Sublethal damage, potentially lethal damage, and chromosomal aberrations in mammalian cells exposed to ionizing radiations

1nr. .I. Rodrarion Ondog\ Bd. Phas.. Vol. 21. pp. 1457-1469 Pnnted I” the U S.A. All r,gh,s resewed.

Copyright

0360-3016191 $3.00 + .@I D 1991 Pergamon Press plc

??Special Feature

SUBLETHAL DAMAGE, POTENTIALLY LETHAL DAMAGE, AND CHROMOSOMAL ABERRATIONS IN MAMMALIAN CELLS EXPOSED TO IONIZING RADIATIONS JOEL S. BEDFORD, Department

of Radiological

Health Sciences,

D.

PHIL.

Colorado State University,

Fort Collins, CO 80523

Sublethal and potentially lethal damage are abstract terms that originated and were defmed without reference to molecular and subcellular entities in which such radiation damage was registered. The establishment of a cause-and-effect relationship between chromosome fragment loss and cell killing by ionizing radiations, along with a substantial body of knowledge in radiation cytogenetics, allows a definition of these terms in a context where hypotheses become testable and progress toward a better understanding of these phenomena is more likely. Accordingly, the simplest hypothesis which best fits the observations is as follows. Most aberrations are exchange types requiring an interaction to form the exchange between two broken regions of a chromosome or chromosomes, that is, a break-pair. Very few single breaks fail to rejoin or restitute, so the vast majority are sublethal. Any such sublethal break may become a potentidly lethal break-pair if another sublethal break occurs within some range where it is possible for the two to interact. The proportion of break-pairs in which a mis-repair event results in a lethal acentric fragment-producing exchange, can be altered depending on treatment conditions. Such conditions change the balance between “PLD repair” and “PLD fixation.” Studies on the control of radiosensitivity have focused on differences in repair processes, but large differences in radiation response may just as well occur with identical repair processes in operation but with different conditions of fixation. Ionizing radiation, Repair, Chromosomal aberrations, Cytogenetics, Sublethal damage, Potentially lethal damage, Mammalian cells. INTRODUCTION

are stimulated to proliferate immediately, or very shortly after irradiation, survival is generally lower than that observed if some time is allowed to elapse before stimulation (15, 29, 39). Modifications and conditions which alter cell survival as measured by these operations have been examined at great length, and much useful information has been obtained. Since cell killing is the damage end-point measured, these observations are directly pertinent to concerns of radiotherapy. Basic studies at the molecular level have sought to identify specific molecular lesions caused by radiations that can lead to cell death, or other forms of damage, and to understand the precise nature of repair processes which cells use as they attempt to cope with such damage. Much has also been learned by the latter approach, and it has the intuitive attraction that an understanding of basic mechanisms may be more likely to be useful in the long run. A major difficulty in the interpretation of experiments addressing molecular repair mechanisms, however, has been the uncertainty as to whether the particular molecular damage being measured is in any way involved with cell killing. The

Sublethal and potentially lethal radiation damage repair are operationally defined in terms of alterations in the proportion of cells surviving depending on conditions under which the treatments are given (4, 6, 15, 19, 23, 24, 29, 39, 48, 50, 62). Sublethal damage, as the name indicates, is not lethal to cells, but can interact with similar damage from further radiation treatment to produce lethal damage. The survival curve shoulder is indicative of a damage accumulation process (23, 24). The recovery from such damage can be determined by measuring the response of cells surviving a radiation dose following further radiation treatment given at some time after the first. If damage existing in cells surviving the earlier radiation treatment is able to repair before the second treatment, then the second treatment will be less effective in producing additional cell killing. Potentially lethal radiation damage is damage which under one set of conditions is lethal but under another is not (6, 15. 29, 39, 48, 55). A commonly cited example is the observation that if non-cycling or slowly cycling cells

Acknowledgements-This paper is dedicated with great affection and thanks to Dr. Frank Ellis. It was in his radiotherapy unit that I worked on my doctoral degree, with the help of Eric Hall and Ray Oliver, and got my start in radiobiology. Some of the work described in this paper was supported by grants CA 18023, CA 4950 1, and CA 09236 awarded by the National Cancer Institute,

DHHS. The general theme of this “mini-review” arose out of material the author has developed and modified and presented over several years in the form of a refresher course at the annual meeting of ASTRO. Accepted for publication 14 June 1991. 1457

1458

I. J. Radiation Oncology 0 Biology 0 Physics

questionable relevance of DNA single strand breaks to mammalian cell killing by ionizing radiations is a good illustration of this point. Radiation cytogenetics has an even richer history than either “DNA repair” or “cellular radiobiology.” While its contributions to radiobiology have been large indeed, their importance has not always been fully appreciated. It now seems likely that the production of chromosomal aberrations is far more important than any other process involved in cell killing, mutation induction, or oncogenic transformation by ionizing radiation. If this is true, then radiation cytogenetics provides a very useful observation window for viewing a relatively early stage in the development of molecular damage which eventually leads to the expression of cell lethality or other forms of radiation damage. The present paper will attempt to review some of the high-points regarding the pertinence of radiation cytogenetits as a framework in our understanding of the nature of sublethal and potentially lethal damage and its repair in mammalian cells following exposure to ionizing radiations. By far the most important issue here is the central role of radiation-induced chromosomal aberrations in cell killing. Since all of the arguments to be developed hinge on the truth of this assertion, a discussion of the most important points of evidence is necessary. Veterans in the field will recognize that most of the observations and arguments presented below are not new. What has been lacking in the literature, however, is a current perspective of the ways in which many isolated and sometimes apparently conflicting observations can be viewed as a reasonably consistent picture, the only such picture we have at this stage.

CHROMOSOMAL CELL KILLING:

ABERRATIONS AND CAUSE AND EFFECT?

Evidence for a cause and effect relationship between the production of chromosomal aberrations and cell killing has been presented on many occasions, but the evidence is largely circumstantial (3. 8, 10, 11, 15, 19, 20, 25. 49). For example, close correlations have been drawn between the mean lethal dose and the dose necessary to produce an average of one grossly visible aberration per cell in the first mitosis after irradiation (15, 21, 25, 26, 49). For any such correlation, of course, it is entirely possible that the structures or processes whose damage leads to grossly visible chromosomal aberrations and those for cell killing could be entirely different while their separate radiosensitivities are identical. If this were the case, a perfect correlation would be found, but one would also expect that some cells killed by the radiation should have no chromosomal aberrations, while some cells which survive should have aberrations. The elegant series of experiments carried out by Joshi and Revel1 and their colleagues (34, 52) were designed precisely to examine this question. To avoid complicating second-order effects, which will be discussed later, they chose to use low passage normal diploid hamster cells irradiated

November 1991, Volume 21, Number 6

Fig. 1. Examples of the formation of some chromosome-type and chromatid-type aberrations appearing at the first mitotic metaphase following exposure to ionizing radiation. Chromosome-type asymmetrical exchanges yield acentric fragments including both chromatids of the chromosome or chromosomes. Examples shown in this diagram are the dicentric plus its acentric fragment (asymmetrical interchange) and the interstitial deletion (asymmetrical intra-arm intrachange) shown in the light chromosome along with another unaffected (dark) chromosome. Interstitial deletions are mostly acentric rings but are usually quite small so the “hole” in the ring cannot be seen. The other category of asymmetrical exchange is the centric ring plus its acentric fragment (asymmetrical inter-arm intrachange), which is not shown in this diagram. It is seen at much lower frequencies than the aberrations shown. Only two chromosomes (non-homologous) are shown per cell since that is the minimum required for an interchange.

