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A critical appraisal of clonogenic survival assays in the evaluation of radiation damage to normal tissues
Radiotherapyand Oncology, 1 (1984) 241-246 Elsevier
24l
RTO 00032
A critical appraisal of clonogenic survival assays in the evaluation of radiation damage to normal tissues A d a m Michalowski M R C Cyclotron Unit, Hammersmith Hospital, Ducane Road, London I'II12 OHS, U.K.
(Received 10 March 1983, revision received 13 July 1983, accepted 9 September 1983)
Key words: Radiationdamage; Normal tissues; Clonogenicsurvival
Summary
Assessment of radiation damage to normal tissues in terms of dose-response curves for infinite proliferative potential ("survival curves") does not take into account the decrease with increasing dose of the multiplication rate of the clonogenic and non-clonogenic (radiation-sterilized) cells which may be implicated in the expression of the damage to tissue function or gross appearance. While radiation selectively lowers the chance of successful mitotic divisions, other anticancer agents may in addition interfere with different cellular processes. Comparisons of effectiveness of radiation with that of other modalities should not therefore be limited to analysis of survival curves. Assays of cell "survival" in self-renewing normal tissues in situ often define properties of a non-random sample of the clonogens. The nature of repair associated with the post-irradiation delay in performance of transplantation assays for normal clonogenic cells remains unclear. Dose-response relationships for functional impairment or gross damage to tissue, especially those obtained by irradiation with many fractions, do not necessarily yield to interpretation in terms of clonogenic cell "survival".
Introduction
In the evaluation of radiation effects much weight has been given to the proliferative survival of clonogenic cells defined as retention of the ability for indefinite proliferation. It is the purpose of this paper to examine the application of dose-response curves for clonogenic survival to the measurement and interpretation of radiation damage to normal tissues.
0167-8140/84/$03.00 9 1984 ElsevierSciencePublishers B.V.
How valid are clonogenic assays in the assessment of radiation effects?
Dose-survival curves for cultured cells may be generated with considerable precision which is somewhat deceptive with respect to the total survival of cells integrated over time. This is because the size of colonies derived from survivors markedly decreases with increasing radiation dose [2,16], as does the average number of cells in abortive (nonviable) clones [11,16]. Figure 1 brings together the
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Fig. 1. Dose-response curves for clonogenic survival and for relative average cell number of HeLa cells in surviving and abortive colonies. Data from Puck and Marcus [16]. 1.0
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3 6 9 DOSE (flY) Fig. 2. Dose-response curve for clonogenic survival of exponentially growing V79 cells irradiated with single variable doses of 250 kV X-rays. The accompanying lower curve labelled "cell number" illustrates the dose-dependence for the ratio of total number of cells per Petri dish and number of viable colonies in a parallel dish treated identically and incubated for the same length of time, when the value of the ratio at zero dose is taken to be unity. The bars represent standard errors of the means. Data from A. Michalowski, B. M. Cullen and H. C. Walker (1981, unpublished).
three dose-response relationships from the classical paper by Puck and Marcus [16]. It shows the wellknown radiation survival curve and two other lines tracing the dose-dependent impairment of the proliferative performance of both surviving and radiation-sterilized ("killed") cells. Because the rate of cell multiplication decreases with increasing dose of radiation, survival curves systematically underestimate the damage to proliferative function which is expressed during the post-irradiation period of macroscopic colony formation. This conclusion is further illustrated in Fig. 2 which shows X-ray survival curve for cultured V79 cells and the accompanying radiation dose-response curve for cell number, demonstrating that the actual proliferation of irradiated cells is impaired more profoundly than their infinite capacity for reproduction. The point has been elaborated by Joshi et al. [10] who showed that the number of cells per clone derived from a survivor varied over more than one order of magnitude with single X-ray doses of ~<3.8 Gy. The authors therefore distinguished three categories of surviving cells according to their post-irradiation growth rate which was "normal", "slow" or "very slow". The relative contribution of the slow