cheek pouch. In: Effects of Radiation on Cellular Proliferation and Differentiation, pp. 475— TANNOCK, I. F., 1972. Oxygen diffusion and the distribution 491 (IAEA, Vienna). of cellular radiosensitivity in tumours. British Journal of Radiology, 45, 515-524." DENEKAMP, J., 1970. The cellular proliferation kinetics of THOMLINSON, R. H., and GRAY, L. H., 1956. The hisanimal tumours. Cancer Research, 30, 393-400. tological structure of some human lung cancers and the DENEKAMP, J., and THOMLINSON, R. H., 1971. The cell possible implications for radiotherapy. British Journal of proliferation kinetics of four experimental tumours after Cancer, 9, 539-549. acute X-irradiation. Cancer Research, 31, 1279-1284. ELKIND, M. M., SUTTON, H., and MOSES, W. E., 1961. TUBIANA, M., 1971. The kinetics of tumour cell proliferaPostirradiation survival kinetics of mammalian cells tion and radiotherapy. British Journal of Radiology, grown in culture. Journal of Cellular and Comparative 44, 325-347. Physiology, 58, Supplement 1, 113-134. TUBIANA, M., FRINDEL, E., and MALAISE, E., 1968. La cinetique de proliferation cellulaire dans les tumeurs FRINDEL, E., VASSORT, F., and TUBIANA, M., 1970. Effects of animales et humaines-influence d'une irradiation. In: irradiation on the cell cycle of an experimental ascites Effects of Radiation on Cellular Proliferation and Diftumour of the mouse. International Journal of Radiation ferentiation, pp. 423-452 (IAEA, Vienna). Biology, i 7, 329-337. HERMENS, A. F., 1973. Variations in the cell kinetics and VAN PEPERZEEL, H. A., 1970. Patterns of tumour growth after irradiation, M.D. Thesis, University Amsterdam the growth rate in an experimental tumour during (Het Nederlands Kankerinstitut, Amsterdam). natural growth and after irradiation. M.D. Thesis, 1972. Effects of single doses of radiation on lung Amsterdam (Radiobiological Institute TNO, Rijswijk). metastases in man and experimental animals. European HERMENS, A. F., and BARENDSEN, G. W., 1969. Changes of Journal of Cancer, 8, 665-675. cell proliferation characteristics in a rat rhabdomyosarcoma before and after X-irradiation. European Journal WINTER, W. A., 1972. Strahlenbedingte Storung der Zellproliferation bei der durch Isoproterenol induzierten of Cancer, 5, 173-189. Speicheldriisenhyperplasie. Verhandlungen der deutschen HUG, O., and v. SZCZEPANSKI, L., 1969. Cell proliferation Gesellschaft fur Pathologie, 56, 472-476. and radiosensitivity in transplantable animal tumours.
208
Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma By Lore v. Szczepanski and K.-R. Trott Abteilung Strahlenbiologie und Biophysik, Institutfur Biologie der Gesellschaftfur Strahlen- und Umweltforschung,8042 Neuherberg, West Germany (Received January, 1974) ABSTRACT
The proliferation kinetics of the transplanted adenocarcinoma 284 of C3H mice from the first to the sixth day after 600 R and from the first to the eighth day after 1,200 R has been studied by repeated labelling of the tumour cells in vivo with 3H-thymidine and measurement of the labelling index and the percentage of labelled mi to tic cells. The time course of the post-irradiation synchronization was followed during the first day. Later, the mean generation times of the tumour cells were usually prolonged and the spread of the generation times increased. Three to fcur days after irradiation, the tumour increased its growth fraction to twice the normal value by triggering resting cells (Go cells) into cycle.