during G, where only chromosome-type aberrations would be involved. Further, they took advantage of the fact that the putatively lethal aberrations, which are essentially all asymmetrical exchange-types, result in the formation of acentric fragments that formed micronuclei visible by phase contrast microscopy in at least one daughter cell following the first post-irradiation mitosis. Examples of some of these aberration types are illustrated, by way of review, in Figure 1. By repeated microscopic observations of many individual live cells, they recorded the proportion with or without micronuclei and continued the observation to determine the corresponding colony forming ability for each cell. Nearly all irradiated cells generating one or more micronucleus failed to continue proliferating, whereas nearly all that did not generate micronuclei did continue to proliferate. Their experimental procedure and conclusion is illustrated in Figure 2. Thus, they showed what was essentially a one-to-one correspondence for these endpoints. Since parallel experiments to measure aberration frequencies in fixed cells under the same conditions showed that nearly all the putatively lethal aberrations were being detected as micronuclei, the one-to-one correspondence can be extended to acentric-fragment-generating chromosome-type aberrations. As mentioned earlier, most of these were asymmetrical exchange types. The generation of micronuclei through acentric fragment loss at mitosis after irradiation of cells in G, or G, results

Chromosomal aberrations, sublethal, and potentially lethal damage 0 J. S.

BEDFORD

1459

While these are extremely interesting, they may be viewed for the moment as second-order effects superimposed upon the primary or first-order process operating in normal diploid cells. It is important to recognize, however, that in certain (rare?) instances other processes of cell killing (e.g., apoptosis) cannot be easily ignored. NEARLY ALL LETHAL ABERRATIONS REQUIRE TWO BREAKS FOR THEIR FORMATION

Fig. 2. The protocol and result of the experiments of Joshi and Revel1 and their colleagues to follow the effect of chromosome fragment loss on the colony forming ability of individual normal diploid hamster ref. (34)).

cells

irradiated

in G,,

(From

Joshi

et al.,

in a genetic loss for both daughter cells since both chromatids are involved in the chromosome-type aberrations which form. The process of fragment loss at the first mitosis after irradiation of rat kangaroo cells in G, with a dose of 4 Gy is illustrated in Figure 3. The magnitude of this microscopically visible genetic loss has been estimated to involve at least 8 or 10 housekeeping-type genes for even the smallest readily visible acentric fragment (15). For a normal diploid cell, then, the corresponding alleles on the homologous chromosome would have to satisfy completely all housekeeping requirements of the cell, with virtually no important heterozygosity or gene dosage effects operating to avoid the loss of proliferative capacity. As mentioned above, acentric fragments derive (mostly) from asymmetrical exchange aberrations, and the other portion of such exchanges are often dicentrics which can form bridges at anaphase if the two centromeres of each chromatid migrate to opposite poles during anaphase. While it has been postulated that bridging dicentrics may play a prominent role in cell killing, this does not appear to be true for euploid cells irradiated to doses corresponding to about the first decade or so of cell killing (15, 52). Tetraploid cells show about twice the aberration frequency per unit dose as their diploid counterparts, but they are not more radiosensitive (e.g., (52)). Apparently, they are better able to tolerate the fragment loss because of the redundancy as observed, for example, in yeast many years earlier (42). This does not mean that aberrations are no longer killing the cells; it just takes more of them (or more serious ones) to do so. Examples of such apparent inconsistencies regarding precise quantitative correlations between aberration induction and cell killing are not uncommon, especially with highly aneuploid cell lines.

As illustrated by the x-ray dose-response curves in Figure 4 for low passage normal human diploid fibroblasts irradiated in Gl, chromosome-type terminal deletions are much less frequent, especially with increasing dose, relative to the more abundant interstitial deletions (mostly small acentric rings which are asymmetrical intra-arm intrachanges) and dicentrics (which are asymmetrical interarm interchanges). Each of the latter two types of aberration requires the production of two-breaks. These may restitute and disappear without a trace, or they may misrejoin with about equal probability (2), to form either symmetrical (mostly non-lethal) exchanges or asymmetrical (mostly lethal acentric fragment generating) exchanges. The “two-hit” basis for exchange aberration formation is very important regarding the origin of the survival curve shoulder and the nature of sublethal damage. One general explanation has invoked “enzyme kinetics” amounting to a dose-dependent repair saturation coupled with a damage fixation process to explain the survival curve shoulder. Since aberrations are primarily responsible for cell killing, and most of these require two radiation-induced breaks for their formation, it then becomes essential to explain how they arise from a repair saturation phenomenon. No satisfactory explanation has been given. A decrease in the number of initial chromosome breaks that rejoin or mis-rejoin with a corresponding increase in the number that do not rejoin because of a repair saturation with increasing dose, should result in a gradual dose-dependent increase in the relative proportion of terminal deletions and a relative decrease in the proportion of exchange aberrations. As illustrated in Figure 4, this is precisely the opposite of what has been repeatedly observed. TWO NEARBY INTERPHASE CHROMOSOME BREAKS ARE POTENTIALLY LETHAL: ONE BY ITSELF IS (ALMOST ALWAYS) SUBLETHAL

Since most lethal aberrations are exchanges whose formation involves the mis-rejoining of two breaks in fairly close proximity, such pairs of breaks when they occur are “potentially lethal. ” One may reasonably imagine that the broken chromosome ends in closest proximity would be those belonging to the chromatin of the same original strand. Consequently, the most likely fate for any break would be restitution. Judging from data on the initial frequency of chromosome breaks in diploid human G, fibro-

I. J. Radiation Oncology 0 Biology 0 Physics

November 1991, Volume 21, Number 6

Fig. 3. An illustration of chromosome fragment loss during mitosis and the formation of a micronucleus. The fragment without a kinetochore and spindle microtubule attachment fails to migrate to either pole during anaphase. The fragment is not incorporated into the nucleus of either daughter cell and forms a micronucleus in one of them. Rat kangaroo cells were irradiated (4 Gy) in G, and cells were fixed in situ at a time corresponding to their first postirradiation mitosis. Microtubules were stained by treating the cells with biotinylated mouse anti-beta tubulin followed by rhodamine labelled avidin, then chromosomes were stained with chromomycin A3 and the cells were viewed and photographed under a fluorescence microscope. Panel A. metaphase; B. anaphase; C, telophase; and D, a daughter cell containing a micronucleus. The demonstration was carried out, and photographs were kindly supplied by Ms. Maria Muhlmann-Diaz.

blasts as viewed following premature chromosome condensation (PCC) compared to the frequency of terminal deletions after cells reach mitosis, it can be estimated that less than 1% of the initial chromatin breaks (and probably
growth conditions necessary to measure the changes in cell survival attributed to “cellular PLD repair,” must alter the proportion of these break-pairs in which any form of exchange (mis-repair) occurs. The ratio of symmetrical (nonlethal) to asymmetrical (lethal) exchanges has been shown to be 1 .O in human-hamster hybrid cells irradiated in monolayers regardless of whether sub-culture was immediate or delayed (2), although it is well known that the

Chromosomalaberrations, sublethal, and potentially lethal damage 0 J. S. BEDFORD

0 Inlerstilial

2.5 -

A

Dicentrics

0 Terminal = 6

Welions

-

and Rmgs

--

Dclcl~ons

----

2.0 -

Dosc(Gy)

Fig. 4. X-ray dose-response curves for the production of chromosome-type aberrations generating acentric fragments in low passage AG1522 diploid human fibroblasts. Non-cycling G, monolayer cultures were irradiated, and then incubated for 24 hours before sub-culture and incubation until fixation at the first post-irradiation mitosis. (From Comforth and Bedford, ref. (15)).

