Changes of cell proliferation characteristics in a rat rhabdomyosarcoma before and after X-irradiation

Europ.07. CancerVol. 5, :pp. 173-189. Pergamon Press 1969. Printed in Great BHtain

Changes of Cell Proliferation Characteristics in a Rat Rhabdomyosarcoma Before and After X-Irradiation A. F. HERMENS and G. W. BARENDSEN

Radiobiologiral Institute TaYO, Lange Kleiweg 151, Rijswijk Z.H., The Netherlands

INTRODUCTION

THE GROWTH of a solid tumour is mainly determined by cell production and cell loss. The rate of cell production is a function of the fraction of proliferating cells and the cell cycle time, both of which can be studied by autoradiographic methods [1]. The rate of cell loss can at present only be derived from the difference between the rate of cell production and the rate at which the total cell population increases [2]. In a n u m b e r of experimental solid tumours the relationship between the volume increase and cell proliferation has been shown to depend on tumour size [3-6]. The results of these studies indicate that with increasing turnout volume, the fraction of proliferating cells may decrease, while in addition the cell cycle time m a y change. Furthermore it was shown that the fraction of proliferating cells as well as the cell cycle time are not constant parameters throughout a tumour, but that these parameters m a y vary at different locations in the tumour [7]. Irradiation m a y :induce changes in turnout growth rate, which must be due to changes in cell proliferation characteristics. Knowledge of the cell cycle parameters before and after irradiation is required to interpret the effect of Submitted for publicath)n 17 December 1968. Accepted 27 December 1968. 173

this treatment. Primarily, irradiation of a tumour induces loss of the capacity for unlimited proliferation in a fraction of the t u m o u r cell population. M a n y of these ceils can still divide a few times before they die and are eliminated. Consequently, the tumour volume does not decrease immediately after irradiation. I f the dose of radiation is large enough, the rate of production of new cells eventually becomes smaller than the rate of elimination o f cells from the tumour, and as a result the tumour volume will decrease. If no cure is attained, the rate of cell production by cells, which have retained the capacity for unlimited proliferation, will finally exceed the rate of removal of dead cells and the tumour will resume growth. The sequence of changes occurring in a tumour after irradiation during regression and recurrence can only be interpreted through studies of all the factors involved, including the fraction of cells which have retained the capacity for unlimited proliferation, the fraction of cells which divide only a limited number of times, the distribution of cell cycle times and the mean life time of cells which no longer proliferate. The studies described in this paper were undertaken to obtain information about the relative importance of these factors in an experimental rhabdomyosarcoma transplantable in an inbred strain of rats.

174

A.F. Hermens and G. W. Barendsen

MATERIALS AND M E T H O D S (a) Tumours The solid turnouts employed, are transplantable rhabdomyosarcoma's, derived by a special selection procedure. These tumours have very reproducible growth properties and can be investigated by various techniques to be described. The rhabdomyosarcoma originated in December 1962 in an irradiated rat of the inbred WAG/Rij strain. The tumour was found to be transplantable in this strain. From the ninth passage, in December 1963, a tumour was excised and with a cell dispersion technique, a mono,cellular suspension was prepared [8]. These cells were cultured in vitro in a medium consisting of Hanks' balanced salt solution, Eagle's amino acids and vitamins (MEM), 6% newborn calf serum, inactivated by heating to 56°C during one hour, and 100 i.u. penicillin per ml. Subsequently, a single clone of ceils was removed from a culture dish and these cells were implanted into the flank of a rat, where they grew into a tumour. From this tumour a cell suspension was prepared again and the cells cultured in vitro. A selected clone from one of these cultures was then implanted subcutaneously into the flank of a rat to grow a tumour. This selection procedure was repeated several times and in J u l y 1966 resulted in a tumour, denoted R-l, which can be transplanted reproducibly from the in vitro system to the in vivo system and conversely. The cells derived from this R-1 tumour have been propagated in tissue culture since 1966 and are denoted R-1 cells. In the present investigation R- 1 cells cultured in vitro were trypsinized in order to make a cell suspension which was used to inoculate female WAG/Rij rats, 7-8 weeks old. Each animal received one inoculation, containing 106 cells, per flank. T h e tumours arising from the inoculated cells were allowed to grow to a volume of 1.0-2.0 cm s. Subsequently the tumours were irradiated with a dose of 2000 tad of X-rays or assigned to control groups. During the experiments all animals were kept under the same conditions and supplied with food pellets and water ad libitum. During the interval between labelling of the animals and excision of the tumours, the room temperature was kept at 20°(I and the light intensity at 600 Ix. (b) Assay of the capadty of tumour cellsfor unlimited proliferation The selection procedure described in the previous section has yielded a tumour which

can be assayed with respect to the proliferative capacity of the constituent cells by a direct plating technique. From unirradiated and irradiated tumours, cell suspensions can be prepared and known numbers of cells can be plated in culture dishes. With cells from unirradiated tumours, plating efficiencies of 354-10% were obtained. From a comparison of the plating efficiency of cells derived from an irradiated tumour with that of controls, the 'surviving fraction' of clonogenic cells present in the treated tumour can be calculated. Details of the experimental procedure have been described elsewhere [9, 10]. (c) Irradiations Irradiations were carried out with a 300 kV, 10 mA Philips Mtiller X-ray generator. The half value layer of the radiation was 2"0 m m Cu and the dose rate at the centre of the tumour was approximately 500 rad/min. Prior to irradiation the animals were anaesthetised by injection of 0.045 mg of Veterinary Nembutal* per g body weight. The animals were shielded in a lead box from which the tumour protuded through a slit. The slit was made wide enough to prevent obstruction of the blood supply of the tumour. Nevertheless, as discussed later, measurements of surviving fractions of cells and of growth delay after a dose of 2000 rad of X-rays indicate that the fraction of anoxic cells in tumours irradiated in these conditions may have been larger as compared with tumours which were irradiated with a collimated beam. (d) Measurements of tumour volumes T u m o u r volumes before and after irradiation were derived by measuring with vernier callipers three dimensions of the tumour, which can easily be palpated through the skin. From these data tumour volumes were calculated for a sphere or a spheriod, depending on the shape of the tumour. Relative volumes are computed from the ratio of the volume at a given time and the volume on the day of irradiation. For all points of growth curves, relative volumes of at least five tumours were averaged. (e) Autoradiography In order to study the proliferation parameters of cells in turnouts, animals were injected intraperitoneally with a single dose of 1~c *Pentobarbital Natrium (Veterinair) was obtained from Abbott S.A., Brussels, Belgium.