growers to the total number of survivors increased from 7% to 70% between single X-ray doses of 0.2 Gy and 3.8 Gy. Moreover, at the end of the observation period the number of cells in both normally and slowly growing colonies had decreased with increasing dose of radiation. Joshi et al. [10] also emphasized the uncertainties surrounding the conventional methods of identifying survivors in fixed and stained cell cultures which may influence the parameters of the clonogenic survival curve. While survival curves obtained after single doses of radiation underestimate the degree of proliferative impairment, they may also underestimate the efficiency of recovery processes contributing to the sparing which accompanies fractionation. Littbrand interpreted his in vitro experiments to the effect that repair between two fractions involved not only clonogenic capacity but also radiation-induced prolongation of the intermitotic time [13]. In addition, dose fractionation resulted in an increase
243 of the rate of cell reproduction in abortive clones. The unlimited proliferative potential of those cells which survive a radiation insult is evidently important in rapidly proliferating tissues. However, in slowly turning over cell lineages such infinite potential may not be required during the adult lifetime of the animal. It is therefore advisable, especially in slowly proliferating tissues, to take account of those irradiated cells which fail to produce clones of some defined size (usually 1>50 cells) and are classed as non-viable, but which are nevertheless capable of increasing their number by one order of magnitude or even more [10]. These cells are disregarded in the assessment of radiation effects based on survival curves. For all the above reasons, when considering cell kinetic sequelae of irradiation it seems advantageous to relate to dose a full spectrum of clonal rates of net increase in cell number [19]. This is a more objective and precise way of quantifying effects of radiation on the proliferative function than the traditional binary approach which sets an arbitrary threshold for colony size, ignores all clones failing to reach it, and neglects size differences between colonies which surpass it. Even the recent refinement suggested by Joshi et al. [10] who distinguished three categories of proliferative survivors does not make full use of the information on radiation dose-dependent changes in growth rates within each category and amongst non-survivors (stop growers). The widespread use of survival curves in assessment of damage to normal tissues caused by anticancer modalities other than radiation, either alone or in combination with the latter, awaits careful justification. With radiation the rationale for measurements of reproductive survival is provided by the fact that, with a few exceptions, proliferation is by far the most radiosensitive cell function. With other anticancer modalities, proof that reproduction is the most sensitive function is badly missing. For this reason, all comparisons of radiation effects with those of other agents, which are based on survival curves, are of limited or even dubious significance unless the associated gross (or functional) damage is shown to be identical. Since this is by no
means invariably the case, there is a need to devise appropriate complementary ways of comparing effects of ionizing radiations with those of other modalities such as drugs and hyperthermia. This last point may be illustrated by referring to differential effects of local hyperthermia and of irradiation, on two epithelial compartments of mouse intestinal mucosa. Following heat treatment at 43~ the loss of radioactivity from selectively labelled villous cells was seen to be greater than the loss of crypts [8]. Also, scanning and transmission electron microscopy demonstrated preferential thermal damage to the enterocytes accompanied by exaggerated extrusion of the cells at the villous tips [3,4]. It was concluded that the non-proliferative, irreversibly postmitotic cells on the villi are more susceptible to thermal injury than the proliferating crypt cells. This is in contrast to the relatively more slowly developing effects of ionizing radiation [3,8,15]. In other words, while the crypt-survival assay is a useful method of quantifying the acute effect of radiation in the intestine, it is insufficient in the assessment of heat damage, as it overlooks the very early loss of epithelial lining of the small intestine. Observations of this type with other tissues and modalities may turn out to be a rule rather than an exception. When systematic comparisons of effects of radiation with those of other agents by means different from the clonogenic assays become available, an abundance of new types of radiobiological data will probably emerge.