The development of optimal dose-fractionation schedules in radiotherapy requires knowledge of the behaviour of the tumour and normal tissues during the fractionation intervals. In addition to recovery from sublethal radiation damage and reoxygenation, which have already been studied extensively, cell division and recycling of resting cells might be important factors determining the optimal interval between the individual dose fractions. Therefore, we investigated the proliferation pattern of a mouse mammary carcinoma during the first week after single exposures of 600 R and 1,200 R in a study parallel to tumour cure experiments on the optimal spacing of fractions of 600 R and 1,200 R in the same tumour (to be published elsewhere). By that means we hope to find out about the role each of the above-mentioned factors might play in the establishment of optimal fractionation schemes. MATERIAL AND METHODS
The experiments were performed on the murine adenocarcinoma 284 growing in vivo. Female C3H mice were 10-12 weeks old when they entered the experiment. They were fed on Altromin pellets and provided with water ad libitum before and during the experiment. The tumour was serially transplanted by subcutaneous implantation of 2 mm pieces into the right scapular region. It grew to a diameter of 8-10 mm within 12-14 days. The animals were anaesthetized with hexobarbital before irradiation. Fifteen animals were irradiated at a time in a jig with the body shielded by 5-2 mm lead and only the tumour exposed to 300 kV X
rays, filtered by 0-6 mm Cu and 1 -0 mm Al. The exposure rate, at a distance of 51-5 cm was 60 R/minute. The cell-cycle parameters were studied by counting the ratio of labelled to total mitoses at various times after a single injection of 3H-thymidine ( 3 H-TdR). Percent labelled mitoses (PLM) versus time curves were established in unirradiated tumours as well as in tumours which at various times before injection had been irradiated with single exposures of 600 R and 1,200 R. The tumour-bearing animals received 30 /uCi 3H-TdR i.p. (specific activity 2 Ci/mM) at various intervals after irradiation to study the progression of the labelled cells into subsequent mitoses. Every 30 minutes during the first 12 hours and every hour up to 36 hours after labelling, two animals from each group were sacrificed; the tumours were removed and fixed in 4 per cent formalin. They were embedded in paraffin wax and 5ft sections were prepared for autoradiography by dipping in Kodak NTB 2 emulsion. After two weeks' exposure at 8=C the sections were developed and stained with hemalum-eosin. A hundred mitoses were counted in each tumour to define the percentage of labelled mitoses. In some slides, the mitotic index was established by counting 3,000 cells. To establish the fraction of proliferating cells in the tumour at various times after irradiation tumours were repeatedly labelled from three hours to six days after 600 R and from three hours to eight days after 1,200 R. 30 /xCi 3H-TdR was injected i.p. into the animals at the respective times one, twice, three or four times at two-hourly intervals. An hour later two animals from each group were sacrificed and the tumours removed. In the autoradiographs 3,600 cells were analysed in each of the tumours to calculate the labelling index. The experimental points in the figures give the mean value derived from two tumours. RESULTS
The tumour grows rapidly and reaches a diameter of about 1 cm 14 days after transplantation. The volume doubling time of tumours of this size was
200
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1975
Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma
3-0 days. After irradiation with 600 R and 1,200 R, the tumours regressed rapidly and after 48 hours their volumes were only 15-20 per cent of their preirradiation sizes. These did not change considerably until four to six days after irradiation. Regrowth started six days after 600 R and was not measurable before day 8 after 1,200 R. In earlier passages, the tumour had a very regular structure with chords and lobules surrounded by connective tissue (Fig. 1). Only the marginal zone of one or two cell layers in the lobules were labelled after a single i.p. injection of 3 H-TdR (Hug and
FIG. 1. Adenocarcinoma of C3H-mice. Autoradiograph of the well differentiated part of the untreated tumour after a single i.p. injection of 3 H-TdR. One hour after application the labelled cells are seen only in the marginal zones of the tumour lobules (Szczepanski e£ at. 1971). > 800
Szczepanski, 1969; Szczepanski, Hug and Spangenberg 1971 for detailed description of the tumour histology). The cells in the central part of the lobules were not proliferating and also were morphologically different from the cells of the proliferating zone. With increasing size of the lobules necrotic foci appeared in their centres. This regular structure has not been observed so clearly in more recent passages but its general features are still preserved. If the interval between labelling and killing of the animal is prolonged, the migration of labelled cells through the region of resting cells into necrotic regions takes place. Three days after labelling some dying cells can already be recognized in the centre of the lobules whereas after four days most labelled cells are found in the necrotic region (Fig. 2). The proliferation parameters of the untreated tumour taken from the per cent labelled mitosis (PLM) curve (Fig. 3) are listed in Table I. These
HOURS AFTER TdR
FIG. 3. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into unirradiated animals. ( • experimental points, • A computed curve). ( curve fitted by eye).
TABLE I PROLIFERATION PARAMETERS OF THE UNTREATED C3H ADENOCARCINOMA
Doubling time of tumour volume Labelling index (all histologically "normal" tumour cells included)
f 2. Adenocarcinoma of C3H-mice. Autoradiograph of the well differentiated part of the untreated tumour after a single i.p. injection of 3 H-TdR. Four days later most labelled cells are found in the centralnecrotic regions of the lobules (Szczepanski et al., 1971). x800. FIG.
