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Radiotherapy and Oncology 39 (1996) 155-165

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Relationship between DNA damage, rejoining and cell killing by radiation in mammalian cells

ML N6iieza, T.J. McMillanb, M.T. Valenzuelaa, J.M. Ruiz de Almod6var*a, V. Pedrazaa aLaboratorio & Investigaciones Mkdicas y Biologia Tumoral, Departamento de Radiologia y Medicina F&a, Universidad de @ana&,

18071 Granada. Spain bThe Institute of Cancer Research, Radiotherapy Research Unit, Sutton, Surrey. SM2 5NG. UK

Received 23 June 1995; revised 31 January 1996; accepted 12 February 1996

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The prevailing hypothesis on the mechanism of radiation-induced cell killing identifies the genetic material deoxyribonucleic acid (DNA) as the most important subcellular target at biologically relevant doses. In this review we present new data and summarize the role of the DNA double-strand breaks (dsb) induced by ionizing radiation and DNA dsb rejoining as determinants of cellular radiosensitivity. When cells were irradiated at high dose-rate, two molecular end-points were identified which often correlated with radiosensitivity: (1) the apparent number of DNA dsb induced per Gy per DNA unit and (2) the half-time of the fast component of the DNA dsb rejoining kinetics. These two molecular determinants, not mutually exclusive, may be linked through a common factor such as the conformation of DNA.

Keywords: Radiosensitivity; DNA double-strand breaks; Initial damage; Rejoining

1. Introdwtion

Although experimental error, cell selection and tech- nical factors may all contribute to variation in radiosensitivity between cell lines, it is difficult to avoid the conclusion that, when measured at a clinically realistic dose rate, radiosensitivity varies widely among human tumor types [81],

However, it is not clear why cells differ in their sensitivity to ionizing radiation. This question is important both in cancer therapy - where normal and tumor cells differ in their sensitivities - and in the field of car- cinogenesis, where environmental exposure to radiation hazards may have different effects on different individuals. Radiotherapy is an important modality of treatment used in more than half of all cancer patients. It would, therefore, be useful to know why cells differ in their radiosensitivity.

l Corresponding author, Tel: +34-58-244056; Fax: +34-58-242865.

Experimental studies have shown that the surviving fraction at 2 Gy (SF2) measured in vitro can predict response to in vivo irradiation [30,91-931. It is therefore important to continue to develop rapid ways to predict tumor. radiosensitivity and normal tissue tolerance. In recent years, clonogenic assays have been widely used to measure cell radiosensitivity. A problem with these clonogenic assays is the time required to determine sensitivity, since it is not always acceptable to delay a treatment decision in radiotherapy for more than 2-3 weeks while radiosensitivity is analyzed in the laboratory. Alternative measures of sensitivity have recently been developed using different DNA damage assays, including the pulsed-field gel electrophoresis (PFGE) technique. This assay is used to quantify the apparent number of DNA double-strand breaks (dsb) as well as dsb rejoining, but tells us nothing about the accuracy of these processes. In contrast, it is sensitive at doses relevant to cell survival and can measure heterogeneity of damage or repair.

The prevailing hypothesis on the mechanism of

0167-8140/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII: SOl67-8140(96)01732-X

156 M.I. Ntiiiez et al. /Radiotherapy and Oncology 39 (1996) 155-165

radiation-induced cell killing identifies the genetic material deoxyribonucleic acid (DNA) as the most important subcellular target at biologically relevant doses. Two main hypotheses have been proposed to explain differences in radiosensitivity among cells: (1) cells may vary in the amount of damage induced by a given dose of radiation [l 1,43,49,65,74,96] and (2) cells may differ in their capacity to repair radiation damage to DNA [29,43,45,52,53,78,96,101]. Within any chosen system the relative importance of these two factors may vary so that neither may explain radiosensitivity in every case.

Radiation damage to mammalian cells can be divided into two categories: (1) lethal damage, which is unrepairable and leads irrevocably to cell death and (2) sublesions resulting from sublethal or potentially lethal damage, which under normal circumstances can be repaired in hours unless additional damage is added or interaction of sublesions takes place. The fate of cells that have acquired sublesions depends on competing processes of repair and fixation. The fixation process is conceived as one of binary interaction between sublesions. This is the component of radiation damage that can be modified by postirradiation environmental conditions.