Pitfalls of elonogenic assays performed on normal tissues The clonogenic assays performed on normal tissues irradiated in vivo also have intrinsic shortcomings. From the technical point of view, the assays fall into two categories. One is a group of tests in which the cells are irradiated and enumerated in vivo in the same host. Included in this group are the endogenous spleen colony formation [18], the clonogenic endpoint applied to the growing cartilage [12], the macrocolony assay for epidermal [20] and intestinal stem cells [22], crypt counts performed in
244 irradiated stomach [5] as well as the small [23] and large intestine [25], seminiferous tubule survival [24] and recently the renal tubule survival assay [26]. The second group of tests involves transplantation of irradiated cells. To this category belong spleencolony formation [17] and the more recent assays for the follicular cells of the thyroid [6], mammary gland epithelium [7], and the parenchymal cells of the liver [9]. With regard to the first group of assays, the shortcomings may be summarized as follows [15]. The close mutual proximity (high density) of clonogenic cells in animal tissues is such that it precludes in situ assessment of their proliferative survival with small total doses of radiation. Consequently, a single exposure has two aims: firstly, reduction of viable cell number per unit of volume analogous to dilution of cell suspension in in vitro assays and secondly, production of a measurable response. What is being characterized in terms of survival curve parameters is a sample of 1% of the initial number of clonogenic cells, preselected by heavy irradiation. The question arises, "is the selection procedure random or not?" One may expect that with photons the selection will favour the cells residing in the less sensitive cell cycle phases. One may note that in all split-dose experiments there is a large sparing effect and wonder whether or not the cells are selected in situ for their capacity for repair in the interval employed. There exist also some positive findings pointing to heterogeneous radiosensitivity of stem-cell populations [14]. There is a possibility therefore that when different radiations to be compared are used for dilution as well as for measurement of their effectiveness, the survival curves may reflect differences due to both radiation quality and non-random sampling. An isoeffect for clonogenic cell survival would then have a limited meaning, because the properties of survivors may be different and not necessarily representative of the >~99% of cells in the population. The second group of clonogenic assays, that involving transplantation, is free from the consequences of the overcrowding of clonogenic cells, as the cells can be diluted at will in a test tube. The disadvantage of these tests derives from the neces-
sity of converting the solid tissue into a single cell suspension and placing the cells in a foreign microenvironment. The problem here is that one cannot exclude a priori an interaction between the injury imparted by radiation and that due to the handling of the cells. The possibility of such an interaction has long overshadowed experiments concerned with repair of potentially lethal radiation damage. More specifically, it has been suggested that dispersion of the cells normally growing in close mutual contact results in diffusion of a hypothetical Q factor into the medium, and that the consequent decreased intracellular concentration of the factor is responsible for a diminished capacity for repair of radiation damage [la]. Conversely, assuming that there are no artifacts inherent in the transplantation assays performed immediately after irradiation, it is important to elucidate the nature of the mechanisms underlying the conspicuous repair of radiation damage in situ, and particularly to find out whether the repair is intra- or intercellular (co-operative). The former is usually assumed to be the case, e.g. when estimating the zero-dose number of clonogenic cells by back-extrapolation of the survival curves obtained by means of in situ assays. As new clonogenic assays of each type are likely to be developed for quantifying damage suffered by cells irradiated in vivo, we must learn to recognize and take account of possible artifacts inherent in 9 the approach in order to make full use of it. For reasons given above, both categories of clonogenic assay may be more pertinent to the subsequent functional impairment (or gross injury) by radiation in rapidly turning over cell lineages than in those proliferating more slowly.
How close is the relationship between clonogenic survival and ultimate radiation damage? Both types of assay resulting in survival curves for normal tissues are by their very nature restricted to clonogenic cells which, according to the common notion, form a minority in any self-renewing normal tissue of the adult. This means that, in order to explain all radiation effects in the majority of the