absolute number of exchanges per unit dose is considerably greater (and survival considerably lower) for immediate

sub-culture. Opposing processes of “fixation” and “repair”* of potentially lethal damage (PLD) have been postulated and discussed in abstract terms for many years (e.g., (22, 48, 50, 61)) along with the idea that the balance or relative contributions of the two processes operating together would determine the overall survival of cells after irradiation. Nevertheless, in recent times much of the emphasis in the interpretation of PLD and its modification has been on the repair process. The involvement of chromosomal exchanges in cell killing would equate restitution with repair, asymmetrical exchange with lethal mis-repair, or “lethal damage fixation” and symmetrical exchange with non-lethal mis-repair or “non-lethal damage fixation.” The increase or decrease in survival following a given radiation dose, depending on changes in treatment conditions, must therefore arise either from a change in the proportion of chromosome break restitution relative to exchange within break-pairs where exchange is possible, or from a change in the absolute number of break-pairs in which exchange is possible. Thus, several possibilities exist that might explain the underlying changes in the conversion of potentially lethal damage (break-pairs) to lethal damage *The term repair is sometimes interpreted to mean restoration to the original condition, although restoration to a working condition is literally and practically a more accurate definition. Fix-

1461

(asymmetrical exchanges) corresponding to changes in treatment conditions. For example, the arrangement and/or state of chromatin in the nucleus may change with different culture or growth conditions to alter either the proportion of all breaks that exist in pairs where exchange may occur, or the proximity of breaks within existing breakpairs such that the probability of exchange versus restitution would change. What seems less likely is that repair (restitution) by itself would occur to a greater or lesser extent under the alterations in conditions typical of experiments demonstrating ‘‘PLD repair. ” This is especially pertinent to arguments comparing the induction and disappearance of molecular radiation lesions with PLD and its repair. Dr. B. Loucas in my laboratory has shown recently, for example, that anisotonic salt treatment (0.5 molar NaCl in Dulbecco’s PBS) either immediately before or after irradiation of G, human diploid fibroblasts actually increuses the number of interphase chromosome breaks expressed as PCC breaks compared to that in cells treated in isotonic PBS. The hypertonic salt treatment itself produces no chromosome breaks, and it has been shown that the number of DNA single or double strand breaks is not altered (at least in the more “biological” dose ranges used) by the salt treatment (36, 41). The number of PCC breaks expressed per unit dose immediately after irradiation of these cells in PBS was estimated (17) to be only about 15% of the number of DNA double strand breaks (dsbs), but since the PCC break frequency increased by more than 50%, they would then represent some 20 to 25% of the dsbs with the hypertonic salt treatment. As Raaphorst and Dewey have shown, the frequency of radiation-induced chromosome-type exchange aberrations seen after cells reach mitosis is also increased by hypertonic salt treatment, whereas the ratio of different aberration types remains about the same, indicating that break rejoining to form exchanges is not inhibited. In fact, the frequency of exchanges (formed by mis-rejoining) is enhanced (51). As these authors point out, changes in fixation (22) may be the key factor underlying the increased chromosomal radiosensitivity accompanying treatment with anisotonic salt treatments (51).

A LINEAR INCREASE IN SINGLE BREAKS WITH DOSE LEADS TO A CURVILINEAR INCREASE IN BREAK-PAIRS, i.e., SUBLETHAL DAMAGE ACCUMULATION RESULTS IN A SHOULDERED SURVIVAL CURVE As discussed above, a pair of interphase chromosome breaks in sufficiently close proximity would constitute a potentially lethal lesion, but for the most part neither individual break by itself (nor any other single break) would be ation is taken to mean a process that converts the damage to a state no longer subject to modification.

1462

I. J. Radiation

Oncology

0 Biology 0 Physics

lethal. Such single lesions are literally sublethal, as well as being sublethal regarding their involvement in the survival curve shoulder. The number of individual interphase chromosome breaks capable of interacting to form an exchange, if sufficiently close to another, increases linearly with the radiation dose; but the number of break-pairs where such interaction can occur would clearly not do so, as discussed, for example, by Neary (46), Savage (53), and also by Lea nearly half a century ago (37). The number of break-pairs would increase in proportion to the square of the radiation dose if each break were produced independently of any other. This quantitative dose-squared dependency can be appreciated from the following simplified argument. Supose after irradiation a cell nucleus of radius R (volume ? ;nR3) contained n breaks produced independently of each other and in direct proportion to the radiation dose, D. If one break must lie within a range r of another to interact, then any second break that occurs within the volume of a sphere of radius r(volume $IT~, centered around a particular break could lead to an exchange. Since the fraction of the total volume of the nucleus taken up by the smaller sphere of radius r is ($rr3)l(~nR3) = (r/R)3 the number of second lesions in the smaller sphere would be (n - 1) ’ (r/ R)3 or, if n is larger than about 20 or so, -n$r/R)3. The same consideration must be given for each break in turn, and the result summed (n times) to give the total number of break-pairs within interaction range produced by independent electron tracks, aY,, which is (n)*(n)*(rR)3, or

aY, = n*(r/R)‘.

November

1991, Volume 21, Number 6

rectly proportional to the number of electron tracks, and consequently, the dose, that is, as mpD. These would have to be added to the break-pairs from the proximity of single breaks each originating from an independent track, which we already determined was &,D2. So the total yield of potentially lethal break-pairs, Y,, is

Y, = a,D

+ &,D2.

Eq. 2

We have discussed at some length the fact that only two options are open for the vast majority of these break-pairs. Either they rejoin to restitute or they n&-rejoin to form an exchange, with half of the exchanges (asymmetrical) being lethal in normal diploid cells. Very few fail to rejoin at all since (at least for normal human cells) relatively few terminal deletions occur. t The proportion of potentially lethal break-pairs that do form lethal asymmetrical exchanges may differ depending on the ability of different cells or the same cells under different conditions to preferentially restitute breaks in break-pairs, but the fact that only a proportion form asymmetrical exchanges under a given set of conditions will change the yield of potentially lethal breakpairs Y, to Y,, the yield of those that actually form so

Y, = a,D

+ l&D*.

Eq. 3.

Since the number of lethal events per cell, Y, is only an average number (not every cell will have exactly YL) a close approximation to the proportion with exactly 0, 1, 2, etc., is given by the Poisson formula

Eq. 1

e-‘LI*X

p, =

---y-9

x.

We have already said that n is directly proportional to the radiation dose, D, that is, n = k,D, so n2 (r/R)3 = D*k*(r/ R)3 = &,D* where pi, = k2 (r/R)3. Thus, the number of break-pairs produced by independent electron tracks is proportional to the square of the dose. The dose received from a typical orthovoltage x-ray machine or a gamma-ray source is delivered by ionizations that occur along the tracks of electrons. For all intents and purposes, the smallest dose that can be received by a cell nucleus corresponds to the passage of one electron track, but in traversing the nucleus quite a few ionizations occur along it. Of the order of a few hundred ionizations deposit a dose of some 0.2 to 0.5 cGy in the nucleus for one such track. It is obvious that there would be some chance that two breaks close enough to form a break-pair might occur along even one (each) electron track. The number of break-pairs from this “one-track” mechanism would be di-

This is, essentially, the biology underlying the dual theory of radiation action elegantly described by Kellerer and Rossi (35), and is also similar to considerations reported

tFollowing a dose of 6 Gy to contact inhibited Go normal diploid fibroblasts, fewer than 10% of all aberrations are terminal deletions for delayed sub-culture conditions, but about 20% are terminal deletions for immediate sub-culture. There may be a

small shift in either the proportion that do not rejoin or the proportion of incomplete exchanges under these conditions. For certain other cells somewhat higher proportions of terminal deletions have been observed (e.g., 3).