Double-strand breaks in DNA are the most common type of radiation lesions that lead to mammalian cell death [27,41,44,67]. Mammalian cells are proficient in the repair of DNA damage, but not all radiation lesions undergo repair. The molecular basis of the DNA dsb rejoining process is not clear, but presumably is dependent on the postirradiation chemical processes [ 12,13,67]. Al- though there is evidence associating proficiency of DNA dsb rejoining with cell lethality, it is clear that this represents a mechanistic oversimplification [lo]. It is important to underline that DNA double-strand break rejoining could be equivalent to repair if done properly and to misrepair when the lesion cannot be repaired correctly. Mammalian cells repair the majority of double- strand breaks and it seems likely that failure to repair subclasses of dsbs, possibly in critical genomic sites, results in cell death following irradiation. There can be lit- tle doubt that such extensive repair of a variety of DNA lesions will lead to a natural selection process in which the least repairable lesion will preferentially lead to cell killing [al]. Sites with multiple local lesions [89] or some subclasses of dsb, possibly in critical genomic sites, are candidates for cell death after irradiation.

The search for explanations for the variations in cellular radiosensitivity has involved different cell systems whose results may not always be comparable. However, on the basis of the cellular model used, the data obtained by several authors can be classified into four groups: (a) radiosensitive mutant/parental cell line, (b) cells irradiated in the presence/absence of various radiomodifiers, (c) radiosensitive human syndromes and (d) human tumor cell lines.

We will discuss separately (1) the role of initial induced DNA damage and (2) the influence of dsb rejoining data cellular radiosensitivity, using the values of surviving fraction at 2 Gy as the measure of the cell sensitivity to the killing effects of ionizing radiation.

2. Inltlal DNA damage

The chain of events leading from exposure to radiation to cell death involves DNA damage. This damage may be either direct or indirect by free radicals produced by secondary chemical reactions around the DNA, often involving the water lying close to DNA. After damage has been induced, many processes remove and repair the damage in an attempt to restore the genetic sequence to its original state. At the physicochemical level, damage involves ionization of atoms within the DNA molecules with consequent disruption of interatomic bonds and deformation of other abnormal covalent bonds between the constituents of DNA and chromatin. There is com- petition between rapid chemical restitution of damage and its fixation by the interaction of oxygen. The damage remaining at this stage, termed initial damage, may be lethal to a cell if not repaired. The level of initial damage is usually measured in cells treated on ice, in which enzymatic repair does not occur.

Familiarity with the dose-response relationship is necessary to better understand the link between DNA dsb induced by radiation and cell survival. Some studies have reported a non-linear dose-response relationship for dsb induction [65,67,68,97]. This has been inter- preted as a consequence either of the exhaustion of a chemical repair process or as a cooperative process of damage induction [67,68]. In contrast, a number of authors have found a linear relationship between the yield of dsb and dose [5,12,17,26,27,47,49,54,76]. This controversial point may also reflect differences in the methodology used [37]. In an attempt to shed light on this issue, Ward, on the basis of data on the chemical process that occur in cells during irradiation, concluded that, in the presence of oxygen, the number of DNA dsb is linearly related to dose [90]. Recent data support this conclusion [36,74,95]. It is worth emphasizing that most of the effect of oxygen on cell survival can be explained in terms of modifications in the level of induced damage [36,90,95].

3. The role of initial DNA damage as a determinant of cell radIosl?nsItlvlty

The question now is: are differences between cell lines in the amount of initial damage responsible for differences in radisensitivity? To answer this question we will analyze the reports separately, according to the cell system chosen, since this can be critical to the results obtained. The logic behind this is as follows.

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(1) The amount of damage to DNA can be affected by differences in the concentration of substances that in- teract with free radicals during irradiation. A large pro- portion of the results obtained were from experiments in which both cellular radiosensitivity and DNA damage were measured in the presence of radiomodifiers [66-68,76,95].

(2) It has been suggested that differences in molecular damage can be attributed to the chromatin conformation. This might give scavenging molecules different degrees of access to damaged DNA [55,56,58,90].