245 remaining cells solely in terms of the variables defining the survival curves, one has to invoke the existence of a fixed relationship between the clonogens and the remainder. This relationship is generally assumed to result from the fact that if all clonogens are sterilized by irradiation, the nonclonogenic population will proceed to decline at a rate given by the normal steady-state rate of turnover of these cells. Should some clonogens "survive", i.e. retain reproductive integrity, the extent to which the decline of the non-clonogens continues before being reversed depends on both their own pre-irradiation (normal) turnover rate and on the repopulation rate of the "surviving" clonogens. Hence, the time-course and the extent of radiation damage is taken to be governed primarily by normal kinetic parameters, and these are biological rather than radiobiological in nature (although this may not be invariably the case [19]). Since irradiation leads to a disturbance in tissue kinetics as injury begins to be expressed and homeostatic responses ensue, it follows that interpretation of results on damage caused by fractionated treatment in terms of single dose-response curves for clonogenic survival is justified only if the duration of the entire course of exposures falls within the time-limits of the steady-state equilibrium. This prerequisite should be rigorously observed in any such analysis. In addition, there is a specific radiobiological line of thought to th: same effect. Dose-sparing associated with fractionation is due exclusively to a combination of Elkind-type repair and the physiological rate of cell proliferation only when the entire course of exposures is completed while the population in question is still obeying the steady-state kinetics. If the treatment extends beyond that time, at least three additional variables come into play, all three due to accelerated proliferation of the "surviving" clonogenic cells. Higher than normal rate of birth of these cells ("repopulation" as distinct from maintenance proliferation in steady-state conditions) further decreases effectiveness of irradiation as judged by the absolute number of "survivors". However, a rising proliferating fraction would lower the amount of potentially lethal dam-
age repaired, and thus acts to aggravate radiation effects. Finally, shortening of the intermitotic time, with the consequent changes in the make-up of the clonogenic population with respect to contribution of various cell cycle phases and in the resultant radiosensitivity, modifies the values of parameters defining the single dose-survival curve. These changes are due to the call for "survivors" to proliferate faster, as distinguished from selection by radiation of cells residing in the less sensitive cell cycle phases followed by "redistribution" [21]. Information on how these and other events proceed in time following initial irradiation is incomplete, and therefore the data on effects of fractionated treatment are likely to provide clinically useful hints rather than insights into mechanisms. In particular, dose-response relationships for functional (or gross) endpoints of radiation damage caused by fractionated treatment are not interpretable in terms of clonogenic "survival". The events listed above contribute to the influence the treatment time has on the outcome of repeated exposures, and their importance will be greater the faster the physiological turnover of the cell lineage in question and the more sensitive the homeostasis of cell number. It is therefore important to state explicitly the overall time of treatment when referring to values of the parameters characterizing both the clonogenic and functional (or gross) dose-response relationships derived from multifraction irradiation. The above views on the complex inter-relationship between the level of clonogenic "survival" and the severity of functional impairment (or gross le2 sions) are at variance with arguments [lb] that the two quantities are joined together by a close causal link. While recognizing that cell "killing" by radiation plays a crucial role, we have argued for further studies on a number of factors which govern the process of translation of the initial proliferative impairment into the ultimate functional deficiency or macroscopic injury [19]. Such studies may lead to the practical knowledge of how to influence this translation process by postirradiation manipulations, with the aim of lessening the severity of functional or gross radiation damage.
246 Acknowledgements I am grateful to Dr. T. Alper and Dr. T. E. Wheldon for extensive discussions on the subject matter of the paper, and to Dr. B. M. Cullen and Dr. S. B. Field for their help in preparing the manuscript. The work has been partly supported by contract BIO-D-474-81-UK from the Commission of the European Communities.
11 12 13
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References 15 1 Alper, T. Cellular Radiobiology, (a) p. 199; (b) pp. 3 5. Cambridge University Press, Cambridge, 1979. 2 Beer, J . Z . Heritable lesions affecting proliferation of irradiated mammalian cells. Adv. Radiat. Biol. 8: 364~417, 1979. 3 Carr, K. E., Hume, S. P., Marigold, J. C. L. and Michalowski, A. Scanning and transmission electron microscopy of the damage to small intestinal mucosa following X irradiation or hyperthermia. Scanning Electron Microsc. I: 393-402, 1982. 4 Cart, K. E., Hume, S. P., Marigold, J. C. L. and Michalowski, A. Scanning electron microscopy of early effects of hyperthermia on mouse small intestine (Abstract). Strahlentherapie 158: 380, 1982. 5 Chen, K. Y. and Withers, H.R. Survival characteristics of stem cells of gastric mucosa in Call mice subjected to localized gamma irradiation. Int. J. Radiat. Biol. 21: 521-534, 1972. 6 Clifton, K. H., DeMott, R. K., Mulcahy, R. T. and Gould, M . N . Thyroid gland formation from inocula of monodispersed cells: early results on quantitation, function, neoplasia, and radiation effects. Int. J. Radiat. Oncol. Biol. Phys. 4:987 990, 1978. 7 Gould, M. N. and Clifton, K.H. The survival of mammary cells following irradiation in vivo: a directly generated single dose survival curve~ Radiat. Res. 72: 343- 352, t977. 8 Hume, S. P., Marigold, J. C. L. and Michalowski, A. The effect of local hyperthermia on the non-proliferative, compared with proliferative, epithelial cells of the mouse intestinal mucosa. Radiat. Res. 94:252 262, 1983. 9 Jirtle, R. L., Michalopoulos, G., McLain, J. R. and Crowley, J. Transplantation system for determining the clonogenic survival of parenchymal hepalocytes exposed to ionizing radiation. Cancer Res. 41: 3512-3518, 1981. 10 Joshi, G. P., Nelson, W. J., Revell, S. H. and Shaw, C.