201
3-0 days 12-5%
Mitotic index
0-37%
Median duration of the Gi phase
4-7 hours
Median duration of the S phase
6-8 hours
Median duration of the G2 phase
1 -9 hours
Median cell cycle time
13-6 hours
Growth fraction
*••> / o
Cell loss factor
0-5
VOL. 48, No. 567
Lore v. Szczepanski and K.-R. Trott were derived from the curve calculated by the computer program of Steel and Hanes (1971). This curve fits the experimental points in the untreated tumour very well. For irradiated tumours, the fit of the computer PLM curve to the experimental data
HOURS AFTER TdR 3 H INJECTION
FIG. 7. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals four days after 600 R. (# experimental points, A- — —A computed curve.) HOURS AFTER TdR- 3 H
INJECTION
4. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals a day after 600 R. (•experimental points, A A computed curve). FIG.
HOURS AFTER TdR- 3 H
INJECTION
8. The percentage of mitoses that times after injection of 3 H-TdR after 1,200 ( • experimental points, A FIG.
HOURS AFTER T d R ^ H
INJECTION
FIG. 5. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals two days after 600 R. ( • experimental points, A A computed curve.)
HOURS AFTER TdR- 3 H
are labelled at various into animals four days R. A computed curve.)
HOURS AFTER TdR
INJECTION
FIG. 6. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals three days after 600 R. ( • experimental points, A -A computed curve.)
FIG. 9. The percentage of mitoses that are labelled at various times after injection of 3 H-TdR into animals six days after 1,200 R. ( • experimental points, AA computed curve.)
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1975
Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma is good in some cases (Figs. 6, 7, 9 and 10). However, in others the fit of the computer curve to the experimental points is poorer (Figs. 4 and 8). In the PLM curve obtained following injection of the label four days after 1,200 R (Fig. 8), the experimental data suggest a double peak 15 hours and 23 hours after labelling rather than the second peak calculated by the computer 17 hours after labelling. In Fig. 4, the experimental points in the trough between the peaks indicate a broad plateau lasting for nearly ten hours. The generation time was prolonged at all the times of observation after 600 R and also eight days after 1,200 R. A slight shortening was seen only four and six days after 1,200 R. In addition to the lengthening of the median generation time, we also found an increased spread in generation times in all cases. In Table II the computed cell cycle parameters at various times after irradiation are listed together with the data taken from the curves fitted by eye.
Summarizing the data in Table II, minor changes in the cell-cycle times could be found within the first week after irradiation. The length of the DNA synthetic period is remarkably constant but there is a tendency for this to become shorter after irradiation. Also, the G2-period is very constant, but was observed to double 24 hours after 600 R. The Giphase showed the greatest variations, partly due to scatter in the later experimental points. Therefore, these values have to be taken with reservation. There were considerable fluctuations in the mitotic index after irradiation (Fig. 11). Twelve hours after irradiation it was essentially zero after 1,200 R and 0-1 per cent after 600 R. It then increased for three days after 600 R when it had reached the control level again. The following day, the mitotic index was about twice the control value, to which it decreased again six to eight days after irradiation. The post-irradiation overshoot in mitotic index after 1,200 R was observed one to three days later than after 600 R.
8
sts
8d after 1200 R Oill
I --y 1 v
60
s -" 40
;
HOURS AFTER TdR^H
INJECTION
FIG. 10. The percentage of mitoses that are labelled at various 3 times after injection of H-TdR into animals eight days after 1,200 R. ( • experimental points, A.- - -A. computed curve.)
TIME (DAYS)