where P, is the probability that exactly x events will occur when the mean is IL. A normal diploid cell will survive only if it contains no such aberration, and the probability of this happening (the fraction surviving) when the mean is Y, is

Po=S=

e-(yL) V,>”= e-(~d , 0.

or since Y, = ou,D + &D*

Chromosomal

aberrations,

sublethal,

and potentially

Fig. 5. Diagrammatic illustration of the linear-quadratic increase in potentially lethal chromosome break-pairs with increasing dose and the build-up of sublethal single breaks (along the top of the diagram from left to right) when a second dose follows the first with no time delay. The bottom portion of the diagram shows the disappearance (restitution) of most of the breaks with time leaving cells that survive with no lethal exchanges or developing and with no potentially lethal break-pairs or sublethal breaks remaining. These surviving cells will then respond to a second dose as if they had not been irradiated. Therefore, the overall survival for the starting population will be proportional (in a logarithmic way) to (aD + pD2).2 = 2aD + 2PD2 rather than a(2D) + p(2D)2 = 2aD+4PD2 obtaining when there is no time separation between the two equal dose fractions of D each. (See text for further

details).

by Neary (46). The formation of chromosome exchanges has been discussed by others in much greater detail (see 53). DOSE-FRACTIONATION OR DOSE-RATE EFFECTS OCCUR IF SUBLETHAL SINGLE BREAKS DISAPPEAR DURING THE TIME REQUIRED TO DELIVER THE TOTAL DOSE A qualitative appreciation of the mechanism underlying dose-fractionation and dose-rate effects on cell killing is easy to see if the latter results from the formation of chromosomal exchange aberrations which require an interaction of two independent lesions. If each lesion heals or restitutes with time, then for sufficiently low dose rates, no two independently-produced lesions would ever be present in the same cell at the same time and no killing could occur since no interaction would be possible. This idea is sufficient for a qualitative understanding of dose-rate effects due to repair processes. For a more quantitative appreciation, it may be helpful to follow the argument presented in the following paragraph and illustrated in Figure 5. Figure 5 illustrates the status of a cell regarding single chromosome breaks and break pairs immediately after a

lethal damage 0 J. S. BEDFORD

1463

dose, D, (with hypothetical values of or and BP), and, along the top portion of the diagram, after a dose 2D given in 2 fractions of size D each with no time between fractions. The number of lesion pairs increases as a linearquadratic function of dose (again, along the top of the diagram from left to right) but if some time elapses before the second dose, D, then rejoining may have occurred, and the cells will either contain a “fixed” potentially lethal (i.e., lethal) lesion or will be undamaged both with respect to potentially lethal and sublethal lesions, as illustrated along the bottom portion of the diagram. The second dose will then have the same killing effect on the survivors as the first dose, rather than the greater effect obtaining when the second dose is given immediately after the first. The reduction in the number of chromosome exchanges when an 8 Gy dose was delivered to plateau phase cultures of normal human AG1522 cells in either 1 fraction or up to 16 fractions with 6 hours between fractions is shown in Figure 6A. Figure 6B shows the change in cell survival over a range of doses under similar conditions when these cells were irradiated using either single acute doses or in dose fractions 4, 2, 1, or 0.5 Gy per fraction with 6 hours between fractions. For direct comparison with figure 6A the surviving fractions for a total dose of 8 Gy only in figure 6B should be noted. Survival increased in the same way that the aberrations per cell decreased as the number of fractions increased from 1 to 16. There has been some rather extensive argument concerning the nature of the survival curve shoulder, mostly centered around “damage accumulation” on the one hand and “repair saturation” on the other. In fact, damage accumulation, per se would apply in either case since something must accumulate for anything to become saturated. The major problem with various ideas of repair saturation in connection with survival curve shoulders that have been entertained for the past 25 years is that they generally assume that a single lesion, if unrepaired, will kill a cell. If chromosome aberrations kill cells and most of them are exchange-types, how does one get an exchange with only one unrepaired lesion? Can an undamaged chromosome in teract with a damaged one to produce an exchange? Most of the arguments have been rhetorical, but one experimental approach has been reported recently that directly addresses these questions. By fusing irradiated and unirradiated mitotic cells in such a way that the damaged and undamaged genomes could be identified and would mix shortly after fusion, Comforth was able to show that virtually no exchanges form between irradiated and unirradiated chromosomes, although mixed exchanges do form if both genomes are irradiated (18). One possibility that has not been entertained in connection with notions of a repair saturation-origin of the survival curve shoulder is that there may be sufficient differences in the molecular processes leading to restitution (repair) and exchange (mis-repair) so that a saturation or deficiency in the “repair” process might occur without saturation of a “different” exchange or mis-

1464

I. J. Radiation Oncology 0 Biology ??Physics

Fig. 6. The left panel (A) illustrates the decrease in yield of chromosome exchange-type aberrations (rings and dicentrics, and interstitial deletions) at the first mitosis following irradiation of non-cycling G, normal human fibroblasts (AG1522) to a total dose of 8 Gy but with the dose given in either 1, 2, 4, 8, or 16 fractions with 6 hours between fractions. The frequency of terminal deletions does not change even with the dose spread over the longest time (90 hours) with the smallest dose per fraction (0.5 Gy). For doses per fraction of 1 Gy or smaller the yield of exchange aberrations does not change much either. The right panel (B) shows survival curves for the same cells under the same conditions but each total dose was broken up into smaller and smaller fractions with 6 hours between fractions in all cases. Cell cycling during treatments was not a problem in these experiments since the cells were irradiated in contact-inhibited monolayers (G,) and were not sub-cultured until 24 hours after the last fraction to insure that all repair or fixation of damage that was going to occur

had occurred before making the assessment of aberration yield or cell survival. (From Bedford and Comforth, ref. (4).)

repair process. In any case, it appears that at least two damaging events must occur for each exchange.

WHAT ABOUT CELLS THAT ARE NOT IRRADIATED IN Gl OR GO, ARE NOT DIPLOID OR ARE NOT NORMAL? The above discussion has focused on the simplest underlying case of normal diploid cells irradiated during the G, period of the cell cycle, or in G,. This situation is simplest because irradiation of G, or G, cells with ionizing radiation results in the production of chromosome-type aberrations in which mitotic chromosomes are affected at the same locus of both chromatids and both daughter cells are, therefore, affected (see Fig. 1). For irradiation of cells in S or G,, chromatid-type aberrations are produced in which chromosomes are not affected at the same locus on both chromatids of mitotic chromosomes (except for isochromatid types), and there is always a chance, depending on the segregation of chromatids at mitosis, that one of the two daughter cells will be entirely normal. For example, a simple chromatid-type deletion should not be lethal at all, because one daughter cell would have a complete genome and should, therefore, be able to form a colony. Several chromatid exchange type aberrtions would also have a probability of not being lethal if the normal chromatids of both affected chromosomes segregated to the same pole at

November 1991, Volume 21, Number 6

T

I 0

2

I

I.

I

4

6

I

I.