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A. Discrimination of slow growth from non-survival among small colonies of diploid Syrian hamster ceils after chromosome damage induced by a range of X-ray doses. Int. J. Radiat. Biol. 42:283 296, 1982. Jung, H, Postirradiation growth kinetics of viable and nonviable CHO cells. Radiat. Res. 89:88 98, 1982. Kember, N. F. Cell survival and radiation damage in growth cartilage. Br. J. Radiol. 40: 496-505, 1967. Littbrand, B. Multiplication of tumor cells in vitro after oxic or anoxic exposure to Roentgen radiation. Acta Radiol. Thor. Phys. Biol. 9:337 352, 1970. Lu, C. C., Meistrich, M. L. and Thames, H . D . Survival of mouse testicular stem cells after gamma or neutron irradiation. Radiat. Res. 81: 402-415, 1980. Michalowski, A. Effects of radiation on normal tissues: hypothetical mechanisms and limitations of in situ assays of clonogenicity. Radiat. Environ. Biophys. 19:157 172, 1981. Puck, T. T. and Marcus, P.I. Action of X-rays on mammalian cells. J. Exp. Med. 103: 653-666, 1956. Till, J. E. and McCulloch, E.A. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14: 213-222, I961. Till, J. E. and McCulloch, E . A . Early repair process in marrow cells irradiated and proliferating in vivo. Radiat. Res. 18: 96-105, 1963. Wheldon, T. E., Michalowski, A. S. and Kirk, J. The effect of irradiation on function in self-renewing normal tissues with differing proliferative organisation. Br. J. Radiol. 55: 759-766, 1982. Withers, H . R . The dose-survival relationship for irradiation of epithelial cells of mouse skin. Br. J. Radiol. 40: 187-194, 1967. Withers, H.R. The four R's of radiotherapy. Adv. Radiat. Biol. 5: 241-271, 1975. Withers, H. R. and Elkind, M . M . Dose survival characteristics of epithelial cells of mouse intestinal mucosa. Radiology 91: 998-1000, 1968. Withers, H. R. and Elkind, M . M . Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. J. Radiat. Biol. 17: 26l 267, 1970. Withers, H. R., Hunter, N., Barkley, H. T. and Reid, B. O. Radiation survival and regeneration characteristics of spermatogenic stem cells of mouse testis. Radiat. Res. 57: 88-103, 1974, Withers, H. R. and Mason, K.A. The kinetics of recovery in irradiated colonic mucosa of the mouse. Cancer 34: 896903, 1974. Withers, H. R. and Mason, K . A . Radiation response of renal tubule cells (Abstract). Int. J. Radiat. Oncol. Biol. Phys. 8, Suppl. 1: 72, 1982.
24l
RTO 00032
A critical appraisal of clonogenic survival assays in the evaluation of radiation damage to normal tissues A d a m Michalowski M R C Cyclotron Unit, Hammersmith Hospital, Ducane Road, London I'II12 OHS, U.K.
(Received 10 March 1983, revision received 13 July 1983, accepted 9 September 1983)
Key words: Radiationdamage; Normal tissues; Clonogenicsurvival
Summary
Assessment of radiation damage to normal tissues in terms of dose-response curves for infinite proliferative potential ("survival curves") does not take into account the decrease with increasing dose of the multiplication rate of the clonogenic and non-clonogenic (radiation-sterilized) cells which may be implicated in the expression of the damage to tissue function or gross appearance. While radiation selectively lowers the chance of successful mitotic divisions, other anticancer agents may in addition interfere with different cellular processes. Comparisons of effectiveness of radiation with that of other modalities should not therefore be limited to analysis of survival curves. Assays of cell "survival" in self-renewing normal tissues in situ often define properties of a non-random sample of the clonogens. The nature of repair associated with the post-irradiation delay in performance of transplantation assays for normal clonogenic cells remains unclear. Dose-response relationships for functional impairment or gross damage to tissue, especially those obtained by irradiation with many fractions, do not necessarily yield to interpretation in terms of clonogenic cell "survival".
Introduction
In the evaluation of radiation effects much weight has been given to the proliferative survival of clonogenic cells defined as retention of the ability for indefinite proliferation. It is the purpose of this paper to examine the application of dose-response curves for clonogenic survival to the measurement and interpretation of radiation damage to normal tissues.
0167-8140/84/$03.00 9 1984 ElsevierSciencePublishers B.V.
How valid are clonogenic assays in the assessment of radiation effects?