3.1. Radiosensitive mutant/parental cell lines

Many analyses of the molecular basis of radiosensitivity have been done in radiosensitive mutants isolated in the laboratory. In many cases radiosensitive strains showed the same amount of initial DNA damage after irradiation as the parental line [50]. The same conclusion has been reached by other authors using rodent cell lines [12,31,37,39,45,46]. Yasui et al. [lOO] have studied the nuclear organization in the radiation sensitive xrs-5 cell line and its parental CHO Kl cell and have suggested that the different DNA attachment organization in the xrs-5 cells may play a role in modulating radiation sensitivity. The differences in radiosensitivity between mutants isolated to date and parental cells could not be attributed to differences in DNA dsb induction. Iliakis et al. [38] have studied rat embryo cells (REC), REC transformed by the H-ras plus v-myc on-

cogenes (3.7) and immortalized REC (mycREC), and found that the 3.7 cells are relatively resistant to ionizing radiation in comparison with the non-transformed parent cells or mycREC cells. Both cell lines show a similar amount of DNA dsb per Gy.

Although it is not easy to draw a general conclusion, most studies published to date show that the initial radiation-induced DNA damage is the same in radioresistant as in radiosensitive variants. Considering the large number of steps that follow the irradiation of a cell is not surprising that damage induction is not always different between pairs of radiosensitive and radioresistant cells. Differences in cellular radiosensitivity may arise from a molecular determinant other than the yield of dsb produced per radiation dose unit.

Results obtained in our laboratory (Figs. la and lb) from a cell line and its corresponding radioresistant mutant (A2780 and A2780 cp [4]) support this conclusion. Differences in radiosensitivity could be due in this case to differences in glutathione content (Fig. lc).

3.2. Cells irradiated in the presence or absence of radiomodifiers

To assess the contribution of DNA dsb to cell killing, it would be reasonable to vary the amount of damage by irradiation under different conditions and then to test whether the dsb dose-response curves still correlate with cellular survival. It is believed that two-thirds of the damage to DNA in mammalian cells occurs via the hy-

0 10 20 30 40 !

Dose (Gy) z ; 200-

Dose (Gy)

T

q A2780

0 A2780cp

Fig. 1. (A) Acute cell survival curves for A2780 and A2780 cp. Survival data have been calculated by fitting the experimental data to the lineal quadratic model. (B) Relationship between dsb and dose for A2780 and A2780 cp cell lines. The number of DNA induced by radiation has been calctilated by fitting the distribution of DNA fragment size data after PFGE to a mathematical model detailed in Ref. [74]. (C) Relative measure of GSH for A2780 and A2780 cp. The GSH content was estimated by flow cytometry as indicated by Fahey and Newton 125) and Rice et al. [71].

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droxyl radical. Substances that bind these free radicals during irradiation, i.e. oxygen and hydrogen donors, are compounds such as those containing sulfhydryl groups. These molecules are generally termed radiomodifiers. Studies of the influence of radiomodifiers [66-68,76,95] suggest clearly that the yield of dsb may reflect competi- tion between oxygen and chemical repair reactions. Alaoui-Jamali et al. [l] studied the radiosensitivity of the wild-type MCF-7 human breast cancer cell line and compared it with that of radioresistant subline MCF-7 ADR@. They found that GSH depletion by BSO treatment sensitizes ADRo cells, but not WT cells, to killing ,by radiation. Thus the level of dsb induced by radiation generally varies with radiomodifier status in such a way that may be sufficient to account for the entire radiomodifier effect seen in clonogenic assays.

3.3. Radiosensitive syndromes in humans

Further information has come from the study of DNA damage-inducible responses in mammalian cells derived from humans with diseases or syndromes that make them inherently more sensitive than is normal to radiation. Ataxia-telangiectasia (AT) is one of the best- studied members of a group of human genetic disorders characterized by hypersensitivity to radiation [82]. The sensitivity of AT lymphocytes and cultured flbroblasts to the lethal effects of X-rays is a hallmark of the disease [59]: ataxia-telangiectasia cells are three to four times more radiosensitive than normal cells at high and low dose-rate exposure [6,16]; however, the level of initial damage in AT fibroblasts is similar to that in normal fibroblasts [62,70,98].

3.4. Human tumor cell lines

To elucidate the connections between molecular damage and cell survival, Ward [90] examined the mechanism of production of radiation damage and postulated that, under oxic conditions, (1) the number of DNA dsb is linearly related to dose and (2) although the chromatin structure may be different in radiosensitive cells, making the DNA more accessible to radical attack, the yields of molecular damage are probably independent of tumor cell type. Thus, Ward considers that sensitive cells may be repair-deficient.

The argument presented by Ward [90] indicated that variations in yields of molecular damage to DNA per dose in oxic cells are probably constant. However, results published by others do not present a consistent pic- ture to support Ward’s hypothesis.