11. Mitotic index at various times after radiation doses of 600 R and 1,200 R. FIG.
TABLE II THE CELL PROLIFERATION PARAMETERS (IN HOURS), AFTER IRRADIATION
Ts
TGI
Time after irradiation Control 1 dav after 600 R 2 davs after 600 R 3 days after 600 R 4 days after 600 R 4 days after 1,200 R 6 days after 1,200 R 8 davs after 1,200 R
PLM curve fig3 4 5 6 7 8 9 10
Tc
TG2
Computer
Fitted by eye
Computer
Fitted by eye
Computer
Fitted by eye
Computer
Fitted by eye
4-7 6-8 6-4 6-5 8-2
4-3 9-2 7-5 6-5 8-1
6-8 6-5 6-5 64 6-3
7-0 6-7 6-8 6-4 6-2
1-9 3-5 2-4 1-9 1-7
2-0 3-4 2-2 2-0 1-8
13-6 17-6 160 15-2 16-9
13-3 19-3 16-5 151 161
20 19 23 31 36
2-1 3-7 10-2
3-7 4-3 10-0
6-3 51 6-3
6-5 5-5 6-8
2-1 2-4 2-3
2-0 2-2 2-2
121 11-8 19-8
12-2 12-0 190
28 26 38
C.V.* (%)
*Coefficient of variation in the distribution of the generation times calculated by the computer program of Steel and Hanes (1971). 203
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48, No. 567 Lore v. Szczepanski and K.-R. Trott
1200 R
60
TdR^H
INJECTIONS
TdR-^
12. Labelling index after one, two, three, or four injections of tritiated thymidine (interval two hours) at various times after radiation doses of 600 R. FIG.
INJECTIONS
13. Labelling index after one, two, three, or four injections of tritiated thymidine (interval two hours) at various times after radiation doses of 1,200 R. FIG.
TABLE III
TABLE IV
CELLS IN S PHASE AND FLUXES INTO AND OUT OF S PHASE AT VARIOUS TIMES AFTER IRRADIATION WITH 600 R
CELLS IN S PHASE AND FLUXES INTO S PHASE AT VARIOUS TIMES AFTER IRRADIATION WITH 1,200 R
Time after 600 R (h)
Control 3 6 12 24 48 72 96 144
CellsO ' in S o
12 11 2-5 1-5 8 13 23 23 17
Inflow (% Per h) 5-0 0-3 0-5 0-8 2-8 3-2 4-2 4-2 3-7
Time after 1,200 R
Outflow* (% Per h)
(h)
5-0 3-0 0-2
Control 3 6 12 24 48 72 96 144 192
Cells in S phase °o
12 2 1 1 6 10 16 23 25 16
Inflow into S (° o per h) 5-0 0-2 0-4 0-8 2-3 2-3 3-7 3-7 3-2 2-5
*As explained in the text, outflow could be calculated during the first six hours only.
The labelling index (LI) after repeated injections of 3 H-TdR for six hours with the first injection given at various times after 600 R is shown in Fig. 12. In the unirradiated tumour, the labelling index rapidly increased from 12-5 per cent to 42 per cent. When the first injection was given three hours after 600 R, the curve was essentially horizontal at 11 per cent. When the first injection was given six and twelve hours after 600 R, a slight rise in the labelling index was seen, but the values were all at or below 5 per cent. When the repeated labelling was started 24 hours after 600 R the labelling index rose from 8 per cent to 25 per cent and 48 hours after 600 R
from 14 per cent to 33 per cent. Seventy-two hours and ninety-six hours after 600 R the starting point at 23 per cent was well above the control value, rising to nearly 50 per cent within six hours. Six days after 600 R the curve was similar to the control curve rising from 16 to 38 per cent. From the slope of these curves the inflow of cells into S-phase can be calculated. At early times, when the individual curves overlap, the flow of cells out of S-phase can be calculated from the difference between the inflow of cells into S-phase and the simultaneous change in the labelling index (LI) after pulse labelling. These data are presented in Table III.