Tumour cell proliferation before and after X-irradiation

175

CROSS-SECTION OF AN EXPERIMENTAL RNABDOMYOSARCOMA .

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SH-thymidine* per gram body weight. The animals of control groups were injected at time t = 0 , where as the animals with the irradiated tumours were injected at four days (t=4) after irradiation, when the tumours, after an initial continued growth and subsequent regression, have attained volumes equal to those of the controls at time t = 0 (Fig. 2). Subsequently, at regular time intervals of up to 40 hr after injection of *H-thymidine, the tumours were exci.,;ed from animals anaesthetised with ether. After excision the tumours were fixed in Bouin's solution, and by standard histological techniques, serial cross sections of 3~t thick were prepared and used to make autoradiographs by the dipping film emulsion technique [11]. Two types of film emulsions have been used, the G-5 and the K-2 Nuclear Research Emulsions.~'. The second type of emulsion has finer grains and is therefore more suitable for the study of histological aspects of cells in mitosis, especially in the irradiated tumours. The exposure time of the autoradiographs was 6 weeks for the G-5 emulsion and 8 weeks for the K-2 emulsion. The autoradiographs were processed by a standard photographical processing technique and *SH-thymidine with a s[ecific activity of ] "9 c/m.mole was obtained from Schwarz Bioresearch Inc., Orangeburg, N.Y., U.S.A. -~Obtained from Ilford Nuclear Research, Essex, England.

stained with hematoxylin and eosin immediately after fixation and washing in distilled water. In these autoradiographs the numbers of labelled and non-labelled cells in interphase were scored in individual areas measuring 100×100 t? each, localized at intervals of 100 ~t, in two zones perpendicular to each other, which pass through the centre of the tumour cross section (Fig. 1). Interphase cells which morphologically showed signs of pyknosis, lysis or severe nuclear distortion were excluded. This fraction did not exceed 10% in the control tumours and 20% in the irradiated tumours. For each turnout the numbers of labelled and non-labelled cells in mitosis (metaphases+ anaphases) were scored separately in an area of about 500-1000 tt wide, parallel to the circumference of the turnout cross section and in a circular area with a diameter of about 1000-2000 !~, located in the centre of the turnout (Fig. 1). Per tumour cross section at least 100 cells in mitosis were scored in each of the areas. The cell cycle time of tumour cells proliferating in vitro was estimated from R-1 cell cultures grown on cover slips in Petri dishes containing 4 ml of culture medium. Single cells were plated and allowed to grow for 6 or 10 days in an incubator flushed with a mixture of air and 2 . 5 % C O , at 37°C. After 6 or 10 days colonies had developed with an average number of 80 or 1200 cells respectively.

176

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Growth curves of R-1 rhabdomyosarcoma's and the reeurrente after a dose of 2000 tad of 300 kV X-rays. Part A, curve 1 : Growth curve of control turnouts. The relative tumour volume of 1" 0 corresponds to an average tumeur volume of 1.5 cm8, Part A, curve 2: Growth curve of turnouts irradiated at t=0. Part A, curve 2': Hypothetical curve extrapolating the growth of the recurrent tumour parallel to the growth of an untreated tumour. Part B: Variation of the fraction of donogenic cells as a function of the time after irradiation.

3H-thymidine was then added to the medium to a final concentration of 0" 1 pc 3H-thymldine per ml medium. After 15 min incubation the radioactive medium was discarded. The cells were then first washed with medium at 37°C containing 10 I~g/ml non-radioactive thymidine and subsequently with standard medium at 37°C. Finally the cells were supplied with standard medium at 37°C and incubated for time intervals of up to 36 hr. During this period of incubation, coverslips were taken at regular intervals from the Petri dishes and dipped in Bouin's solution to fix the cells. Autoradiographs were then prepared by coating the coverslips with a G-5 Nuclear Research Emulsion and exposure for 10 days. Ceils were considered to be labelled, if the number of reduced silver grains over the nucleus was larger than four in the case of the K-2 emulsion and larger than five in the case of the G-5 emulsion. RESULTS (a) Growth o f the tumour and survival o f its constituent cells

Some of the morphological properties of the

tumour which are of importance with respect to the tumour growth, have been reported previously [7]. In particular it may be noted that in tumours with a volume of less than 3 g, massive necrosis is generally not observed. In Fig. 2A, the growth curves for unirradiated and irradiated tumours are presented. Curve 1, obtained for unirradiated tumours, shows that the growth is not exponential. The growth rate decreases with increasing tumour volume. At the time of irradiation, at t=0, the tumour volume doubling time (T~) is about 6 days, while at t = - - 1 5 , T a = l . 5 days and at t = + 1 5 , 2"==10 days. Curve 2 was obtained by irradiating at t=0, a number of tumours with a dose of 2000 rad 300 kV X-rays. During the first two days after irradiation the tumours continue to grow slowly and subsequently from t = 2 on, a decrease in volume is observed. The minimum relative volume, attained at 6-8 days after irradiation, is equal to 0.75, and at day t=12, the volume is equal to the volume at t = 0 . The growth rate of the recurrent tumour does not differ significantly from the unirradiated controls of the same volume. It can be con-

Tumour cell proliferation before and after X-irradiation cluded that the dose of 2000 rad of X-rays results in only a relatSively small decrease of the tumour volume and in a short growth delay of 12 days, i.e. only two times the volume doubting time of 6 days of unirradiated tumours of this size. In Fig. 2B, fractions of clonogenic cells in tumours are presented, which were measured at various intervals after irradiation with a dose of 2000 rad of 300 kV X-rays. From each tumour a cell suspension was prepared and plated in four Petri dishes, according to techniques described in more detail elsewhere [10]. After 12 days the clones were fixed and stained. The fraction of clonogenic cells in an irradiated tumour was calculated in relation to the plating efficiency of cells from unirradiated control turnouts of the same volume. Each point represents the value for a single tumour. The vertical bars indicate the confidence intervals calculated fi'om the variation between the numbers of clones consisting of more than 50 cells on the four dishes. The results show that a dose of 2000 rad X-rays reduces the fraction of clonogenic cells to a value of about 0.8 % relative to unirradiated tumours. The term 'elonogenic cells' is more appropriate here than 'surviving ceils' because at time intervals exceeding a [~w hours after irradiation,

cell proliferation and cell loss may modify the fraction of cells, capable of unlimited proliferation in the tumour. The results presented in Fig. 2B show further that during three days after irradiation the fraction of ceils capable of unlimited proliferation remains approximately constant. Starting after day 4, a rapid increase is observed and at day 9, the fraction of clonogenic ceils in the irradiated tumours is equal to that in an unirradiated tumour. Neither the small increase beyond 100% between t = 11 and t = 14, nor the small decrease between t=16 and t=18, can be considered as significantly different from the 100% value. Comparison of the growth curve 2 of Fig. 2A and the curve of Fig. 2B shows that, although the effect of 2000 rad X-rays on the tumour volume is relatively small, important changes occur in the cell population with respect to cell proliferation and cell loss. (b) Labelling patterns and cell cycle characteristics Fractions of labelled cells were calculated from the numbers of labelled and non-labelled cells in interphase, counted in alternating areas of 104~~ in tumour cross sections (Fig. I). The mean number of cells scored per area of 1041~ is equal to about 150 ceils for untreated

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in Fig. 1. Part A is an example of the pattern of labelled cells in untreated turnouts. Part B is an example of the pattern of labelled cells in tumours, 4 days after treatment with 2000 rad of X-rays.