Kelland et al. [43], Peacock et al. [62], McMillan et al. 1491 and others [74,96] compared initial damage in a number of human tumor cells of differing sensitivities and reported significant differences in levels of initial damage. Also the work of Radford et al. [67-701, has

shown that the level of initial DNA damage measured by neutral filter elution correlates with cell killing after irradiation under a variety of conditions and in cells of differing radiosensitivity. Peacock et al., working with human tumor cell lines, have found that sensitive cells incur a larger number of lesions per Gy than do resistant cells [62,63]. Initial damage (i.e. damage prior to enzymatic repair) therefore appears greater in sensitive cells. Some of the other studies of this topic are listed in Table 1 which shows that this opinion has been supported by others. It therefore now seems clear that: (1) there is a linear relationship between DNA dsb and dose [73], and (2) the high radiosensitivity of certain human tumor cell lines may result from high levels of initial radiation- induced DNA damage. Our results in human breast cell lines [74] support the view that initial damage is a major determinant of cell radiosensitivity. It has been argued that apparent differences in damage induction may be simply due to DNA conformation differences affecting behaviour in the assays. However, if this is the case then, as Ward points out [90], the differences in conformation may also affect the true level of dsb.

4. DNA double-strand break rejoining

Double-strand breaks in DNA are associated with killing in mammalian cells, as damage to both strands of the DNA is assumed to deprive the repair system of the template required for efficient and accurate restoration of the molecule. The importance of dsb in cell killing has been demonstrated by the isolation of radiosensitive mutant cell lines partially defective in their ability to repair this type of lesion. Jeggo et al. [41] found that radiosensitivity in xrs mutant cell lines is accompanied by a reduction in the rate of dsb rejoining and by an increase in the fraction of dsb remaining unrejoined after prolonged incubation at 37°C. It was recently found that complementation of the radiosensitive phenotype in such mutants by the introduction of Ku80 cDNA is accompanied by restoration of proficiency DNA dsb rejoining to almost that of parental cells, as measured both by a neutral single-cell microgel electrophoresis technique (Comet assay) and by PFGE. These results provide further biochemical evidence of the involvement of Ku protein in the repair of DNA dsb [42,72].

Studies of the DNA dsb rejoining kinetics during postirradiation incubation of cells have fitted the experimental data to a mathematical model defined by a monoexponential function [2,8,9,85], or more frequent- ly, to a combination of two monoexponential functions (biphasic model) that reflect the existence of two different components - fast and slow - of the kinetic process [10,15,16,28,38,69,80,87]. Frankenberg-Schwager and colleagues have stressed a most interesting feature of these kinetics [28], showing that the proportions of

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Table I Molecular bases of radiosensitivity in human tumor cell lines. The role of initial damage

Measurement technique Cell lines Comments Reference

Neutral Bher elution

Neutral falter elution

Neutral falter elution

Asymmetric field inversion gel electrophoresis

Neutral filter elution/ neutral comet assay

Pulsed-field gel electrophoresis

Neutral filter elution


Five human cervix carcinoma cell lines

Two tumor cell lines (neuroblastoma and bladder carcinoma) and three tibroblast lines Nine human tumor cell lines (lung, bladder, neuroblastoma, rhabdo- myosarcoma, melanoma)

Five human tumor cell lines (head and neck) and one normal human Iibro- blast line. Six human tumor cell lines (melanoma glioma, adenocarcinccarcinoma, prostate and cervical carcinomas)

Five human breast cancer and one human bladder carcinoma cell lines.

Twenty primary cultures obtained from ovarian cancer and malignant melanoma. Eight human tumor cell lines (neuroblastoma, lung, melanoma, medulloblastoma, bladder and cervix).

dsbs induced after irradiation correlates with cellular radiosensitivity. The most radiosensitive lines incurred more breaks than the most radioresistant lines. In tumor cell lines radiosensitivity was determined by the initial incidence of lesions. No difference was found in the tibroblasts (including AT). Differences in radiosensitivity may be due at least in part to different levels of damage induction, but some lines may vary in their tolerance of damage due to differences in biological characteristics such as repair capacity. There was no correlation between initial DNA dsb formation and radiosensitivity.