Dose-survival curves for cultured cells may be generated with considerable precision which is somewhat deceptive with respect to the total survival of cells integrated over time. This is because the size of colonies derived from survivors markedly decreases with increasing radiation dose [2,16], as does the average number of cells in abortive (nonviable) clones [11,16]. Figure 1 brings together the
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Fig. 1. Dose-response curves for clonogenic survival and for relative average cell number of HeLa cells in surviving and abortive colonies. Data from Puck and Marcus [16]. 1.0
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3 6 9 DOSE (flY) Fig. 2. Dose-response curve for clonogenic survival of exponentially growing V79 cells irradiated with single variable doses of 250 kV X-rays. The accompanying lower curve labelled "cell number" illustrates the dose-dependence for the ratio of total number of cells per Petri dish and number of viable colonies in a parallel dish treated identically and incubated for the same length of time, when the value of the ratio at zero dose is taken to be unity. The bars represent standard errors of the means. Data from A. Michalowski, B. M. Cullen and H. C. Walker (1981, unpublished).
three dose-response relationships from the classical paper by Puck and Marcus [16]. It shows the wellknown radiation survival curve and two other lines tracing the dose-dependent impairment of the proliferative performance of both surviving and radiation-sterilized ("killed") cells. Because the rate of cell multiplication decreases with increasing dose of radiation, survival curves systematically underestimate the damage to proliferative function which is expressed during the post-irradiation period of macroscopic colony formation. This conclusion is further illustrated in Fig. 2 which shows X-ray survival curve for cultured V79 cells and the accompanying radiation dose-response curve for cell number, demonstrating that the actual proliferation of irradiated cells is impaired more profoundly than their infinite capacity for reproduction. The point has been elaborated by Joshi et al. [10] who showed that the number of cells per clone derived from a survivor varied over more than one order of magnitude with single X-ray doses of ~<3.8 Gy. The authors therefore distinguished three categories of surviving cells according to their post-irradiation growth rate which was "normal", "slow" or "very slow". The relative contribution of the slow growers to the total number of survivors increased from 7% to 70% between single X-ray doses of 0.2 Gy and 3.8 Gy. Moreover, at the end of the observation period the number of cells in both normally and slowly growing colonies had decreased with increasing dose of radiation. Joshi et al. [10] also emphasized the uncertainties surrounding the conventional methods of identifying survivors in fixed and stained cell cultures which may influence the parameters of the clonogenic survival curve. While survival curves obtained after single doses of radiation underestimate the degree of proliferative impairment, they may also underestimate the efficiency of recovery processes contributing to the sparing which accompanies fractionation. Littbrand interpreted his in vitro experiments to the effect that repair between two fractions involved not only clonogenic capacity but also radiation-induced prolongation of the intermitotic time [13]. In addition, dose fractionation resulted in an increase
243 of the rate of cell reproduction in abortive clones. The unlimited proliferative potential of those cells which survive a radiation insult is evidently important in rapidly proliferating tissues. However, in slowly turning over cell lineages such infinite potential may not be required during the adult lifetime of the animal. It is therefore advisable, especially in slowly proliferating tissues, to take account of those irradiated cells which fail to produce clones of some defined size (usually 1>50 cells) and are classed as non-viable, but which are nevertheless capable of increasing their number by one order of magnitude or even more [10]. These cells are disregarded in the assessment of radiation effects based on survival curves. For all the above reasons, when considering cell kinetic sequelae of irradiation it seems advantageous to relate to dose a full spectrum of clonal rates of net increase in cell number [19]. This is a more objective and precise way of quantifying effects of radiation on the proliferative function than the traditional binary approach which sets an arbitrary threshold for colony size, ignores all clones failing to reach it, and neglects size differences between colonies which surpass it. Even the recent refinement suggested by Joshi et al. [10] who distinguished three categories of proliferative survivors does not make full use of the information on radiation dose-dependent changes in growth rates within each category and amongst non-survivors (stop growers). The widespread use of survival curves in assessment of damage to normal tissues caused by anticancer modalities other than radiation, either alone or in combination with the latter, awaits careful justification. With radiation the rationale for measurements of reproductive survival is provided by the fact that, with a few exceptions, proliferation is by far the most radiosensitive cell function. With other anticancer modalities, proof that reproduction is the most sensitive function is badly missing. For this reason, all comparisons of radiation effects with those of other agents, which are based on survival curves, are of limited or even dubious significance unless the associated gross (or functional) damage is shown to be identical. Since this is by no
means invariably the case, there is a need to devise appropriate complementary ways of comparing effects of ionizing radiations with those of other modalities such as drugs and hyperthermia. This last point may be illustrated by referring to differential effects of local hyperthermia and of irradiation, on two epithelial compartments of mouse intestinal mucosa. Following heat treatment at 43~ the loss of radioactivity from selectively labelled villous cells was seen to be greater than the loss of crypts [8]. Also, scanning and transmission electron microscopy demonstrated preferential thermal damage to the enterocytes accompanied by exaggerated extrusion of the cells at the villous tips [3,4]. It was concluded that the non-proliferative, irreversibly postmitotic cells on the villi are more susceptible to thermal injury than the proliferating crypt cells. This is in contrast to the relatively more slowly developing effects of ionizing radiation [3,8,15]. In other words, while the crypt-survival assay is a useful method of quantifying the acute effect of radiation in the intestine, it is insufficient in the assessment of heat damage, as it overlooks the very early loss of epithelial lining of the small intestine. Observations of this type with other tissues and modalities may turn out to be a rule rather than an exception. When systematic comparisons of effects of radiation with those of other agents by means different from the clonogenic assays become available, an abundance of new types of radiobiological data will probably emerge.