178

A. F. Hermens and G. IV. Barendsen Table 1. Fractions of labelled cells in the periphe(y and the centre of unirradiated and irradiated tumours excised at different times after pulse labelling Percentage labelled cells* Time of excision after pulse labelling (hr)

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14"34-2"9 -14.64-4.7 13.54-3.'7 13-64-4"3 20.54-6.2

*Standard errors have been calculated from the variations between values obtained for at least

6 areas of 100 I~× I00 I~. tumours and equal to about 100 cells for irradiated tumours labelled at t----4. These data were used to construct labelling patterns of the turnout cross sections by plotting the fraction of labelled cells as a function of the location in the turnout. In Fig. 3, examples are presented of labelling patterns ofunirradiated tumours and irradiated tumours, labelled at t----0 and t----4 respectively and excised two hours after injection of 8H-thymidine. It can be concluded that in untreated tumours, excised 2 hr after labelling (Fig. 3A), large fractions of labelled cells occur more frequently near the periphery, whereas generally smaller fractions are found in the centre of the tumour. A similar pattern, but somewhat less pronounced, has been described previously for smaller tumours [7]. In Table 1, column 2 and 3, mean values of fractions of labelled cells, computed from data obtained for the periphery and the centre respectively, are tabulated for unirradiated turaours excised at different time intervals after pulse labelling. It can be concluded that in the periphery the fractions of labelled cells increase with time after pulse labelling, whereas no significant increase is observed in the centre. The increase of the fraction of labelled cells in the periphery with the time interval after labelling may be due to two phenomena namely proliferation of labelled cells and elimination of non-labelled cells. Further evaluation of these factors depends on results to be obtained from experiments in which continuous labelling is employed. The labelling pattern of an irradiated turnout, injected with 3H-thymidine at t = 4 and t + 4 and excised 2 hr post-labelling, is shown in Fig. 3B. It can be seen that, although considerable variations in fractions of labelled cells are observed the systematic difference

between the centre and the periphery of the tumour shown in unirradiated tumours, is not present in the irradiated tumour. Mean values of fractions of labelled cells for the periphery and the centre of tumours, irradiated at t = 0 and excised at t---4 at different time intervals after pulse labelling, are presented in column 4 and 5 of Table 1. It can be concluded that the differences between the values for the periphery and the centre are much smaller for the irradiated tumour as compared with the controls. Furthermore no significant increase of fractions of labelled cells as a function of time after pulse labelling is shown either for cells present in the periphery or for the centre of the irradiated tumours. In order to derive the fractions of proliferating cells and the rates of cell proliferation from labeUing indexes (fractions of labelled cells), cell cycle parameters were estimated for cells proliferating in the centre and near the periphery of unirradiated and irradiated tumours, according to the method described by Quasfler and Sherman [12]. For this purpose fractions of labelled cells in mitosis were determined as a function of the time after pulse labelling. The results obtained for unirradiated tumours, labelled at t=0, are presented in Fig. 4. The fraction of cells in the population which are in mitosis is 0.65%. The m a x i m u m percentages of labelled cells in mitosis observed in the first wave of the curves A and B of Fig. 4 are equal to 97% and 89%, respectively. The m a x i m u m values for the corresponding second waves are equal to 70% and 52%. From these curves mean values of the period of DNA synthesis ( T s ) , the time which the cells spend in G2+M, and of the cell cycle time (Te) can be estimated. These estimations involve uncer-

Tumour cell proliferation before and after X-irradiation

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Fig. 4. Variations of the fractions of labelled cells in mitosis as a function of time after pulse labelling in untreated tumours. Each point represents the fraction of labdled mitoses computedfrom a total of 1O0 metaphases+ anaphasesobserved. For each time interval slides of at least two tumour crosssections were used. The curve of part A representsdata obtainedfrom peripheral areas of turnout cross sections. The curveof part B representsdata obtainedfrom centralparts of tumour crossseaions.

concluded that Tc in the centre of the tumour is about 7 hours longer than Tc in the periphery, mainly due to a longer G1 period. As discussed by Quastler [13] and Steel [4], the cell cycle parameters derived from this analysis, apply to cells observed in mitosis. It is not certain that these cells represent a random sample of the population of proliferating cells present in the tumour at any one time. The cell cycle parameters presented in Table 2 are therefore weighted towards those of cells which have short cycle times. In Fig. 5 the fractions of labelled mitoses as a function of the time interval after labelling, are presented for tumours irradiated with 2000 rad of X-rays on t=0 and excised at t=4. The fraction of cells of the total tumour cell population which are in mitosis, is 0-62%.

tainties, due to the fact that the cell cycle parameters are subject to variations, as evidenced by the observation that, after the first wave, the percentage of labelled mitoses does not decrease to zero and that the second wave does not reach values close to 100%. The values of Ta~+u, T s and Tc are presented in Table 2. TGz+u has been derived as the time interval between labelling and the midpoint of the ascending part of the first wave. T s has been derived as the time interval between the midpoints of the ascending and descending parts of the first wave. Tc has been derived as the time interval between the midpoints of the ascending parts of the first and the second wave of labelled mitoses. TG1 has been derived from T~I-=Tc--Ts--TG~+~. From the values presented in Table 2, it can be Table 3.

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Tumour cell proliferation before and after X-irradiation

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tumour spend in G~-+-M phase is slightly longer as compared with cells in unirradiated tumours.

