Biochimica et Biophysica Acta, 739 (1983) 17-26 17 Elsevier Biomedical Press

BBA91151

EFFECT OF APHIDICOLIN ON DE NOVO DNA SYNTHESIS, DNA REPAIR AND CYTOTOXICITY IN y-IRRADIATED HUMAN FIBROBLASTS

IMPLICATIONS FOR THE ENHANCED RADIOSENSITIVITY IN ATAXIA TELANGIECTASIA

P A U L J. SMITH and MA L C OL M C. PATERSON

Radiation Biology Branch, Health Sciences Division, Chalk River Nuclear Laboratories, Atomic Energy of Canada Limited, Chalk River, Ontario KOJ 1JO (Canada)

(Received May 18th, 1982)

Key words: DNA synthesis; DNA repair," Gamma-irradiation; Ataxia telangiectasia," Aphidicolin

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase ct and consequently of de novo DNA synthesis in human cells. We report here that in y-irradiated normal human cells, aphidicolin (at 5 I~g/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. -/-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase ct is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.

Introduction

Ataxia telangiectasia is a rare hereditary multi- system disorder in man (for review see Ref. 1). Affected individuals are cancer prone and also show striking clinical hypersensitivity to conven- tional radiotherapy administered for the treatment of solid malignancies. Cultured cells from ataxia telangiectasia patients also show abnormally re- duced colony-forming ability when exposed to ionizing radiation [2,3] or radiomimetic chemicals

Abbreviations: DMSO, dimethylsulphoxide; Syn r, Syn ° and Syn s represent phenotype designations for the resistant, normal or sensitive responses, respectively, of de novo D N A synthesis to inhibition by a given agent.

[4,5]. One class of AT cell strains, designated e x r - , demonstrate genetically determined defects in DNA repair functions as evidenced by reduced levels of y-ray-induced DNA repair synthesis or unscheduled DNA synthesis and the slow removal of radiogenic base or sugar damage [1]. A second class, designated exr +, demonstrate no abnormali- ties for the above gross measures of excision repair capacity [1]. Although there is heterogeneity in the expression of repair deficiencies, all ataxia telangiectasia strains studied have been found to be consistently radiosensitive, suggesting that other metabolic defects may be involved in directing cell lethality.

Recently, a new laboratory feature of AT cell strains has been observed [6-10], namely the resis-

0167-4781/83 /0000-0000 /$03 .00 © 1983 Elsevier Biomedical Press

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tance of de novo DNA synthesis to the inhibitory action of X- or 3,-irradiation. This laboratory phe- notype appears to be due to the disruption of normal interactions between DNA repair func- tions and the replication complex [8,11]. Alterna- tively, it has been suggested that the primary genetic defect in ataxia telangiectasia is not located in the repair of DNA damage per se, but involves the inability of AT cells to restore an altered chromosomal protein structure arising from the damage itself [9] or from the repair of this damage [12]. The latter view is consistent with a model in which AT cells demonstrate increased cell killing following irradiation, due to the premature con> mitment of cells to either S phase or mitosis [13] before DNA repair processes have acted to restore DNA structural integrity and coding fidelity. In its most simple form this model suggests that the toxic effect of radiation on AT cells may be re- duced by the imposition of an artificial block to DNA synthesis and cell growth during the critical post-irradiation period, Ideally, the blocking agent used should neither be cytotoxic nor have effects on DNA repair functions, and this report presents an attempt to investigate the above prediction using the antibiotic, aphidicolin.

Aphidicolin is a tetracyclic diterpene tetraol, isolated from Cephalosporium aphidicola and is an inhibitor of replicative DNA synthesis and conse- quently cell growth in eukaryotic cells (for review see Ref. 14). The antibiotic is a specific inhibitor of the polymerase a and is without effect on polymerase /3 or 7 [15,16]. The polymerase c~ of animal cells is involved in the replication of chro- mosomal DNA whereas the polymerases/3 and y are most likely involved in the repair of nuclear DNA and synthesis of mitochondrial DNA, re- spectively [ 17].

There are conflicting reports on the effect of aphidicolin on DNA repair functions in human cells, thus the role of polymerase c~ in repair processes is still under debate. Studies have largely been confined to the repair of ultraviolet-induced D N A damage. Aphidicolin has been reported to inhibit the excision of pyrimidine dimers in HeLa cells [18], and repair replication in HeLa cell G 2 nuclei in vitro [19] or in permeabilized human peripheral blood lymphocytes [20] with supplied exogenous deoxynucleoside triphosphates. In con-

trast, aphidicolin was not found to inhibit ultra- violet-induced repair synthesis in vivo either in the nuclear DNA [21] or the mitotic chromosomes of HeLa cells [22].

The controversy over the effects of aphidicolin on DNA repair appears to arise from a combina- tion of factors, including the difficulty in dis- tinguishing unambiguously between DNA pre- cursor incorporation due to either de novo DNA synthesis or repair synthesis, wide variation in the aphidicolin concentrations employed and potential cytotoxic effects of prolonged exposure to the antibiotic.

At the chromosomal level, aphidicolin increases both the spontaneous and ultraviolet-induced levels of sister-chromatid exchanges in Chinese hamster cells [23], possibly reflecting effects at the level of the replication fork. Aphidicolin induces moderate levels of chromatid-type aberrations in both G o and G 2 human lymphocytes and also interacts synergistically with X-irradiation to increase the yields of chromosome-type aberrations in G O and of chromatid-type aberrations in G 2 [24]. Interest- ingly, irradiated G O cells treated for short periods with aphidicolin also give rise to chromatid-type aberrations, a finding which parallels that seen in X-irradiated G o lymphocytes from persons af- fected with ataxia telangiectasia [25].