Pitfalls of elonogenic assays performed on normal tissues The clonogenic assays performed on normal tissues irradiated in vivo also have intrinsic shortcomings. From the technical point of view, the assays fall into two categories. One is a group of tests in which the cells are irradiated and enumerated in vivo in the same host. Included in this group are the endogenous spleen colony formation [18], the clonogenic endpoint applied to the growing cartilage [12], the macrocolony assay for epidermal [20] and intestinal stem cells [22], crypt counts performed in
244 irradiated stomach [5] as well as the small [23] and large intestine [25], seminiferous tubule survival [24] and recently the renal tubule survival assay [26]. The second group of tests involves transplantation of irradiated cells. To this category belong spleencolony formation [17] and the more recent assays for the follicular cells of the thyroid [6], mammary gland epithelium [7], and the parenchymal cells of the liver [9]. With regard to the first group of assays, the shortcomings may be summarized as follows [15]. The close mutual proximity (high density) of clonogenic cells in animal tissues is such that it precludes in situ assessment of their proliferative survival with small total doses of radiation. Consequently, a single exposure has two aims: firstly, reduction of viable cell number per unit of volume analogous to dilution of cell suspension in in vitro assays and secondly, production of a measurable response. What is being characterized in terms of survival curve parameters is a sample of 1% of the initial number of clonogenic cells, preselected by heavy irradiation. The question arises, "is the selection procedure random or not?" One may expect that with photons the selection will favour the cells residing in the less sensitive cell cycle phases. One may note that in all split-dose experiments there is a large sparing effect and wonder whether or not the cells are selected in situ for their capacity for repair in the interval employed. There exist also some positive findings pointing to heterogeneous radiosensitivity of stem-cell populations [14]. There is a possibility therefore that when different radiations to be compared are used for dilution as well as for measurement of their effectiveness, the survival curves may reflect differences due to both radiation quality and non-random sampling. An isoeffect for clonogenic cell survival would then have a limited meaning, because the properties of survivors may be different and not necessarily representative of the >~99% of cells in the population. The second group of clonogenic assays, that involving transplantation, is free from the consequences of the overcrowding of clonogenic cells, as the cells can be diluted at will in a test tube. The disadvantage of these tests derives from the neces-
sity of converting the solid tissue into a single cell suspension and placing the cells in a foreign microenvironment. The problem here is that one cannot exclude a priori an interaction between the injury imparted by radiation and that due to the handling of the cells. The possibility of such an interaction has long overshadowed experiments concerned with repair of potentially lethal radiation damage. More specifically, it has been suggested that dispersion of the cells normally growing in close mutual contact results in diffusion of a hypothetical Q factor into the medium, and that the consequent decreased intracellular concentration of the factor is responsible for a diminished capacity for repair of radiation damage [la]. Conversely, assuming that there are no artifacts inherent in the transplantation assays performed immediately after irradiation, it is important to elucidate the nature of the mechanisms underlying the conspicuous repair of radiation damage in situ, and particularly to find out whether the repair is intra- or intercellular (co-operative). The former is usually assumed to be the case, e.g. when estimating the zero-dose number of clonogenic cells by back-extrapolation of the survival curves obtained by means of in situ assays. As new clonogenic assays of each type are likely to be developed for quantifying damage suffered by cells irradiated in vivo, we must learn to recognize and take account of possible artifacts inherent in 9 the approach in order to make full use of it. For reasons given above, both categories of clonogenic assay may be more pertinent to the subsequent functional impairment (or gross injury) by radiation in rapidly turning over cell lineages than in those proliferating more slowly.