T h e m a x i m u m percentages of labelled cells in mitoses for the first arid the second wave in the periphery are equal 1:o 88% and 83% respectively and in the centre 83% and 62% respectively. The cell cycle', parameters summarized in Table 2 have been derived in the same way as described for unirradiated tumours. It can be concluded that the cell cycle times, for cells in the periphery of the irradiated tumours as well as for cells in the centre, are considerably shorter than corresponding values for unirradiated tumours. T h e differences are mainly due to variations in the values of TG1. The time which the; cells in the irradiated

(c) Cell cycleparameters of R-1 cells culturedin vitro In Fig. 6 the fractions of labelled cells in mitosis are presented as a function of the time after pulse labelling in vitro. Each point represents the mean value derived from counting all mitoses in a number of clones containing between 1000 and 1400 cells each, growing on a Petri dish. Similar data were measured for clones containing an average of 80 cells. The values derived for Te, Tal, T s and TG~+~ have been summarized in Table 2.

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182

A. F. Hermens and G. W. Barendsen

DISCUSSION (a) Cell survival and growth delay after irradiation The work presented in this paper is part of a comprehensive study concerning the reactions of the experimental rhabdomyosarcoma and its constituent cells, to various treatment regimes of fast neutrons and X-rays. The purpose of the present study is to find an interpretation of the apparent discrepancy between two effects induced by a dose of 2000 rad X-rays, namely, a decrease of the surviving fraction of cells to 0.8% and a decrease in tumour volume by only a factor of 0.7, followed by a rapid recurrence [9]. Theoretical aspects of the influence of irradiation on cell proliferation and tumour growth have been discussed by various authors, in particular with respect to changes in the growth fraction [14--16]. The results presented in this paper demonstrate that the changes induced by irradiation are more complex than has been anticipated. In our experiments effects of a single dose of 2000 rad were investigated, because a dose of 1000 rad causes only a temporary decrease in growth rate, but no distinct volume decrease, while a dose of 3000 rad produces a very large decrease in volume and results in such a high frequency of cell death, that studies of proliferation parameters are very difficult. The time interval of four days post-irradiation was investigated first, because at this time the tumour volume has started to decrease significantly from its maximum at two days postirradiation and has reached a volume equal to that at t=0. Results pertaining to other time intervals will be reported in a subsequent paper. The growth delay of 12 days, induced by 2000 rad. X-rays in the present experiments, is smaller than the value of 18 days measured for the same R-1 rhabdomyosarcoma, irradiated in a different experimental arrangement, described elsewhere [10]. The value of 12 days was derived as the time interval required for the tumour to regrow to the volume at the time of irradiation. In the experiments which yielded a value of 18 days, growth delay was estimated as the time interval required for the irradiated tumor to regrow to twice the volume at the time of irradiation, from which the doubling time of unirradiated tumours was subtracted. From curve 2 of Fig. 2A it can be deduced that the latter method applied to the present data, results in an estimate of the growth delay of 13 days, which is not significantly different from the value of 12 days derived by the first method. The difference between

the two values for growth delay of 13 and 18 days respectively, is presumably due to a difference between the oxygenation conditions of the tumours in the different irradiation arrangements. Although the slit in the lead shield used, was wide enough, the blood flow in the tumour protruding through the slit may have been impaired as a result of a decrease in temperature of the tumour below the rat body temperature. The assumption of an increased tumour hypoxia, relative to tumours irradiated without shielding of the rat, is supported by the results of measurements of surviving fractions of cells for tumours irradiated in three different conditions shown in Table 3. The level of hypoxia in the tumour at the time of irradiation does not cause uncertainties in the interpretation of the results however, because the actual number of cells capable of unlimited proliferation, is known from the measurements. As noted earlier the growth delay of 12 to 13 days, caused by 2000 rad X-rays in the present experiments is equal to only twice the doubling time of 6 days for unirradiated tumours of the same volume. If the very simple assumptions are made that before irradiation all cells in the tumour contribute to the growth with a cell cycle time of 6 days, and that the irradiation only reduces the fraction of cells capable for unlimited proliferation and finally that cells in which this capacity is impaired are lost from the tumour very rapidly, then it might be concluded that 2000 rad has reduced the fraction of surviving cells to a fraction of 0.25. Figure 2B shows that these assumptions are not valid because the surviving fraction is reduced to 0.008. If the assumption is made that the cells in the tumours irradiated at t----0, start to multiply at a rate corresponding to that of unirradiated tumours of smaller size, e.g. as indicated by curve 2', which is drawn parallel to curve 1, then the conclusion might be drawn that the surviving fraction of cells is equal to about 4%. Comparison of this value with the curve of Fig. 2B, shows that this assumption is not valid either, and it must be concluded that the recurrence of the irradiated tumour is a more complex phenomenon. This curve shows that the fraction of cells capable for unlimited proliferation, remains constant during about 3 days after irradiation. This does not imply of course that no proliferation of cells occurs, but rather that, after an unknown period of mitotic delay, a large fraction of the cells that have lost the capacity for unlimited proliferation, may divide a few times and consequently the ratio of the fractions of clonogenic and

Tumour cell proliferation before and after X-irradiation

183

(b) Cell proliferation characteristics before and after irradiation

contains m a n y non-proliferating cells, i.e. 'resting but viable' cells, and if the cell cycle times of the proliferating cells show large variations and are relatively long compared with the minimum cell cycle time possible under optimal conditions, then after irradiation the surviving ceils may repopulate the tumour at a rate which is larger than anticipated from the proliferation characteristics before irradiation. These conditions appear to prevail in slowly growing solid tumours in animals and man. The cause for such an increase in proliferation of surviving ceils may be complex and might depend on improvement of the supply of nutrients or oxygen [22] or on changes in spatial relationships with other ceils due to cell loss. In order to obtain further insight in the cellular phenomena occurring after irradiation in the R-1 rhabdomyosarcoma, the cell proliferation characteristics, which were determined from the results of labelling experiments, will be analysed.