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Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma The analogous curves and data after 1,200 R are shown in Fig. 13 and Table IV. In contrast to the curves after 600 R, there was a very low level of labelling index already three hours after 1,200 R which did not change after repeated injections. Twenty-four plus four hours after 1,200 R there seems to be a rather sudden increase in the labelling index from 7 to 20 per cent within two hours. The other curves are similar to those after 600 R with the only exception that they are delayed by about one day. Taking all data presented in Figs. 3-13 and Tables II-IV together, the proliferation behaviour of the cells of the tumour after irradiation during the following six days can be described as follows: The exposure of 600 R induced a block at the end of the G2-phase preventing the proliferating cells from entering mitosis and the Gi-phase. Accordingly the mitotic index fell very rapidly to very low values. However, as long as there were still cells in the Giphase the inflow into the S-phase and the outflow into the G2-phase took place as before. About three hours after irradiation, the Gi-pool was exhausted and no more cells entered the S-phase whereas the progression of cells from S-phase into the G2-phase proceeded at nearly normal speed until the S-pool also was nearly empty. Twelve hours after 600 R over 80 per cent of all proliferating cells accumulated in the G2-phase. The release from this block occurred between 12 and 24 hours after 600 R. During this time of blocked proliferation, the volume of the tumour decreased rapidly. During the mitotic delay period a great deal of cell death was observed in the marginal zones of the tumour lobules. The first wave of DNA synthesis occurred about 24 hours after 600 R. Forty-eight hours after irradiation, the proliferation seemed to be nearly normal again—only the cell cycle time was prolonged by three hours. Three days after irradiation, a sudden increase in the growth fraction occurred, which after 600 R amounted to nearly a doubling of the pre-irradiation growth fraction and which lasted for two days. Six days after 600 R the pre-irradiation proliferation pattern was attained again, whereas the tumour was still half of its pre-irradiation size. Essentially similar interpretation can be given to the data after 1,200 R. Differences concern mainly the appearance of a block of DNA-synthesis in cells in S-phase and a block at the Gi/S border which lead to a rapid decrease of the labelling index within three hours to 2 per cent. The increase in growth fraction is delayed about 1 day and lasts about three days. Eight days after 1,200 R the growth fraction
is nearly normal again, but the median generation time is still six hours longer than before irradiation.
DISCUSSION
In the transplantable adeno-carcinoma of the C3H mouse profound changes in the proliferation pattern took place after irradiation. After a period of synchronization lasting for about 24-48 hours and a period of nearly normal proliferation a transient but extensive increase in the growth fraction was observed. Mammary carcinomas of the C3H mouse are a very commonly-used experimental system for the study of the effects of irradiation. The kinetic parameters taken from the PLM curve and tabulated in Table I compare well with those published by others {e.g. Denekamp, 1970; Begg, 1971). The tumour has a rather low growth fraction of only 25 per cent as a result of its well-organized structure. Proliferation takes place in a small rim near the connective tissue which separates the individual lobules. Proliferation ceases at distances where, according to calculations by Thomlinson and Gray (1956) and by Tannock (1972) enough oxygen, to allow normal proliferation, should still be available. There is a continuous migration of cells from the proliferation zone near the blood vessels through the compartment of resting cells with gradually decreasing oxygen concentration into regions of obvious necrosis. The turn-over time of the non-proliferative compartment in the unirradiated tumour seems to be short, since two to three days after pulse labelling, labelled cells can already be found in the necrotic region. A more indirect approach to quantitative evaluation of cell flow can be made by comparing the curves of increasing labelling index after repeated 3H-TdR-injections before irradiation and after one week following 600 R when proliferation may be supposed to be normal again. In the unirradiated tumour the labelling index increases from 13 to 43 per cent within six hours, and in the regrowing tumour from 17 to 38 per cent within the same period of time. This rise in the labelling index is due to: (1) the inflow of unlabelled cells into S-phase; (2) mitotic division of labelled cells; (3) elimination of unlabelled cells from the tumour. The steeper rise of the LI in controls compared to the regrowing irradiated tumours is mostly due to loss of unlabelled cells from the tumour at a rate of more than 1 per cent/hour, whereas it can be assumed that in the regrowing tumour no preferential loss of unlabelled cells occurs.