They found differences in initial numbers of dsb using the neutral filter elution but there was no correlation with radiosensitivity. dsb induction was similar among these cells when measured with comet assay. Using a mathematical model based on the DNA fragment size distribution in the gel line to calculate dsb/Gy per DNA unit, a statistically significant relationship was found between damage induced and cellular radiosensitivity. They did not find any relationship between the initial frequencies of DNA dsbs and SF2

Statistically significant correlations were found between cellular radiosensitivity (SF2) and the slope of the dose-response curves for molecular DNA damage (dsb) as measured by PFGEb.

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Bdsb, double-strand break. bPFGE, pulsed-field gel electrophoresis.

the two components are dose-dependent. This dose de- pendency slows the overall rate of dsb rejoining as the dose increases, although each of the two components shows a dose-independent rate of dsb rejoining. We recently obtained evidence in support of this finding in two cell lines of different radiosensitivity [53]. The multiple phases may reflect the repair of different types of lesion, with the residual damage as a final subset of the lesions [81]. Therefore, it makes sense to classify DNA dsb induced by ionizing radiation according to their rejoining kinetics [23]. We would distinguish: (1) type I, dsb as those rejoined by the fast component of the kinetic process, (2) type II, dsb as those rejoined by the slow component and (3) type III, dsb as breaks that are not rejoined at the end of the incubation time (residual damage) (Fig. 2). This phenomenon has been inter- preted in terms of the mechanism of dsb formation and the enzymatic mechanism of repair. Type I dsb would thus be DNA dsb produced by the attack of a single OH radical [79], or a break rejoined by enzymes which are constitutively expressed in mammalian cells [7,35,69];

type II DNA dsb may be associated with dsb produced by multiple radical attack [89], or may represent dsb rejoined by the induced products of specific genes involved in the repair of more complex types of DNA lesion

lo-a”““““.““. I ” ” ” ” 0 4 8 12 16 20 24

Time (hours)

Fig. 2. Different types of DNA dsb (type I, type II and type III) induced by ionizing radiation in a human tumor cell line (MCF-7 BB).

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[7,32]. Finally, type III DNA dsb may be severe lesions formed by single hit events which cannot be repaired at any dose-rate [75,81].

5. Tbe role of DNA dsb rejoining as a determinant of eelluIar radiosensitivity

5.1. Radiosensitive mutant and parental cell lines

Jeggo investigated xrs-6 and XR-V 15B, two members of the IR complementation group 5 isolated from the hamster CHO and V79 cell lines, respectively, and found that these cell lines arc highly sensitive to ionizing radiation and that their ability to rejoin radiation-induced DNA double-strand breaks is impaired [41]. Similar results have been found by Kysela et al. using the same cell lines irradiated with different radiation qualities [46]. Iliakis et al. [40] studied the repair-deficient cell line xrs-5 and its parental cell line, CHO, and noted that the rejoining half-times were longer in xrs-5 than in CHO. The fraction of unrejoined dsb decreased with decreasing doses of radiation and the author proposed that this mechanism underlies the increased sensitivity to radiation in xrs-5 cells. In the radiosensitive mutants (xrs-1 and xrs-5) studied by Dahm-Daphi et al. [20], the number of dsb declined with half-times that were longer for xrs-1 and xrs-5 as compared with CHO Kl parental cells. The fraction of non-repairable dsb was higher in mutant cells than in Kl cells. Yasui et al. [99] have studied CHO Kl and its mutant xrs-5 cell lines; they found different morphological organization in the nuclear periphery of xrs-5 cells which, in turn, correlates with the radiation sensitivity of the cells compared with its parental line. This factor may play a role in the defective repair of X-ray induced DNA double strand breaks in xrs-5 cells.

Other authors have studied Chinese hamster V79 cells and their radiosensitive mutants XR-V9B [ 1021 and XR- VI 5B [19,45]. The increased radiosensitivity of the mutants was manifested both as a lower fraction of dsb rejoined by the fast repair component and as a longer persistence of unrejoining dsb during postirradiation incubation. In comparisons of the kinetics of dsb rejoining in radiosensitive mutants irs- 1, irs-2 and irs3 vs. the V79 parental line, Cheong et al. [12] found no differences in the induction of DNA dsb/Gy per Dalton and, further reported that the repair of DNA dsb proceeded in all cell lines with similar kinetics, suggesting that a deficiency in induction or rejoining of DNA dsb is unlikely to be the cause of increased radiosensitivity. Debenham et al. 1221 have shown the irs-1 mutant to have a lower repair fidelity than its parent line. The same mechanism has been proposed as a determinant of higher radiosensitivity in a mutant strain derived from a human bladder carcinoma cell line [64]. Iliakis et al. [38], who studied rat embryo cells (REC) and REC transformed by the H- ras plus v-myc oncogenes (3.7) and immortalized REC