I

8

IO

*

I I2

Dose (Gy) Fig. 7. X-ray dose survival curves for AG1522 normal human diploid tibroblasts and ATSBI human diploid fibroblasts from a patient with Ataxia-telangiectasia irradiated as contact-inhibited G, monolayers and either sub-cultured immediately (immediate plating) or after 24 hours (delayed plating) to assay for the colony forming ability (survival) of single cells. The dashed line indicates the survival expected if each initial interphase chromosome break revealed by the PCC technique were potentially lethal and none were repaired. (From Comforth and Bedford, ref. (15)).

mitosis. Clearly, then, it would be expected that more “aberrations” per cell of the chromatid-type would be required to yield the same level of cell killing than would be the case for chromosome-types. It is well known, in fact, that cells rapidly become more and more radioresistant as they progress into S and G,. There may be reasons other than the one described above, but a transition from chromosome-type to chromatid-type aberrations would certainly contribute to this. Cells irradiated in late G, and mitosis are not very sensitive to the induction of exchange aberrations in that mitosis immediately after irradiation because the chromosomes are beginning to condense or have already condensed, yet they are radiosensitive with respect to cell killing. This sensitivity is reflected in a high sensitivity with respect to aberrations appearing at the following mitosis. Polyploidy and aneuploidy represent one class of abnormality that affect radiosensitivity. A number of studies have shown tetraploid cells to be more “radioresistant” than diploid cells. As mentioned earlier, tetraploid cells with twice the chromosome and DNA content have twice the aberration frequencies per cell for a given dose com-

Chromosomalaberrations, sublethal, and potentially lethal damage 0 J. S. BEDFORD

\

\

Fig. 8. The initial breakage and rejoining of G, chromosome breaks after an x-ray dose of 6 Gy is shown as studied by inducing premature chromosome condensation (PCC) at various times after irradiation of two low passage cell lines from normal humans (AG1522 and AG6234) and two lines from different patients with Ataxia-Telangiectasia (ATSBI and GM2052). While the initial break frequency and the rate of rejoining were the same for normal and A-T cells, the residual frequency of excess PCC fragments (number of PCCs in irradiated cells minus the number in unirradiated cells after long rejoining times) was much higher in A-T cells. The residual frequency after long incubation times is not due to unrejoined breaks but to mis-rejoined breaks that

yield excess fragments and which later appear in mitosis mostly as interstitial (exchange) deletions. (From Comforth and Bedford, ref. (17)).

pared to their diploid counterpart, so if they were dying by a “dominant” mechanism such as anaphase bridge formation, they would be more sensitive rather than more resistant. If they die from genetic deficiencies resulting from acentric fragment loss, then a calculation similar to that outlined for yeast cell killing by induced recessive lethal events (42, 63) leads to the prediction of greater radioresistance, which is also accompanied by an increased survival curve shoulder. This is what is generally observed, but the situation is not so straightforward for aneuploid cells with many chromosomal rearrangements. For a hypotetraploid cell line with numerous chromosomal rearrangements, Revel1 and co-workers (52) found the same aberration and fragment loss frequencies per chromosome per unit dose as for their diploid and tetraploid counterparts, but the cells were even more radioresistant (broader survival curve shoulder) than the tetraploids. Still, colony forming ability was quantitatively reduced in proportion to the amount of fragment loss. Why, in absolute terms, these aneuploid cells were so tolerant to fragment loss is an extremely interesting but unanswered question. There are several interesting examples of “abnormal” cells where the abnormality specifically affects ionizing radiation sensitivity in a dramatic way. Cells from patients with Ataxia-Telangiectasia (A-T), and of course the patients themselves, are very radiosensitive (9, 15, 19, 38,

1465

Fig. 9. An illustration that the rate of rejoining of interphase chromosome breaks, indicated by the solid lines in both panels (not “fitted” to the data), is in good agreement with the rate of increase in survival with delayed plating (PLD repair) or with increasing time between two doses of 6 Gy each (SLD) which are represented by the data points. Aberration frequency data were available and closely predicted the measured survival for the “zero” time samples shown in both figures. The same was true for the longer time samples where the curves reach plateaus. Aberration data for intermediate times were not available. What the curves show is simply the me at which survival increases from the zero time minimum to the plateau maximum for each data set with the rate constant for PCC break rejoining used to trace the curves; not the rate constants derived from the survival measurements themselves. Cells were low passage AG1522 normal human fibroblasts irradiated while in a contact-inhibited monolayer. For the split-dose experiment, cells were sub-cultmed and plated for the survival assay 24 hours after the last dose, so all PLD repair or fixation that could occur would have occurred under these conditions for both dose fractions. The only differences in survival, then, would be attributable to SLD repair. (From Cornforth and Bedford, ref. (15); Bedford and Comforth, ref. (4)).

40, 45, 57, 58). Dose response curves for normal and A-T cells with immediate and delayed sub-culture are shown in Figure 7. For A-T cells, a given level of cell killing requires only one-third to one-fifth the dose, given as an acute exposure, necessary to achieve that level in wild-type human cells (15, 19, 45, 57). A-T cells are, of course, cytogenetically identical to normal cells. The condition results from a recessive mutation of a gene which maps to chromosome 11, that is, 1lq22-23 (27). Heterozygotes appear to have a normal wild-type radiosensitivity, but they may not be entirely normal, since an appreciable proportion of breast cancer patients appear to be A-T heterozygotes (54). A-T patients themselves (homozygotes), in addition to several obvious clinical disorders, show an immune deficiency and are highly cancer-prone (57). One of the “hallmarks” of A-T cells is a peculiar chromosomal radiosensitivity. If A-T cells are irradiated in G, or G,, they show not only a large increase in chromosometype aberrations but also a high frequency of chromatidtypes at the first post-irradiation mitosis (15, 57, 58). Chromatid-types are not ordinarily seen in appreciable frequencies following irradiation of normal (or wild-type) cells in G, or G,, although they are seen for similar treatment with DNA single-strand breaking or base damaging agents that do not produce prompt DNA double strand

1466

I. J. Radiation

Oncology

0 Biology 0 Physics

breaks. The increased chromosomal radiosensitivity accounts reasonably well for the increased sensitivity with respect to cell killing. A-T cells are not defective relative to wild-type cells in any known molecular repair process related to their radiosensitivity (38, 59). At least two A-T lines tested show the same initial interphase (G,) chromosome (PCC) break frequency and the same break rejoining rate following a dose of 6 Gy, but, compared to wild-type cells, show a much higher frequency of residual excess PCC fragments after long (24 hr) rejoining times (17). This is illustrated in Figure 8. Others have also reported on the breakage of interphase chromosomes using the premature chromosome condensation (PCC) technique (e.g., 30, 47, 60) first discovered by Johnson and Rao (33). The residual excess PCC fragments result mostly from chromosome intrachanges (interstitial or intercalary deletions) in these cells which involve an exchange and rejoining process (15, 17). They are not defective in break rejoining per se. For contact inhibited monolayers of normal human cells, the rate of interphase chromosome break rejoining is the same as the rate of increase in survival with delayed subculture due to PLD repair, and is also the same as the rate of increase in survival under similar conditions with increasing time intervals between split doses due to SLD repair, as illustrated in Figure 9 (7, 15). Nagasawa and Little have also shown a close correlation between the rates of increase in survival and decrease in chromosomal aberration induction as a function of the time of subculture after irradiation of contact inhibited C3HlOTt cells (44). Delayed plating and split-dose experiments show very little PLD or SLD repair for A-T cells (e.g., see Fig. 7), but they are certainly able to “repair” a great deal of the damage measured in terms of DNA dsb’s (38, 59) and interphase chromosome breaks (15) (see Fig. 8). If any mammalian cell were incapable of repairing DNA dsb’s and even one unrepaired dsb were lethal, the D, dose for X-rays would be about 4 cGy (25 dsb/G, human cell/Gy = 1 dsb/cell/0.04 Gy) (31). Similarly, for interphase chromosome breaks that are expressed following premature chromosome condensation the D, dose would be about 17 to 20 cGy as illustrated by the “dashed-line” survival curve in Figure 7 (5 to 6 PCC breaks/cell/Gy = 1 PCC break/cell/ 0.17 to 0.20 Gy). The D, for A-T cells with immediate or delayed sub-culture is about 45 and 55 cGy, respectively, and for normal diploid human AG1522 cells about 87 and 146 cGy. Clearly if either of these lesions underlie cell killing, some repair must occur even in A-T cells, but the discrepancy is not that large if interphase chromosome PCC breaks are the main lesions of interest. Cells that are subcultured immediately (or log phase cells) must repair some PLD though it may be less (or more PLD is “fixed”) than for delayed sub-culture. = n2(r/R)3,