The suggestion that proliferation of cells in an irradiated tumour during regression and recurrence m a y occur at a rate which is larger than the rate of the proliferation in an unirradiated tumour of the same volume, has been made before, based on a comparison of the growth rate of the tumour with measured or assumed survival curves of the cells [9, 16, 19]. T h e data concerning changes of fractions of clonogenic cells with time, provide experimental evidence of this phenomenon but they do not show whether the small volume decrease and the rapid recurrence of this tumour after irradiation is due to an increase in the growth fraction, defined by Mendelsohn [3] and discussed by Tubiana [16], or whether it is due to a shortening of tile cell cycle. In principle, changes in growth fraction can only cause a relatively rapid repopulation of the t u m o u r after irradiation if in the untreated tumour the growth fraction was considerably smaller than 1. Furthermore, changes in cell cycle times can .cause a relatively rapid repopulation only if the cycle times of cells in unirradiatcd tumours were longer than the m i n i m u m cell cycle time possible under the most favourable conditions. For instance, in case the cells in the untreated tumour are all engaged in proliferation and if all these cells have a cell cycle time which is as short as possible under optimal conditions, then no accelerated repopulation of the tumour by surviving cells can be expected after irradiation. These conditions appear to be approximately met by rapidly growing experimental tumours [20, 21]. O n the other hand, if a tumour

(b 1) Unirradiated tumours Labelling experiments with small tumours of 0-3 cm 3 have been described previously and the results are summarized in Table 2, together with those of tumours of I. 5 cm 8 [7]. As noted earlier, considerable variations in fractions of labelled cells are observed, depending on the location in the tumour. Especially in the large tumours, the fractions of labelled cells are larger in the periphery than in the centre. In order to assess the importance of these differences for the growth of the tumour, an estimate of the relative volumes of the peripheral and the central part of the tumour must be made. From Fig. 3 and m a n y similar data for both small and large tumours, it can be derived that the peripheral part with a high labelling index extends to approximately 2 m m from the capsula of the tumour. The histological cross sections of the tumours investigated, have a mean diameter of about 12 mm. For a spherical tumour, calculation shows that a centre with a diameter of 8 m m comprises about one third of the total volume. This value is presented in the first row of Table 2. For a small tumour with a volume of 0.3 cm 3, the central part comprises less than 10% of the total volume. For these small tumours the growth rate will mainly be determined by cells localized in the peripheral part and in Table 2, separate cell cycle parameters for the central part of small tumours have not been included [6]. The total number of cells in a tumour was estimated from a comparison of its DNA content with the DNA content per cell deter-

non-clonogenic cells remains approximately constant. This profiferation of cells that have lost the capacity for unlimited proliferation is also reflected in the fact that growth of the tumour continues for 2 days after irradiation. The curve of Fig. 2B shows further that, starting at approximately the fourth day after irradiation, the fraction of clonogenic cells increases very rapidly and at 9 days after irradiation, this fraction has returned to a value which is equal to that of unirradiated tumours. This rapid increase is due to two factors, namely elimination of non-clonogenic cells from the tumour and proliferation ofclonogenic cells. These data clearly show that the measurements of changes in tumour volume alone provide very little: information about the phenomena occurring inside the tumour at the cellular level.

184

A. F. Hermens and G. W. Barendsen

mined in a suspension containing a known number of cells [8]. These values are presented in the second row of Table 2. The numbers of cells present per unit volume in small and large tumours did not differ significantly. Consequently the decrease of the turnout volume doubling time with increasing tumour volume, as shown in Fig. 2A and presented in the third row of Table 2, is not due to a change in the mean volume per cell. In order to evaluate in more detail the cause of the decrease in growth rate with tumour volume and its relation to the growth fraction and the cell cycle parameters, it is necessary to calculate a number of characteristics of the cell proliferation. These calculations are based on a mathematical analysis described in detail by Steel [2]. In this analysis a potential doubling time T is defined as the 'expected doubling time of the tumour in the assumed absence of cell loss' [2, 23]. T can be calculated from T-

h . Ts* L--T-

(1)

and

/ exp ~ .

kp+q In 2/T. =

Ta2+~

. (2)

Employing an approximation method outlined by Steel [2], k and T can be calculated if To,+,, Ts and L1 are known. In Table 2, the values o f T and k, calculated by this method, are presented for the different tumours in the central and the peripheral parts respectively. From the values of T and Tc it is then possible to calculate a factor ~ according to: I n a = 7Tc (ln 2)

(3)

where a is the number of proliferating cells (denoted P-cells) produced per division. I f a number of assumptions, specified by Steel [24] are made, the fraction of proliferating cells or growth fraction f is equal to: [=a-

1.

mations. Consideration of possible variations in the measured parameters shows however, that the uncertainties in the estimated growth fractions do not exceed 10%. Accordingly, the difference between the growth fractions in small and large tumours respectively is sufficiently large to conclude that a significant reduction has occurred during growth of the tumour from 0.3 cm 8 to 1 •5 cm 3. Estimates of a and f a r e presented inTable 2. The value o f f = 0 . 4 2 for the small turnouts differs little from the value of 0-40 quoted earlier, derived by a different method [7]. W h e n f i s known, the fraction of non-proliferating cells (Q-cells) can be calculated as equal to ( l - - f ) . For the large tumours, estimates of T, a, f and (1--f) can be made for the centre and the periphery separately. By considering the relative volumes of the centre and periphery, it is possible to calculate mean values of a, f and (1--f) for the whole tumour. From the values of T for the centre and periphery another parameter can be calculated namely the rate constant k,+,~ for the production of new cells:

(4)

These assumptions pertain to identical phasing of the cell cycle and the influence of starting conditions. If these assumptions are met, growth of the tumour is exponential. Since the R-1 rhabdomyosarcoma does not grow exponentially, the values of the various calculated characteristics must be considered as approxi*For the definitions of the symbols compare Table 2.

(5)

From the values of kp+o f o r the different parts of the tumour, it is possible to calculate mean values for each turnout, taking into account the relative contributions of the centre and the periphery to the total volume. From the mean ke+Q value derived in this way, a mean value for T can be calculated. The rate constant for cell loss kL can subsequently be calculated from kL = kp+Q-- k

(6)

where k is the rate constant for growth and is equal to k = In 2 / Td (7) (see Table 2). The cell loss factor has been defined by Steel [24] as ,b= kL/ kP+Q.

(8)

A mean life span of non-proliferating cells can be derived if the assumption is made that only Q-cells are eliminated from the tumour and that this loss occurs randomly distributed in time: 1-f T t ~ = kL (9) From the calculated parameters summarized in Table 2, a number of conclusions can be drawn with respect to the growth of unirradiated turnouts.