How close is the relationship between clonogenic survival and ultimate radiation damage? Both types of assay resulting in survival curves for normal tissues are by their very nature restricted to clonogenic cells which, according to the common notion, form a minority in any self-renewing normal tissue of the adult. This means that, in order to explain all radiation effects in the majority of the
245 remaining cells solely in terms of the variables defining the survival curves, one has to invoke the existence of a fixed relationship between the clonogens and the remainder. This relationship is generally assumed to result from the fact that if all clonogens are sterilized by irradiation, the nonclonogenic population will proceed to decline at a rate given by the normal steady-state rate of turnover of these cells. Should some clonogens "survive", i.e. retain reproductive integrity, the extent to which the decline of the non-clonogens continues before being reversed depends on both their own pre-irradiation (normal) turnover rate and on the repopulation rate of the "surviving" clonogens. Hence, the time-course and the extent of radiation damage is taken to be governed primarily by normal kinetic parameters, and these are biological rather than radiobiological in nature (although this may not be invariably the case [19]). Since irradiation leads to a disturbance in tissue kinetics as injury begins to be expressed and homeostatic responses ensue, it follows that interpretation of results on damage caused by fractionated treatment in terms of single dose-response curves for clonogenic survival is justified only if the duration of the entire course of exposures falls within the time-limits of the steady-state equilibrium. This prerequisite should be rigorously observed in any such analysis. In addition, there is a specific radiobiological line of thought to th: same effect. Dose-sparing associated with fractionation is due exclusively to a combination of Elkind-type repair and the physiological rate of cell proliferation only when the entire course of exposures is completed while the population in question is still obeying the steady-state kinetics. If the treatment extends beyond that time, at least three additional variables come into play, all three due to accelerated proliferation of the "surviving" clonogenic cells. Higher than normal rate of birth of these cells ("repopulation" as distinct from maintenance proliferation in steady-state conditions) further decreases effectiveness of irradiation as judged by the absolute number of "survivors". However, a rising proliferating fraction would lower the amount of potentially lethal dam-
age repaired, and thus acts to aggravate radiation effects. Finally, shortening of the intermitotic time, with the consequent changes in the make-up of the clonogenic population with respect to contribution of various cell cycle phases and in the resultant radiosensitivity, modifies the values of parameters defining the single dose-survival curve. These changes are due to the call for "survivors" to proliferate faster, as distinguished from selection by radiation of cells residing in the less sensitive cell cycle phases followed by "redistribution" [21]. Information on how these and other events proceed in time following initial irradiation is incomplete, and therefore the data on effects of fractionated treatment are likely to provide clinically useful hints rather than insights into mechanisms. In particular, dose-response relationships for functional (or gross) endpoints of radiation damage caused by fractionated treatment are not interpretable in terms of clonogenic "survival". The events listed above contribute to the influence the treatment time has on the outcome of repeated exposures, and their importance will be greater the faster the physiological turnover of the cell lineage in question and the more sensitive the homeostasis of cell number. It is therefore important to state explicitly the overall time of treatment when referring to values of the parameters characterizing both the clonogenic and functional (or gross) dose-response relationships derived from multifraction irradiation. The above views on the complex inter-relationship between the level of clonogenic "survival" and the severity of functional impairment (or gross le2 sions) are at variance with arguments [lb] that the two quantities are joined together by a close causal link. While recognizing that cell "killing" by radiation plays a crucial role, we have argued for further studies on a number of factors which govern the process of translation of the initial proliferative impairment into the ultimate functional deficiency or macroscopic injury [19]. Such studies may lead to the practical knowledge of how to influence this translation process by postirradiation manipulations, with the aim of lessening the severity of functional or gross radiation damage.
246 Acknowledgements I am grateful to Dr. T. Alper and Dr. T. E. Wheldon for extensive discussions on the subject matter of the paper, and to Dr. B. M. Cullen and Dr. S. B. Field for their help in preparing the manuscript. The work has been partly supported by contract BIO-D-474-81-UK from the Commission of the European Communities.
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