(mycREC), found that the rejoining kinetics of dsb after exposure to X-rays were similar in all cell lines. Because all lines displayed a fast and a slow component, these authors suggested that the radioresistance of 3.7 cells is conferred by alterations in the expression of potentially lethal damage, i.e. dsb that could be either repaired (cell survival), not repaired, or misrepaired (cell killing); however, the radioresistance induced by this method was cell cycle-dependent. Thus the prolonged delay in G2 and S, observed in irradiated 3.7 cells, would be ex- pected to favor damage repair over damage fixation and may be responsible for the increased radioresistance in S and Gz [13].

5.2. Radiosensitive syndromes in humans

Minor repair defects may occur in the human popula- tion and may be sufficient to cause a small but significant increase in the sensitivity of individuals to DNA-damaging agents. Several cancer-prone disorders, including AT, are associated with increased cellular sensitivity to radiation. In previous studies no differences in DNA residual damage [16,48] have been found, but either low-fidelity of repair [ 18,2 1 ] or a deficiency in the repair of a small fraction of dsb [6,33,60,62,84] have been implicated in the radiosensitivity of AT cells. In the last few years complementation analysis have distinguished at least four complementation groups in AT [51]. The AT gene has recently been cloned [77] and it has been suggested that ATM (AT mutated) is the sole gene responsible for AT. The amino-acid sequence of its encoded protein shows homology with PI-3 kinases suggesting involvement of the ATM gene product in signal transduction, cellular responses to DNA damage, cell cycle control and apoptosis after treatment of AT cells with DNA-damaging agents. If the normal AT protein is involved in DNA repair, as its resemblance to the other proteins suggest, it may help cells recognize damaged DNA so that it can be repaired before the cells divide [77], although the precise function of this ATM gene is, as yet, unknown.

Low fidelity of repair or a deficiency in the repair of a small fraction of dsb could explain the differences in radiosensitivity found in lymphocytes from patients with Alzheirner’s disease [86]. Skin Iibroblast cell lines derived from non-Hodgkin’s lymphoma patients show increased sensitivity to low dose-rate gamma irradiation and some deficiency in DNA repair has been found in these patients [33,34].

Badie et al. [2] recently did DNA repair studies on a tibroblast culture (180BR) established from an acute lymphoblastic leukemia patient. Their work represents the first example in which hypersensitivity to ionizing radiation in human cells was ascribed directly to a defect in the rejoining of radiation-induced double-strand breaks.

5.3. Human tumor cell lines 6. Concbion

Much of the data concerning the relationship between radiosensitivity and DNA dsb rejoining kinetics have been summarized in Table 2. Most of studies included in this table support a close relationship between parameters of the DNA rejoining process and intrinsic cellular radiosensitivity in human tumor cell lines.

Damage to DNA may be correctly repaired, incor- rectly repaired (misrepaired) or completely unrepaired. Our results show that the DNA dsb rejoining process [53] follows biphasic kinetics, with a rapid initial rate followed by a much slower second component. This has been noted previously [9,38] and has been termed a two- component unsaturated dose-dependent process (TDU) [27]. We suggest that misrepair may occur in both the fast and slow rejoining components [53], but must be more likely when rejoining kinetics are slower. Different authors have found a close relationship between cell survival and the rate of DNA dsb rejoining (Table 2). Thus, cells with a rapid rate of rejoining are generally more resistant to ionizing radiation, perhaps because of a higher fidelity of rejoining.