Tumour cell proliferation before and after X-irradiation Comparison of the parameters for small and large unirradiated tumours shows that the mean cell cycle times of the proliferating cells in the small tumours and in the peripheral part of the large tumours respectively, are approximately equal, whereas in the central part of the large tumours, the cell cycle is longer due to an increase of the Gl-period. Furthermore the slow growth of the large tumours as compared with the small tumours is due to a lower factor a, and consequently a lower growth fraction f, in both the central and peripheral areas. This implies that the number of new proliferating cells (P-cells) produced per division is lower in large tumours, resulting in a larger fraction of non-proliferating cells (Q-cells) and a larger cell loss factor. The mean life span of the Q-cells has decreased only slightly, however. It is of interest to note that relatively small changes in the growth fraction f, which depends on the factor a, and in the cell cycle parameters, which determine the potential doubling time T, cause a much larger change in tumour doubling time T~. Variations in the fractions of proliferating cells with turnout volume have been reported for other solid tumours as well [3, 5]. Variations in cell cycle parameters have been reported for an ascites tumour transplantable in mice [20]. In the R-1 rhabdomyosarcoma both factors contribute about equally to the decrease in growth rate. (b 2) Irradiated tumours A similar analysis as described for the proliferation characteristics of unirradiated tumours can be made from the data measured for irradiated tumours, labelled at four days after irradiation. As noted earlier, the cell cycle time measured in these irradiated turnouts during the period of regression, is shorter as compared with the controls. The values for Ta~+u and Ts are not considerably different from those of cells in unirradiated tumours, but the Gl-period is virtually absent. Similar reductions of cell cycle times have been reported by others tbr normal tissues [25, 26]. Furthermore, as shown in Table 1, Table 2 and Fig. 3, the differences between the labelling indexes in the periphery and in the centre are much smaller in the irradiated turnouts as compared with the .controls, due to a markedly larger labelling index in the centre of the irradiated turnout. The rate of decrease in volume of the irradiated turnout can be characterized by a negative doubling time of --96 hr, determined from the tangent to the growth curve 2 of

185

Fig. 2A at t=4. This value is only an approximate measure of the rate at which the total number of ceils in the turnout decreases. The number of cells/cm3 is equal to 5 . 0 x 1 0 s in unirradiated tumours, whereas in the irradiated tumours at four days after irradiation this number is equal to 2.7 × l0 s, i.e. a factor of 0.54 lower as compared with unirradiated tumours. However, between day 4 and 5 after irradiation, the number of cells/cms does not change significantly. It can be concluded that a decrease in cell density has occurred during the first few days after irradiation, but at t=4, the rate of decrease of the volume is a good measure of the rate at which the number of cells decreases. The results of calculations of growth characteristics of irradiated tumours are presented in Table 2. As noted earlier, it should be emphasized that the results of the calculations must be regarded as approximations, because the assumptions discussed by Steel [2, 24] with respect to the applicability of the mathematical analysis are not met. However, these uncertainties do not preclude some qualitative conclusions to be drawn. The potential doubling time of the irradiated turnout is equal to 38 hr. This value is smaller than the corresponding value of large unirradiated turnouts and is very close to the value of small unirradiated tumours. It can be concluded that the rate of cell production in these tumours at four days after irradiation is not much smaller as compared with unirradiated tumours. It is important to note that this cell production in the irradiated turnouts is mainly due to proliferation of cells, which are not capable for unlimited proliferation. Figure 2B shows that at four days after irradiation the fraction of clonogenic cells is still less than 1%. This small fraction of 1% of cells, capable of unlimited proliferation, cannot be distinguished from the 99% of ceils, which still proliferate but will eventually die. The cell loss factor in the irradiated tumour is much larger than in the control turnouts and the calculated mean life span of the Q-ceUs is much shorter. It may be concluded that in the irradiated tumour at four days after irradiation cells die at a high rate, but the volume decreases not very rapidly and not to a large extent because cell production by cells which will die eventually, continues at a relatively high rate. Repopulation of the turnout by cells which have retained the capacity for unlimited proliferation becomes important only after the fourth day post-irradiation and this will be the subject of a subsequent publication.

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A. F. Hermens and G. W. Barendsen

(c) Cells cultured in vitro The cell cycle parameters and growth characteristics of R-1 rhabdomyosarcoma cells in small and large clones in culture were determined in order to relate the changes observed in vitro with changes occurring in growing tumours. The data obtained for small clones, containing an average of 80 cells, show all characteristics of an exponentially growing population, in which all cells proliferate ( f = l ) and very little cell loss occurs (~-----0.05). The doubling time of 17 hr of the cell population is approximately equal to the cell cycle time, which is equal to the potential doubling time of 16 hr. In the large clones, containing an average of 1200 cells, the cell cycle parameters are not significantly different from those of cells in small clones, but the growth fraction is only 39% as compared with 100% in the small clones. Similar observations have been reported by Frindel [27]. It is of interest to note that this decrease with increasing clone size, in the growth fraction of cells cultured in vitro cannot be due to inadequacy of the nutrient or oxygen supply, because the medium of the cultures was changed frequently. Other factors, e.g. lack of space, must be assumed to cause the reduction in growth fraction with increasing population volume. The cell loss factor is slightly larger in large clones as compared with small clones and the mean life time of Q-cells is approximately 200 hr. It can be concluded that the decrease in growth fraction and the increase in the potential doubling time, which is inversely proportional to the rate of cell production, observed during tumour growth, is also observed in cultured cell populations. This may be considered as an indication that neither in the tumour nor in culture it is the supply of oxygen or nutrients which reduces the growth rate, but that the lack of physical space is the limiting factor. The similarity in the decrease in the growth fraction between cell populations in culture and cell populations in the tumours is also of interest because in culture it has been shown that cells in plateau phase of growth fail to repair sub-lethal damage [28]. The problem of whether Q-cells in tumours can repair sub-lethal damage awaits further investigation. Investigation of variations an a-values are in addition of great interest because, as Bresciani [29] has discussed in detail, this factor determines to a large extent whether a cell population will grow indefinitely. (d) Concluding remarks The results presented in this paper suggest a

tentative interpretation of the phenomena occurring at the cellular level after irradiation of the R-1 rhabdomyosarcoma with a single dose of 2000 rad of X-rays. This dose produces a relatively small decrease of the tumour volume by 25% and a rapid recurrence, notwithstanding the fact that more than 99°,o of the cells have lost the capacity for unlimited proliferation. During the first two days after irradiation turnout growth continues, but the growth rate decreases, while starting on the third day the tumour volume decreases as well. At the fourth day after irradiation, the cell loss factor was found to be much larger as compared with unirradiated tumours. This increase in cell loss must have started before the fourth day. Fractions of damaged mitoses, measured as a function of the time after irradiation, show a rapid increase to a maximum of about 90% between 24 and 48 hr after irradiation, with a subsequent decrease at later intervals. This indicates that the cell loss factor increases rapidly during the first two days after irradiation. As a consequence the cell density of the tumour will decrease, followed by a decrease in tumour volume starting on day 3 after irradiation. The extent of this decrease in tumour volume is relatively small however, due to the fact that all P-cells, including those which have lost the capacity for unlimited proliferation, start to reproduce with a cell cycle time which is much shorter than that o f P-cells in unirradiated tumours. Thus a large decrease in tumour volume is prevented by a temporary rapid proliferation of cells which are mainly capable of only a limited number o f divisions. The shortening of the cell cycle o f these cells might result from the increase in space available per cell, caused by the increased cell loss. Starting on day 4 after irradiation, the small fraction of cells capable of unlimited proliferation was found to increase rapidly. This results in a resumption of growth of the tumour at about day 8 after irradiation and a regrowth to the pre-irradiation volume at about day 12. Further investigations of cell cycle times and cell loss factors on day 8 and 12 are in progres~ in order to evaluate whether in the recurrent tumour these parameters and the growth fraction show important changes. The changes in cell cycle parameters observed after a single dose of 2000 rad, invite the speculation that responses of these cells to subsequent treatments with either radiation or chemotherapeutic agents, might be altered as