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The earliest effect of irradiation was synchronization of the proliferating cells of the tumour. After 600 R, the main effect was a G2-block lasting about a generation time which is in good agreement with data from cells in culture (Sinclair, 1968). After higher doses, additional effects on DNA synthesizing cells and cells entering the DNA synthesis phase were observed. Similar effects have been described in vitro, also (e.g. Mak and Till, 1963). During the period of mitotic delay the tumour shrank at a rate considerably greater than that caused by normal cell loss. This fast regression implies that it takes place mainly in the non-proliferating compartment. Typical radiation-induced cell death appears already during the period of mitotic delay but is maximal between 12 and 24 hours. Obviously, death of cells trapped in mitosis is not the only way irradiated tumour cells die, contrary to observations in cell and tissue cultures (Lasnitzki, 1940; Hurwitz and Tolmach, 1969). From the second day after irradiation onwards, the tumour was much smaller than before irradiation but consisted mainly of doomed cells. The growth fraction did not differ appreciably from that in the unirradiated tumour. Therefore, the post-irradiation regression of tumour mass affected the proliferative and non-proliferative compartments at about equal rates. The prolongation of the generation times two to four days after 600 R was mainly due to an extension of the Gi period. (Table II, Figs. 5-7). The very flat trough between the two peaks in Fig. 4 looks to us rather like a synchronization effect where the bulk of the proliferating, partly synchronized cells was still in pre-synthetic stages of the cell cycle at the start of the PLM experiment. In no instance other than four and six days after 1,200 R did we find a shortening of the generation time; in fact, it usually was prolonged by two to six hours. In addition to this, the coefficient of variation of the generation time distribution was increased from 19 per cent to greater than 30 per cent. On the fourth to fifth day after 600 R and the fifth to seventh days after 1,200 R, a marked increase in the growth fraction was recorded. Since the duration of the S-phase in the PLM curves did not change much and the labelling index after continuous labelling increased steadily at normal rates, the labelling index was proportional to the growth fraction. A shortening of the cell cycle (with normal S-phase duration) to such a degree as to account for the increase in the labelling index is ruled out by the PLM curves and the continuous labelling curves which show an inflow from proliferating but not
labelled cells. Disappearance of the Gi-period, which has been suggested by Hermens and Barendsen (1969) to explain their findings, did not occur in our tumour. The data presented above can only be interpreted as an indication of a sudden increase in growth fraction three days after 600 R and four days after 1,200 R up to nearly twice the normal value, while cells in cycle proliferate at normal or slightly slower speed. This effect was transitory and finished about six days after 600 R and eight days after 1,200 R. To explain the mechanisms for this increase in growth fraction there are several theoretical possibilities most of which can be ruled out by our observations : (1) A sudden loss of 60 per cent of cells in the nongrowing fraction three to four days after irradiation is very unlikely and would have been easily detected in our measurements of the tumour weights. (2) The fact remains that the absolute number of proliferating cells doubled within one day. These cells might have come, either from the growth fraction, or from the non-growth fraction. If all cells in the growth fraction divided once and no cell left the proliferative compartment, a doubling of the absolute number of proliferating cells ought to be found. However, such a sudden change in the post-mitotic pathway for one day only at the third post-irradiation division seems very unlikely. (3) On the other hand, we have direct evidence concerning the origin of increase in growth fraction shown by the PLM curve starting four days after 1,200 R (Fig. 8). Whereas the computer tried to fit the data to include a normal second peak, the experimental data suggest two peaks or rather a dip in the second peak. The depression in the percentage of labelled mitoses 16-21 hours after labelling is due to a sudden increase in the absolute number of unlabelled mitoses. The mitotic index, which was 0-33 per cent 14 hours after labelling, increased to 0-66 per cent 18 hours after labelling. Obviously four days after 1,200 R there were at least two populations in the tumours one of which was partially synchronized. The most satisfactory explanation for the dip in the second peak of the PLM curve of Fig. 8 is the assumption that some resting cells had been triggered into cycle in a parasynchronous way which divided first five days after 1,200 R. This is consistent with the overshoot of the mitotic index five days after 1,200 R.
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MARCH 1975