and in this case,

1 = n2

November

1991. Volume 21, Number 6

ARE THE NUMBER OF PCC BREAKS AFTER A MEAN LETHAL DOSE SUFFICIENTLY HIGH THAT EXCHANGES WOULD BE REASONABLY EXPECTED? It has been estimated that two chromosome breaks must be within a range less than about 0.2 p of each other for an exchange to occur (for review see ref. (53)). If we use this 0.2 p, range for an interaction distance, and a cell nuclear radius of 4 p, the number of randomly distributed breaks (each produced by independent electron tracks) necessary for one potential exchange would be about 89.$ Restitution is thought to occur “most of the time,” but the proportion in which it occurs at a potential exchange site is not known. If it occurred 50% of the time at potential exchange sites and only 50% of exchanges are the lethal asymmetrical types which produce acentric fragments, we would expect that at least 4 X 89 = 356 randomly distributed breaks would be necessary for one lethal exchange per cell. For DNA dsb’s this would require a dose of about 14 Gy (356 dsb + 25 dsb/Gy) or for PCC breaks about 59 Gy (356 PCC breaks + 6 PCC breaks/Gy). Neither one of these results is reasonable from known cellular radiosensitivities. Even if we allow that for a single acute x-ray dose that produces an average of one lethal aberration per human G, cell somewhat less than half of such aberrations are expected to result from two lesions produced by two independent electron tracks (o/p = 6 Gy; ref. (15)), the recalculated doses still would be unreasonably large; 9 and 38 Gy, respectively, if DNA dsb’s or PCC breaks were the critical interacting lesions occurring anywhere within the nucleus. Several explanations for this apparent discrepancy are possible. One is that neither DNA dsb’s nor PCC breaks are related to the damage leading to lethal chromosomal aberrations. Other evidence repels us from this explanation but it should be kept in mind. Another possibility is that an assumption underlying the above calculation involving the radio (r/R)3, where r is the lesion interaction range and R is the nucleus radius, is grossly erroneous. The lesion interaction range has been determined independently by different investigators using basically different approaches and is probably not different from 0.2 p by more than factor of 2. On the other hand, there is absolutely no evidence to support the notion that any two lesions, wherever they may occur in the nucleus within this interaction range, may lead to an exchange. Thus, the term including R3 (proportional to the entire nuclear volume) is indeed in question. Evidence from our own laboratory, which will be presented elsewhere, indicates that ionizing radiation induced exchanges may occur only in certain regions of the genome, and data presented nearly 30 years ago suggested that radiosensitive structures for lethal damage may reside almost

1467

Chromosomal aberrations, sublethal, and potentially lethal damage 0 J. S. BEDFORD

exclusively within a shell located just inside the nuclear membrane (e.g., 12, 13). A five-fold reduction in the volume within the nucleus where exchange is possible would change the above calculation such that a dose of only 6.7 Gy would be necessary to produce an average of one lethal aberration per cell from the two track mechanism (leaving one to be produced from the single track mechanism) assuming initial PCC breaks were the important lesions and could only be produced in this “sensitive volume.” This is close to what has been observed experimentally (15). For DNA dsb’s, it would not be reasonable to expect these to occur in only a fraction of the DNA, but their subsequent processing to form an exchange may well occur in only a fraction. DO MUTATIONS AT LOCI OTHER THAN THE “A-T LOCUS” CONFER IONIZING RADIATION SENSITIVITY AND AFFECT PLD AND SLD REPAIR CAPACITY? A sensitive strain of mouse lymphoma cells designated L5178Y S/S was isolated in 1963 by Beer and co-workers (1, 5). Though wild-type strains of these lymphocytic-derived cells are already more sensitive than average for mammalian cells, and direct comparisons of radiobiological properties of the sensitive and wild-type parent are rarely given, the L5178Y S/S cells are extremely sensitive especially during G, (43) with a D, of the order of 25 cGy. Unlike A-T cells, they become considerably more resistant in late S and early G,. No molecular defect connected with their radiosensitivity is known for these cells, but they do apparently show chromatid-type aberrations (as well as increased chromosome-types) after irradiation in G, so they are similar in this respect to A-T cells. Several ionizing-radiation-sensitive mutants of Chinese hamster cells have been isolated in recent years. The first, designated CHO XRl, was isolated by Stamato and coworkers (54). It, too, is very sensitive in G,, with a D, of about 30 cGy and it is defective in PLD and SLD repair (54). It becomes more radioresistant (though not as sensitive as wild-type cells) during late S and G, (54). CHO XR-1 cells also display the peculiar chromosomal radiosensitivity pattern of A-T cells (2), but the genetic defect is not the same, since Giaccia et al. (28) have shown that normal radioresistance with respect to cell killing is restored in hybrids made between A-T and XRl cells. In collaboration with Stamato and Giaccia, Bahari in my laboratory also demonstrated that the defect leading to the increased sensitivity to chromosome-type aberrations and the production of chromatid-types in G, or G, was corrected in A-T/XR-1 (human/hamster) hybrids. Further, in hybrid cells that have lost most of the human chromosomes, those that retain human chromosome 5 retain radioresistance, whereas those that lose human chromosome 5 lose radioresistence, that is, a concordance was established to enable the mapping of the human gene correcting the hamster radiosensitivity defect to human chromosome 5 (28).

A number of other ionizing radiation sensitive hamster cell mutants have been isolated, such as the XRS 5 and XRS 6 cells of Jeggo (32) that are also defective in SLD and PLD repair. A complete review of this important and newly emerging field involving the genetics of mammalian cell radiosensitivity is beyond the scope of the present paper, but other current reviews are available (e.g., 14, 32).

PHYSICS, CHEMISTRY,

AND BIOLOGY

The early days of “quantitative” radiobiology were dominated by considerations surrounding the physics of energy deposition while the biology was relegated to abstract targets of volume, V, and number, n, within which this energy deposition would lead to biological effects, and which, no doubt, biologists would identify in time. A later area of focus was on the production of short-lived free radicals and their action, or the action of some of their products, again upon abstract targets, this time referred to as “-R’s” or “-R*s.” In either case, the targets were viewed in the abstract, largely because it did not matter what they were within the framework of the general concepts. Examples attesting to the productivity and value of these approaches include the early work of Lea (37) and development of the dual theory of radiation action by Kellerer and Rossi (35), which have provided a foundation for our understanding of the nature of RBE, of dose-time factors in radiotherapy and radiation protection, and the development of chemical radiosensitizers and radioprotectors now undergoing (or being developed for) clinical trials in radiotherapy clinics around the world. Although radiation cytogeneticists had argued for many years that chromosomal aberrations were of prime importance in producing biological effects, a number of apparently conflicting observations held many radiobiologists from fully embracing their central relevance. Meanwhile, “molecular” radiation biology focused primarily on methods for measuring the production of various kinds of damage in DNA and its disappearance after irradiation. More recently, principal interests and objectives in molecular radiation biology have turned toward identifying cloning, mapping and determining the protein products of genes involved in DNA repair. Biology in general, and cytogenetics in particular, now holds a position that is stronger than ever in influencing and guiding efforts in these directions. Basic knowledge from classical radiation cytogenetics already serves as an important guide. Treatment of G, or G, cells with agents known to produce appreciable numbers of DNA double strand breaks immediately (e.g., ionizing radiations, bleomycin, restriction endonucleases) are known to result in the formation of chromosome-type aberrations at the first subsequent mitosis, and produce chromatid-types only when cells are treated in S or G, (see Fig. 1). Similar