Tumour cell proliferation before and after X-irradiation

compared with untreated tumours and that these changes might provide a possibility to

187

achieve improved treatment schemes in radiotherapy combined with chemotherapy.

RESUME

Les param~tres de la prolifgration cellulaire ont gtg Jtudigs dans un rhabdomyosarcome transplantable, chez le rat WAG/R~i. La durge des diffgrentes phases du cycle cellulaire est mesurge par la mgthode des mitoses marquges, aprks injection in vivo de thymidine marquge au tritium, pendant la croissance des tumeurs, et aprks irradiation. Les rgsultats sont compargs avec les phgnomknes observgs sur les mgmes cellules, cultivges in vitro. On observe que le temps de doublement Ta des tumeurs tgraoins augmente de 2,5 jours pour les petites tumeurs (0,3 cm3) ~ 6,0 jours pour les grosses tumeurs (2,5 cm3). Cette augmentation est causge partiellement par l' allongement de la durge du cycle des cellules, mais surtout par le fait que le coeffcient de prolifgration diminue et que le hombre des ceUules gliminges augmente. Le pourcentage des ceUules prolifgrantes (growth fraction) diminue de 42% ~ 29%. Le volume des grosses tumeurs (1,5 cm3) irradigespar une dose de 2000 tad de rayons X, diminue, et, dix jours aprks l'irradiation, elles atteignent un volume reprgsentant 75% du volume originel. Quatre jours aprks l'irradiation, le pourcentage de cellules capables de multiplication illimitge n' est plus que 0,8%. La durge du cycle des cellules prolifgrantes est rgduit ~ 12 heures. Le nombre de cellules gliminges dgpasse le nombre de cellules produites, et c' est ld la cause de la rgduction de volume. La durJe moyenne du cycle des cellules du rhabdomyosarcome cultivges in vitro est de 16 heures pour les ceUules prolifgrant dans de petites colonies (de 80 cellules) et de 16,5 heures pour les cellules prolifgrant dans de vastes colonies (de 1200 cellules). Le pourcentage des cellules prolifgrantes (growth fraction) est de 100 pour les petits colonies et atteint une valeur de 39% pour les grandes colonies.

SUMMARY Proliferation characteristics of cells in an experimental rhabdomyosarcoma, transplantable in an inbred strain of WAG/R O"rats, have been studied for unirradiated and irradiated tumours by autoradiography. The cell cycleparameters have been compared with equivalent data for the same strain of cells cultured in vitro. Fractions of labelled cells and labelled mitoses wen' counted as a function of time after pulse labelling with tritiated thymidine. The change,~ in proliferation characteristics of cells in the tumours are discussed in relation to the volume variations of the tumout's before and after irradiation. The results show that the tumowr growth rate decreases with increasing tumour volume. The volume doubling time Ta is 2" 5 days for a tumour volume V of O. 3 cm3 and Ta= 6 days at V:=1.5 cm3. This decrease is due to a change in cell cycleparameters, including a lengthening of the mean cell cyclefrom 20 to 25 hr in parts of the tumour corresponding to a decrease in the rate of production of new cells, as well as to an increase in the rate of cell loss. The growth fraction in small tumours is equal to O. 42 and in the large tumours to 0-29. After irradiation with 2000 rad X-rays of tumours with a volume of 1.5 cm3, tumour volume decreases and on the tenth day it reaches a value of about O. 75 of the preirradiation volume. However, the fraction of cells which after this radiation dose have retained the capacity for unlimited proliferation is equal to only O. 008. Measurements of ceU cycle parameters on the fourth day after irradiation showed that cells present in the tumour at that time proliferate with a shorter cell cycle time of about 12 hr. Cell loss at four days after irradiation exceeded cell production however and caused the volume to decrease. Cell cycle parameters of the same cell line of rhabdomyosarcoma cells cultured in vitro showed that in small clones containing about 80 cells, the mean cell cycle time is 16 hr, the growth fraction is 1.0 and no cell loss occurs, whereas in large clones containing about 1200 cells, the mean cell cycle time is 16.5 hr, the growth fraction is 0-39 and the cell loss factor i~: small.