Post-irradiation proliferation kinetics of a serially transplanted murine adenocarcinoma Taking all the evidence together, we conclude that, depending on the dose of radiation three or four days after irradiation, resting cells are triggered into cycle to increase the growth fraction for some days. The nature of this trigger remains obscure. It is unlikely that it is the oxygen concentration, since the oxygen tension should be rather high at the border of the non-proliferative compartment, already. If it is the reduction in tumour volume or cell number, one would have to explain the fact that the higher dose which leads to more cell destruction takes longer to trigger resting cells. There probably exists some dose-dependent proliferative delay for resting tumour cells forced to recycle. Such a presynthetic delay has been observed in a number of normal cells where resting cells can be triggered into cycle, as for example in liver (Kelly et al., 1957) or in salivary gland (Winter, 1972). In the C3H adenocarcinoma, however, the irradiation is the trigger as well as the delaying factor. The post-irradiation proliferation kinetics of tumours have been studied by several authors, but usually at one arbitrary time point and by means of the PLM curve only. Denekamp and Thomlinson (1971) discussed the problems encountered in the determination of the cell-cycle parameters shortly after irradiation by the PLM method. Since they concluded that only after all doomed cells had been removed this technique revealed reliable results, they started their kinetic analysis only after regrowth had started {e.g. 14 days after 1,500 rads to a C3H mammary carcinoma). They did not find any conspicuous changes in the generation times nor in the growth fraction. Whereas Denekamp and Thomlinson could be certain of studying surviving cells only, most cells in our studies, especially during the first few days, were doomed to die sooner or later from their radiation damage. However, it has been shown in cell culture that the proliferation kinetics of surviving and inactivated cells after irradiation are not significantly different (Elkind, Sutton and Moses, 1961). Brown and Berry (1968) and Brown (1970) studied the PLM curves of induced squamous cell carcinomas in the cheek pouch of Syrian hamsters 24 hours after 500 R and 72 hours after 1,000 R. They did not observe big changes in the growth fraction in the first five days after 500 R, but a slight prolongation of the generation times which was mostly due to an increase in the duration of the Gi-phase. Frindel, Vassort and Tubiana (1970) demonstrated marked changes in the generation times of the XCTC 2472 tumour, in its ascitic form, 20 hours
and 48 hours after exposure to 250 R, resulting from lengthening of the Gi and S phases of the cells. No such changes were seen in the solid form 48 hours after 600 R (Tubiana, Frindel and Malaise, 1968). In contrast to these papers and our data, which did not demonstrate a speeding up of the cell cycle but rather a slowing down after irradiation, Hermens and Barendsen (1969) and Van Peperzeel (1970) recorded a shortening of the generation times in irradiated tumours. Van Peperzeel studied the labelling index in a very fast growing adenocarcinoma of DBA mice at various times after a single dose of 210 rads. At the peak of the post-irradiation synchronization wave of the labelling index (LI = 75 per cent) she injected 3 H-TdR and analyzed the progression of these cells through the subsequent mitoses by the PLM method. The analysis of the PLM curve revealed a shortening of the cell cycle from 14*5 hours to 11 hours and an increase in the growth fraction from 87 to 100 per cent. Hermens and Barendsen (1969) studied the postirradiation proliferation kinetics in a rat rhabdomyosarcoma four days after exposure to 2,000 R when the tumour had reached its minimal volume and the number of clonogenic cells was just about to rise again. They observed the most dramatic change in generation time which anybody has measured so far. While the growth fraction decreased from 30 to 24 per cent, the mean cycle time was shortened from 28 hours to 11 hours in the tumour periphery. This was due to complete disappearance of the Gi-phase of the cycle. Also, during the period of regrowth the growth fraction was usually smaller than in unirradiated tumours. Only eight days after irradiation Hermens (1973) found a rise in the "fraction of cycling cells" above control levels in the tumour centre. During the regrowth period, irradiated human lung metastases tend to grow appreciably faster for some time than before irradiation (Van Peperzeel, 1972; Malaise et al., 1972). In animal experiments it has been shown that this can be due to changes either in the growth fraction or the generation times or in the cell loss factor. In the C3H mouse adenocarcinoma, irradiation leads to an increase in the growth fraction by triggering resting cells, which might be called Go cells, into cycle. Not much is known about the radiosensitivity of Go cells in vitro or in vivo. Several authors have supposed that they might be considerably more radioresistant than cells in cycle (Hug and Szczepanski, 1969; Tubiana, 1971; Reinhold, 1973). This being so, the triggering of Go cells into the cycle by
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radiation may be one mechanism rendering fractionated radiotherapy more effective than single large doses of irradiation. For the tailoring of optimal fractionation schedules for various human tumours, besides knowledge of the time courses of cell synchronization, reoxygeneration and cell multiplication after each dose, the time when triggering of resting cells into cycle occurs should also be taken into account. ACKNOWLEDGMENTS
IAEA symposium on Radiation Induced Carcinogenesis, Athens, pp. 85-95 (IAEA, Vienna). HURWITZ, C , and TOLMACH, L. J., 1969. Time-lapse cine-
micrographic studies of X-irradiated HeLa S-3 cells. I. Cell progression and cell disintegration. Biophysical Journal, 9,607-633. KELLY, L. S., HIRSCH, J. D., BEACH, G., and PALMER, W.,
1957, The time function of 32P incorporation into DNA of regenerating liver; the effect of irradiation. Cancer Research, 17, 117-121. LASNITZKI, I., 1940. The effect of X-rays on cells cultivated in vitro. British Journal of Radiology, 13, 279-283. MAK, S., and TILL, J. E., 1963. The effects of X-rays on the progression of L-cells through the cell cycle. Radiation Research, 20, 600-618.