1468

1. J. Radiation Oncology

0 Biology 0 Physics

treatment in G, or G, with agents that primarily produce base damage (e.g., U.V. light, alkylating agents) or single strand breaks (e.g., “BUdR + light”) yields chromatidtype aberrations (7). How important, then, is the base damage produced by ionizing radiation in normal cells? What about A-T cells and the x-ray sensitive rodent cell mutants? How important is chromatin structure and/or its transcriptional activity in determining ionizing radiation responses? Are virtually all DNA double strand breaks effectively repaired in certain segments of chromatin so exchanges occur only in others (17)? Is the process leading to the formation of chromosome exchanges fundamen-

November

199 1, Volume 2 1, Number 6

tally different from restitution at the molecular level? If so, how? How is tolerance to genetic loss following acentric fragment production modified or altered with the development of various forms of aneuploidy and chromosoma1 rearrangement? To what extent and under what circumstances may processes other than chromosomal aberration production contribute to cell killing by ionizing radiation? These are only a few questions whose answers may provide insights for clinical exploitation that are presently unknown. Recent advances in cell and molecular biology now offer, for the first time, approaches to answer these questions.

REFERENCES 1. Alexander, P.; Mikulski, Z. B. Mouse lymphoma cells with different radiosensitivities. Nature Lond. 192572-573; 1961. 2. Bahari, I. B.; Bedford, J. S.; Giaccia, A. J.; Stamato, T. D. Measurement of the relative proportion of symmetrical and asymmetrical chromosome-type interchanges induced by gamma radiation in human-hamster hybrid cells. Radiat. Res. 123:105-107; 1990. 3. Bedford, J. S.; Mitchell, J. B.; Griggs, H. G.; Bender, M. A. Radiation-induced cellular reproductive death and chromosome aberrations. Radiat. Res. 76573-586; 1978. 4. Bedford, J. S.; Comforth, M. N. Relationship between the recovery from sublethal X-ray damage and the rejoining of chromosome breaks in normal human fibroblasts. Radiat. Res. 111406-423; 1987. 5. Beer, J. Z.; Lett, J. T.; Alexander, P. Influence of temperature and medium on the x-ray sensitivities of leukaemia cells in vitro. Nature Lond. 199:193-194; 1963. 6. Belli, J. A.; Shelton, M. Potentially lethal radiation damage: repair of mammalian cells in culture. Science 1654:4w92; 1969. 7. Bender, M. A.; Griggs, H. G.; Bedford, J. S. Mechanisms of chromosomal aberration production. III. Chemicals and ionizing radiation. Mutat. Res. 23: 197-212; 1974. 8. Bender, M. A.; Wolff, S. X-ray induced chromosome aberrations and reproductive death in mammalian cells. Am. Naturalist 95:39-52; 1961. 9. Bridges, B. A.; Hamden, D. G. Ataxia-telangiectasia-a cellular and molecular link between cancer, neuropathology and immune deficiency. London: John Wiley and Sons, Inc.; 1982. 10. Carrano, A. V. Chromosome aberrations and radiation-induced cell death. I. Transmission and survival parameters of aberrations. Mutat. Res. 17:341-353; 1973. 11. Carrano, A. V. Chromosome aberrations and radiation-induced cell death. II. Predicted and observed cell survival. Mutat. Res. 17:355-366; 1973. 12. Cole, A.; Humphrey, R. M.; Dewey, W. C. Low voltage electron beam irradiation of normal and BUdR-treated L-P59 mouse fibroblast cells in vitro. Nature 199:780-782; 1963. 13. Cole, A.; Meyn, R. E.; Chen, R.; Cony, P. M.; Hittelman, W. Mechanisms of cell injury. In: Meyn, R. E.; Withers, H. R., eds. Radiation biology in cancer research. New York: Raven Press; 33-58. 14. Collins, A.; Johnson, R. T. DNA repair mutants in higher enkaryotes. J. Cell Sci. 6:Suppl. 61-82; 1987. 15. Comforth, M. N.; Bedford, J. S. A quantitative comparison

16.

17.

18. 19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

of potentially lethal damage repair and the rejoining of interphase chromosome breaks in low passage normal human flbroblast. Radiat. Res. 11 I:38545; 1987. Comforth, M. N.; Bedford, J. S. X-ray-induced breakage and rejoining of human interphase chromosomes. Science 222:1141-l 143; 1983. Comforth, M. N.; Bedford, J. S. On the nature of the defect in cells from individuals with ataxia telangiectasia. Science 227:1589-1591; 1985. Comforth, M. N. Testing the notion of the one-hit exchange. Radiat. Res. 121:21-27; 1990. Cox, R.; Mason, K.; Weichselbaum, R. R.; Nove, J.; Little, J. B. The repair of potentially lethal damage in x-irradiated cultures of normal and ataxia-telangiectasia human fibroblasts. Int. J. Radiat. Biol. 39:357-365; 1981. Davies, D. R.; Evans, H. J. The role of genetic damage in radiation-induced cell lethality. In: Lett, J. T., Adler, H., eds. Advances in radiation biology, Vol. 2. New York: Academic Press; 243-340. Dewey, W. C.; Miller, H. H.; Leeper, D. B. Chromosomal aberrations and mortality of X-irradiated mammalian cells: emphasis on repair. Proc. Natl. Acad. Sci. USA 68:667671; 1971. Dewey, W. C.; Noel, J. S.; Dettor, C. M. Changes in radiosensitivity and dispersion of chromatin during the cell cycle of synchronous Chinese hamster cells. Radiat. Res. 52:373394; 1972. Elkind, M. M.; Sutton, H. Radiation response of mammalian cells grown in culture. I. Repair of x-ray damage in surviving Chinese hamster cells. Radiat. Res. 13:556-593; 1960. Elkind, M. M.; Whitmore, G. F. The radiobiology of cultured mammalian cells. New York: Gordon and Breach; 1967. Evans, H. J. Repair and recovery from chromosome damage induced by fractionated x-ray exposures. In: Silini, G., ed. Radiation Research: Proceedings of the Third International Congress of Radiation Research. Amsterdam: North-Holland; 1967:482-501. Evans, H. J. Repair and recovery at chromosome and cellular levels: similarities and differences. Recovery and repair mechanisms in radiobiology. Brookhaven Symp. Biol. 20: 11 l-133; 1967. Gatti, R. A.; Berkel, I.; Boder, E.; Braedt, G.; Charmley, P. Localization of an ataxia-telangiectasia gene to chromosome 11 q 22-23. Nature (Lond.) 336:577-580; 1988. Giaccia, A. J.; Denko, N.; MacLaren, R.; Mirman, D.; Waldren, C.; Hart, I.; Stamato, T. D. Human chromosome

Chromosomal

29.

30.

31.

32.

33.

34.

35. 36.

37. 38.

39.

40.

41.

42.

43.

44.

45.