188

A. F. Hermens and G. W. Barendsen ZUSAM~.NFASSUNG

Die Charakteristiken der Zellproliferation wurden mittels 3H-Thymidin und Autoradiographie bei bestrahlten und unbestrahlen experimentellen Rhabdomyosarkomen untersucht, welche auf Inzucht-WAG[Rij-Ratten transplantiert waren. Ebenfalls wurden der Generationszyklus und seine Teilphasen yon den in Gewebekultur geziichteten RhabdomyosarkomzeUen ermittelt. Die Wachstumskurve des Rhabdomyosarkoms weicht vom exponentieUen Typ ab, und zwar so, daft die Volumenverdoppelungszeit ( T~) zunimmt yon T a = 6 0 Stunden fiir kleine Tumoren (0,3 cm3) bis zu T~=144 Stundenfiir gr6ssere Tumoren (1,5 cm'). Die zeitliche ~nderung der Volumenverdoppelungist nicht nur zum Teil zuriickzufiihren auf eine Zunahme der Zellzyklusdauer yon 20 Stunden in kleinen Tumoren his zu 25 Stunden in grO'sseren Tumoren, sondern auch auf wichtige Faktoren wie die Abnahme der Fraktion proliferierender ZeUen yon 42% bis 29% und die Zunahme der Fraktion eliminierter ZeUen. Nach Behandlung mit 2000 rad Roentgenstrahlen nimmt das Tumorvolumen ab und erreicht nach 10 Tagen einen Mindestwert yon 75% des urspriinglichen Volumens. Dieser anscheinend geringe Effekt ist in scharfem Kontrast zu dem Befund, daft der Prozentsatz iiberlebenderZellen mit unbegrenzter Proliferationsf~higkeit, bestimmt mittels der Zellklonbildungstechnik, nur um 0,08% betrggt. Zellgenerationszeitenmessungen zeigen, daft die yellproliferation ab 4 Tagen nach der Bestrahlung mit verkiirzter gellzyklusdauer stattfindet. Die Elimination abget6teter Zellen iiberschreitet 4 Tage nach der Bestrahlung dennoch die Zellproduktion. Die ZeHgenerationscharakteristiken von in vitro geziichteten Zellen, studiert in kleinen ZeUklonen (80 Zellen pro Klon) und groflen gellklonen (1200 Zellen pro Klon) zeigen, daft die respektiven Zellgenerationszeiten nicht wesentlich voneinander abweichen, und daft die Werte der respektiven Wachstumsfraktionen von 100% b/s 39% abnehmen. IEE~NCES 1. L . F . LAMERTON,Radiation biology and cell population kinetics. Phys. med. Biol. 13, 1 (1968). 2. G. G. STEEL, Cell loss from experimental tumours. Cell Tissue Kinet. 1~ 193 (1968). 3. M. L. MENDETSOHN, The kinetics of tumor cell proliferation. In Cellular Radiation Biologl p. 498. Williams and Wilkins, Baltimore (1965). 4. G . G . STEEL, K. ADAMS & J. C. BARRETT, Analysis of the cell population kinetics of transplanted tumours of widely-differing growth rate. Brit. 07. Cancer 20, 784 (1966). 5. E. FRINDEL,E. P. MALAISe.,E. AI.PEN and M. TUB~'~A, Kinetics of cell proliferation of an experimental tumor. CancerRes. 27~ 1122 (1967). 6. A.F. HEm~ENS and G. W. BARENDSEN,Variation of the labelling pattern and variation in cell cycle time during the growth of an experimental rhabdomyosarcoma in the rat (abstract). Int. 07. radiat. Biol. 1~ 67 (1968). 7. A. F. HEm~ENS and G. W. BAm~NDSEN, Cellular proliferation patterns in an experimental rhabdomyosarcoma in the rat. Europ. jT. Cancer 3~ 361 (1967). 8. H. S. RmNHOLD, A cell dispersion technique for use in quantitative transplantation studies with solid tumors. Europ. jT. Cancer 1, 67 (1965). 9. G . W . BAmENDSEN,Responses of cultured cells, tumours and normal tissues to radiations of different linear energy transfer. In Current Topics in Radiation Research (Edited by M. EB~.RTand A. HOWARD)Vol. IV, p. 332. North-Holland, Amsterdam (1968). 10. G. W. BARENDSEN and J. J. BgOEgSE, Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays--I. Effects of single exposures. Europ. 07. Cancer5~ (1969). To be published. 11. B. MEssmR and C. P. LEBLO~, Preparation of coated radioautographs by dipping sections in fluid emulsions. Proc. Soc. exp. Biol. (N.Y.) 96, 7 (1957). 12. H. O UASTLEg and F. G. SHERMAN, Cell population kinetics in the intestinal epithelium of the mouse. Exp. Cell Res. 17~ 420 (1959). 13. H. QUASTLER, The analysis of cell population kinetics. In Cell Proliferation (Edited by L. F. LA~mgTON and R. J. M. FRY) p. 18. Blackwell Scientific Publications, Oxford (1963).

Tumour cell proliferation before and after X-irradiation 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25. 26. 27. 28. 29.

L. G. LAJTHAand R. OLIVER, Cell population kinetics following different regimes of irradiation. Brit. J. Radiol. 35, 131 (1962). J . F . FOWLER,Radiation biology as applied to radiotherapy. In Current Topics in Radiation Research (Edited by M. EBERT and A. HOWARD) Vol. 11, p. 305. North-Holland, Amsterdam (1966). M. TUBIANA, E. FRINDEL and E. MALAISE, The application of radiobiologic knowledge and cellular kinetics to radiation therapy. Amer. 07. Roentgenol. 102, 822 (1968). H . B . HEWITT and C. W. WILSON, The effect of tissue oxygen tension on the radiosensitivity of leukaemia cells irradiated in .dtu in the livers of leukaemic mice. Brit. 07. Cancer 13, 675 (1959). H. S. REINHOLD, Quantitative evaluation of the radiosensitivity of cells of a transplantable rhabdomyosarcoma in the rat. Europ. J. Cancer 2, 33 (1966). E. MAL~SE and M: TUB~NA, Croissance des cellules d'un fibrosarcome exp~r![mental irradid chez la souris C3H. C. R. Acad. Sci. (Paris) 263-D, 292 (19661). P . K . LALA,Cytokinetic control mechanisms in Ehrlich ascites tumour growth. In Syrup. on Effects of Radiation on Cellular Proliferation and Differentiation p. 463. IAEA, Vienna (1968). G. P. WHEELER, B. J. BOWDON, L. J. WILKOFFand E. A. DuL~-~a)OE, The cell cycle of leukemia L1210 cells in vivo and in vitro. Proc. Soc. exp. Biol. (N.Y.) 126, £,03 (1967). L . M . VAN PUTTEN,Oxygenation and cell kinetics after irradiation in a transplantable osteosarcoma. In Syrup. on Effects of Radiation on Cellular Proliferation and Differentiation p. 493. IAEA, Vienna (1968). G. G. ST~.~L and J. P. M. BENSTED,In vitro studies of cell proliferation in tumours--I. Critical appraisal of methods and theoretical considerations. Europ. J. Cancer 1, 275 (1965). G.G. STEEL, Cell loss as a factor in the growth rate of human tumours. Europ. 07. Car,cer 3, 381 (1967). S. LESHER,Compensatory reactions in intestinal crypt cells after 300 Roentgens of Cobalt-60 gamma irradiation. Radiat Res. 32, 510 (1967). L.F. ]LAMERTON,Cell proliferation under continuous irradiation. Radiat Res. 27, 119 (1966). E. FRINDEL,F. VASSORT,E. MALAISE,H. CROIZATand M. TUBIANA,Etude des param~tres de la prolifSration cellulaire dans une tumeur exp6rimentale poussant sous forme solide ou ascitique. Bull. du Cancer 55, 9 (1968). G. M. HAHN, Failure of Chinese hamster cells to repair sub-lethal damage when X-irradiated in the plateau phase of growth. Nature (Lond.) 217, 741 (1968'.t. F. BR:SSCIANI,Cell proliferation in cancer. Europ. 07. Cancer 4, 343 (1968).

189