We are grateful to Professor O. Hug for his advice, MALAISE, E. P., CHARBIT, A., CHAVAUDRA, N., COMBES, interest, and stimulation throughout this project. We are P. F., DOUCHEZ, J., and TUISIANA, M., 1972. Change in greatly indebted to Dr. G. G. Steel, Sutton, for the comvolume of irradiated human metastases. Investigation of puter analysis of all PLM data. We thank Miss I. Fettinger repair of sublethal damage and tumour repopulation. and Mrs. R. Gessner for their technical assistance. British Journal of Cancer, 26, 43-52. REINHOLD, H., 1973. Radiosensitivity of capillary endothelium. XIII Congreso Internacional de Radiologia, Madrid. REFERENCES BEGG, A. C , 1971. Kinetic and histological changes of a SINCLAIR, W. K., 1968. Cyclic X-ray responses in mammalian cells in vitro. Radiation Research, 33, 620—643. serially transplanted mouse tumour. Cell and Tissue Kinetics, 4, A0X-4W. STEEL, G. G., and HANES, S., 1971. The technique of labelled mitoses: Analysis by automatic curve-fitting. Cell and BROWN, J. M., 1970. The effect of acute X-irradiation on Tissue Kinetics, 4, 93-105. the cell proliferation kinetics of induced carcinomas and their normal counterpart. Radiation Research, 43, 627- SZCZEPANSKI, L. V., HUG, O., and SPANGENBERG, G., 1971. 653. Fraktionierungsstudien an transplantablen Tiertumoren unter zellkinetischen Aspekten. In: Prdoperative TumorBROWN, J. M., and BERRY, R. J., 1968. Effects of X-irradiabestrahlung, ed. O. Hug (Munich: Urban and Schwartion on cell proliferation in normal epithelium and in zenberg). tumours of the hamster cheek pouch. In: Effects of Radiation on Cellular Proliferation and Differentiation, pp. 475— TANNOCK, I. F., 1972. Oxygen diffusion and the distribution 491 (IAEA, Vienna). of cellular radiosensitivity in tumours. British Journal of Radiology, 45, 515-524." DENEKAMP, J., 1970. The cellular proliferation kinetics of THOMLINSON, R. H., and GRAY, L. H., 1956. The hisanimal tumours. Cancer Research, 30, 393-400. tological structure of some human lung cancers and the DENEKAMP, J., and THOMLINSON, R. H., 1971. The cell possible implications for radiotherapy. British Journal of proliferation kinetics of four experimental tumours after Cancer, 9, 539-549. acute X-irradiation. Cancer Research, 31, 1279-1284. ELKIND, M. M., SUTTON, H., and MOSES, W. E., 1961. TUBIANA, M., 1971. The kinetics of tumour cell proliferaPostirradiation survival kinetics of mammalian cells tion and radiotherapy. British Journal of Radiology, grown in culture. Journal of Cellular and Comparative 44, 325-347. Physiology, 58, Supplement 1, 113-134. TUBIANA, M., FRINDEL, E., and MALAISE, E., 1968. La cinetique de proliferation cellulaire dans les tumeurs FRINDEL, E., VASSORT, F., and TUBIANA, M., 1970. Effects of animales et humaines-influence d'une irradiation. In: irradiation on the cell cycle of an experimental ascites Effects of Radiation on Cellular Proliferation and Diftumour of the mouse. International Journal of Radiation ferentiation, pp. 423-452 (IAEA, Vienna). Biology, i 7, 329-337. HERMENS, A. F., 1973. Variations in the cell kinetics and VAN PEPERZEEL, H. A., 1970. Patterns of tumour growth after irradiation, M.D. Thesis, University Amsterdam the growth rate in an experimental tumour during (Het Nederlands Kankerinstitut, Amsterdam). natural growth and after irradiation. M.D. Thesis, 1972. Effects of single doses of radiation on lung Amsterdam (Radiobiological Institute TNO, Rijswijk). metastases in man and experimental animals. European HERMENS, A. F., and BARENDSEN, G. W., 1969. Changes of Journal of Cancer, 8, 665-675. cell proliferation characteristics in a rat rhabdomyosarcoma before and after X-irradiation. European Journal WINTER, W. A., 1972. Strahlenbedingte Storung der Zellproliferation bei der durch Isoproterenol induzierten of Cancer, 5, 173-189. Speicheldriisenhyperplasie. Verhandlungen der deutschen HUG, O., and v. SZCZEPANSKI, L., 1969. Cell proliferation Gesellschaft fur Pathologie, 56, 472-476. and radiosensitivity in transplantable animal tumours.
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