46.

aberrations,

sublethal,

and potentially

5 complements the DNA double strand-break-repair deficiency and gamma-ray sensitivity of the XR-1 hamster variant. Am. Hum. Genet. 47:459-469; 1990. Hahn, G. M. Radiation and chemically induced potentially lethal lesions in noncycling mammalian cells: Recovery analysis in terms of x-ray- and ultraviolet-like systems. Radiat. Res. 64:553-545; 1975. Hittleman, W. N.; Rao, P. N. Premature chromosome condensation. 1. Visualization of X-ray induced chromosome damage in interphase cells. Mutat. Res. 23:251-258; 1974. Iliakis, G. E.; Cicilioni, 0.; Metzger, L. Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis: calibration by means of i2’1 decay. Int. J. Radiat. Biol. 59:343357; 1991. Jeggo, P. A. Studies on mammalian mutants defective in rejoining double strand breaks in DNA. Mutat. Res. 239:1-16; 1990. Johnson, R. T.; Rao, P. N. Mammalian cell fusion. Induction of premature chromosome condensation in interphase nuclei. Nature 226:717-722; 1970. Joshi, G. P.; Nelson, W. J.; Revell, S. H.; Shaw, C. A. X-ray-induced chromosome damage in live mammalian cells, and improved measurements of its effects on their colony forming ability. Int. J. Radiat. Biol. 41:161-181; 1982. Kellerer, A. M.; Rossi, H. H. The theory of dual radiation action. Cur. Top. Radiat. Res. 8:85-158; 1972. Kosaka, T.; Kaneko, I.; Koide, F. Correlation between nonrepairable DNA lesions and fixation of cell damage by hypertonic solutions in Chinese hamster cells. Int. J. Radiat. Biol. 58:417-425; 1990. Lea, D. E. Actions of radiations on living cells, 2nd edition. London: Cambridge Univ. Press; 1955. Lehmann, A. R.; Stevens, S. The production and repair of double strand breaks in cells from normal humans and from patients with ataxia telangiectasia. Biochem. Biophys. Acta 474149-60; 1977. Little, J. B. Repair of sub-lethal and potentially lethal radiation damage in plateau phase cultures of human cells. Nature 224:804-806; 1969. Little, J. B.; Nagasawa, H. Effect of confluent holding on potentially lethal damage repair, cell cycle progression, and chromosomal aberrations in human and ataxia telangiectasia fibroblasts. Radiat. Res. 101:81-93; 1985. McNally, N. J.; Hinchliffe, M.; Soranson, J. The effect of post-irradiation anisotonic treatment on cell survival and repair of DNA damage. Int. J. Radiat. Biol. 57:503-512; 1990. Mortimer, R. K. Radiobiological and genetic studies on a polyploid series (haploid to hexaploid) of Saccharomyces cerevisiae. Radiat. Res. 9:312-326; 1958. Nagasawa, H.; Cox, A. B.; Len, J. T. The radiation responses of synchronous L5178Y S/S cells and their significance for radiobiological theory. Proc. Royal Sot. Lond. B211:2549; 1980. Nagasawa, H.; Little, J. B. Induction of chromosome aberrations and sister chromatid exchanges by X-rays in densityinhibited cultures of mouse lOTi cells. Radiat. Res. 87:538551; 1981. Nagasawa, H.; Little, J. B. Comparisons of kinetics of X-ray induced cell killing in normal, ataxia telangiectasia and hereditary retinoblastoma flbroblasts. Mutat. Res. 109:297308; 1983. Neary , G. J. Chromosome aberrations and the theory of RBE, I. General considerations. Int. J. Radiat. Biol. 9:477502; 1965.

lethal damage 0 J. S. BEDFORD

1469

47. Pantelias, G. E.; Maille, H. D. Direct analysis of radiationinduced chromosome fragments and rings in unstimulated human peripheral blood lymphocytes by means of the premature chromosome condensation technique. Mutat. Res. 149: 67-72; 1985. 48. Phillips, R. A.; Tolmach, L. J. Repair of potentially lethal damage in x-irradiated HeLa cells. Radiat. Res. 29:413-432; 1966. 49. Puck, T. T. Action of radiation on mammalian cells. III. Relationships between reproductive death and induction of chromosome anomalies by X-irradiation of euploid human cells in vitm. Proc. Natl. Acad. Sci. USA 44:772-780; 1958. 50. Raaphorst, G. P.; Dewey, W. C. Fixation of potentially lethal radiation damage by post-irradiation exposure of Chinese hamster cells to 0.5m or 1.5m NaCl. Int. J. Radiat. Biol. 36:303-315; 1979a. 51. Raaphorst, G. P.; Dewey, W. C. Alterations in the radiosensitivity of CHO cells by anisotonic treatments: correlations between cell lethality and chromosome aberrations. Radiat. Res. 79403416; 1979. 52. Revell, S. H. Relationship between chromosome damage and cell death. Ishihara, T., Sasaki, M. S., eds. In: Radiationinduced chromosome damage in man. New York: Liss; 215233. 53. Savage, J. K. R. Sites of radiation induced chromosome exchanges. Curr. Top. Radiat. Res. 6:129-194; 1970. 54. Stamato, T. D.; Weinstein, R.; Giaccia, A.; Mackenzie, L. Isolation of a cell cycle-dependent gamma-ray sensitive Chinese hamster ovary cell. Somat. Cell Genet. 9: 165-173; 1983. 55. Stapleton, G. E.; Bilen, D.; Hollaender, A. Recovery of X-irradiated bacteria at suboptimal incubation temperatures. J. Cell Comp. Physiol. 41:345-357; 1953. 56. Swift, M.; Reitnauer, P. J.; Morrel, D.; Chase, C. L. Breast and other cancers in families with ataxia-telangiectasia. N. Engl. J. Med. 316:1289-1294; 1987. 57. Taylor, A. M. R.; Hamden, D. G.; Arlett, C. F.; Harcourt, S. A.; Lehman, A. R.; Stevens, S.; Bridges, B. A. Ataxiatelangiectasia: A human mutation with abnormal radiation sensitivity. Nature 258:427429; 1975. 58. Taylor, A. M. R. The effect of radiation on the chromosomes of patients with an unusual cancer susceptibility. In: Ishihara, T., Sasaki, M. S., eds. Radiation-induced chromosome damage in man. New York: Liss; 167-199. 59. Van Der Schans, G. P.; Centen, H. B.; Lohman, P. H. M. The induction and repair of double-strand DNA breaks in normal human and ataxia-telangiectasia cells exposed to [‘%o]-y-radiation 4-nitroquinoline-l-oxide or bleomycin. In: Bridges, B. A., Hamden, D. G., eds. Ataxia-telangiectasia: a cellular and molecular link between cancer, neuropathology, and immune deficiency. New York: Wiley; 1982: 291303. 60. Waldren, C. A.; Johnson, R. T. Analysis of interphase chromosome damage by means of premature chromosome condensation after X-ray and ultraviolet irradiation. Proc. Natl. Acad. Sci. USA 71:1137-l 141; 1974. 61. Whitmore, G. F.; Gulyas, S. Studies on recovery processes in mouse L cells. Natl. Cancer Inst. Monogr. 24:141-156; 1967. 62. Winans, L. F.; Dewey, W. C.; Dettor, C. M. Repair of sublethal and potentially lethal x-ray damage in synchronous Chinese hamster cells. Radiat. Res. 52:373-394; 1972. 63. Zirkle, R. E.; Tobias, C. A. Effects of ploidy and linear energy transfer on radiobiological survival curves. Arch. Biothem. Biophys. 47:282-306; 1953.