Table 2 Molecular bases of radiosensitivity in human tumor cell lines. The role of dsb rejoining

MI. Ntiiiez et al. /Radiotherapy and Oncology 39 (19%) ISS-I65 161

It is well established that several factors influence radiosensitivity. We found a linear relationship between the apparent level of initially induced DNA damage (dsb/Gy per DNA unit) and intrinsic cellular radiosensitivity, measured as SF2 [74]. We also found that the half-time of the fast component of rejoining kinetics is related to SF2 [53]. Briefly, our view is that sensitive cells suffer more DNA dsb per unit dose and the process of DNA dsb rejoining is slower than in radioresistant cells. These two observations may be directly linked through a common factor such as the conformation of DNA [3,14,24,55,56,58], which might influence all as- pects of the cellular response to radiation as well as the ability to detect DNA damage. The conformation of chromatin varies during the cell cycle and some studies in synchronized cells have reported differences in cellular radiosensitivity, the initial level of DNA damage and the rate of DNA repair [39,55,88]. It has been suggested that the compactness of the DNA rather than the actual DNA content is related to cellular susceptibility to radiation [14,55,83] and some data have implicated

Measurement technique Cell lines Comments Reference



Asymmetric field inversion gel electrophoresis

Comet assay


Neutral filter elution/neutral comet assay

Pulsed-tield gel electrophoresis


Five human cervix carcinoma cell lines

Twelve human tumor cell lines (head and neck, sarcoma, melanoma and adenocarcinoma) and two normal human cell lines (tibroblast and mesothelial) Five human tumor cell lines (head and neck) and one normal human tibroblast line

Three human tumor cell lines (two head and neck, one melanoma)

Twenty primary cultures obtained from ovarian cancer and malignant melanoma Six human tumor cell lines (melanoma, glioma, adenocarcinoma, prostate and cervical carcinoma)

Eight human tumor cell lines (neuroblastoma, lung, melanoma, medulloblastoma, bladder and cervix). Five human breast cancer and one human bladder carcinoma cell lines

Rejoining of breaks appeared to be rapid and almost complete within 60 min at 37°C for the three resistant lines. Of the sensitive lines, one exhibited a reduced rate of dsb rejoining. Inherent cellular radiosensitivity was found to directly correlate with the rate at which the DNA dsb were repaired. This rate is a critical factor underlying radioresistance in human tumor cell lines. There was a significant correlation between their extent of dsb rejoining after 1 h of repair following 10 Gy of ionizing radiation and cell line radiosensitivity (Do and SF2). The results were compatible with the assumption that most probably the difference in radiation sensitivity, when cell survival is determined, can be attributed to different repair characteristics. A significant direct relationship was observed between SF2 and the percentages of DNA dsbs rejoined 2 h after irradiation. The rejoining rate measured using tilter,elution did not correlate with radiosensitivity. The rejoining rate was similar when measured using comet assay. Statistically significant correlations were found between cellular radiosensitivity (SF2) and the half-time of the slow phase of dsb rejoining. A statistically significant relationship was found between cellular radiosensitivity (SF2) and the half-time of the fast component of the rejoining kinetics.

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162 M.I. N&z et al. /Radiotherapy and Oncology 39 (19%) 155-165

3-

2-

l-

I I I 0 10 20 30 40 50

tl12 (minutes)

Fig. 3. Relationship between molecular parameters of radiosensitivity for six human tmnor cell lines. Both initial damage data (dsb/Gy per DNA unit) and rejoining data (Tin, min) are from previous works [53,74]. The Trn value corresponds to the fast component of the rejoining process. Cells have been irradiated at 45 Gy for the repair study.

chromatin structure as a determinant of radiosensitivity [W.

When our own data from a set of human tumor cell lines were used the values of induced radiation damage (dsb/Gy per DNA unit) against the half-time of the fast component of rejoining kinetics (Fig. 3), a strong relationship between these two molecular determinants of radiosensitivity was clear (r = 0.952, P = 0.004). This might mean that the induction of more damage leads to a slower rate of repair because of the greater strain on the repair systems, although we also obtained evidence that the repair rates within two different cell lines are dose-independent [53]. This finding supports the idea that both molecular parameters of radiosensitivity may be linked through a common factor such as the conformation of DNA.

This work was supported by the Fondo Investiga- ciones Sanitarias FIS 94/l 55 1. A grant from the Funda- cion Cientifica de la Asociacion Espaiiola contra el Cancer greatly aided this work. MI. Nuiiez is supported by the Fundacion Ben&a San Francisco Javier y Santa Candida, Universidad de Granada, M.T. Valen- zuela is supported by AP92 8837784 and T.J. McMillan is supported by the Cancer Research Campaign and the Medical Research Council. We thank Karen Shashok for improving the English in the manuscript.

References

[l] Alaoui-Jamali, M.A., Batist, G. and Lehnert, S. Radiation- induced damage to DNA in drug and radiation-resistant sublines of a human breast cancer cell line. Radiat. Res. 129: 37-42. 1992.

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