The objective of the present study was two-fold: (i) to determine the effects of aphidicolin on de novo DNA synthesis, y-ray-induced repair synthe- sis and the repair of two classes of radiogenic DNA lesions in normal human cells; and (ii) to test the model considered above by determining the survival capacity of y-irradiated AT cells ex- posed to a non-toxic level of aphidicolin which efficiently inhibit de novo D N A synthesis. Our findings indicated that aphidicolin is not inhibi- tory to DNA repair functions, assayed by three independent methods, and does not affect the lethality of y-radiation towards normal or AT cells. We suggest that inhibition of de novo D N A synthesis per se is not sufficient to protect either normal or AT cells from y-ray induced killing.

Materials and Methods

Cell strains and their cultivation The three human skin fibroblasts strains were

derived from one normal donor (GM 38; purchased from the Human Genetic Mutant Cell Repository, Institute for Medical Research, Camden, N J) and two ataxia telangiectasia patients (AT3BI, kindly supplied by Dr. A. Lehmann, University of Sus- sex, UK; AT5BI, kindly supplied by Dr. A.M.R. Taylor, University of Birmingham, U.K.).

Fibroblasts of each strain were maintained in monolayer culture at 37°C in a humidified atmo- sphere of 5% CO 2 in air. The culture medium was Ham's F12 (Gibco, Burlington, Ontario) supple- mented with 10% (v/v) non-inactivated fetal bovine serum, 1 mM glutamine, 100 units penicil- lin G per ml, and 100 /~g streptomycin sulphate per ml. Unless otherwise indicated, all cell culture supplies were purchased from Microbiological As- sociates Inc. (Walkersville, MD).

Aphidicolin The antibiotic was a generous gift from A.H.

Todd (ICI Ltd., Pharmaceuticals Division, Mac- clesfield, U.K.). A stock of 5000 /~g/ml was pre- pared by dissolving 25 mg aphidicolin in 5 ml DMSO such that the highest active concentration of DMSO in the current experiements was 0.1% (v/v) in culture medium. This residual level of DMSO had no effect on the biological or biochem- ical parameters studied.

Cell survival (a) Aphidicolin treatment. Cells were seeded at

the appropriate densities in growth medium con- taining "t-ray-inactivated (500 Gy) feeder cells of the same strain (giving a total of 8.10 4 cells per 9 cm diameter plastic dish (Lux Scientific Corp., Newbury Park, CA)). Following an 18 h attach- ment period, dishes were drained, and growth medium containing aphidicolin at the appropriate dilution was added. After the specified exposure period at 37°C, cultures were washed with phos- phate-buffered saline, fresh growth medium was added and the cultures were incubated, with weekly medium changes, for 18-24 days. Colonies (con- sisting of 50 or more cells) were fixed and stained prior to counting.

(b) y-irradiation. The technique was similar to that described above for aphidicolin treatment. After cell attachment plates were drained and exposed in equilibrium with air (5 ml phosphate-

19

buffered saline per dish) to varying acute doses of 6°Co ,/-rays from a Gamma-beam 150C (Atomic Energy of Canada Limited, Ottawa, Canada) at a dose rate of 0.85-1.10 Gy-min -~. Cultures were then processed as above for colony formation.

Measurement of inhibition and recovery of de novo DNA synthesis

Cells were plated at 2 • 105 cells per 6 cm plastic dish (Lux Scientific Corp.) and incubated for 24 h. Cultures were then incubated in medium contain- ing 0.025 /~Ci/ml [methyl-14C]thymidine (40.8 mCi/mmol, stock specific activity; New England Nuclear Canada, Lachine, Canada) for 18 h prior to treatment. Cultures were then incubated in growth medium containing aphidicolin or ~,-irradi- ated as described above. At specified times, during or after treatment, cells were incubated for 10 rain in growth medium containing 10 #Ci/ml [methyl- 3H]thymidine (80.1 Ci/mmol, stock specific activ- ity). Cultures were washed with phosphate-buffered saline followed by the addition of 1 ml lysis solu- tion (1.5 g 4-aminosalicylic acid sodium salt (BDH Chemicals Ltd., Poole, U.K.)/0.25 g triisopro- pylnaphthalene sulphonic acid sodium salt (East- man Kodak Co., Rochester, NY)/1.5 ml 2- butanol/23.5 ml distilled water). DNAs in the resulting lysate samples were precipitated in ice- cold 10% trichloroacetic acid and collected on Whatman G F / C filters (Fisher Scientific, Montreal, Canada) for liquid scintillation counting in toluene-Scintiprep 2 (Fisher Scientific, Toronto, Canada). Radioactivity in 3H and 14C channels was background and spill-over corrected, and val- ues expressed as disintegrations per min. For each condition the level of DNA synthesis in treated cultures (as a percent of the untreated control) was calculated from:

100 × [ (dpm 3H/dpm '4C) ...... d/(dP m 3H/dp m 14C) . . . . . .

DNA repair assays (a) DNA single-strand breaks and enzyme-sus-

ceptible lesions. This method has been described previously [26]. Exponentially growing cultures of control (14C-labelled) and experimental (3H- labelled) cells were held on ice or irradiated (in a Gamma-cell 220 unit at 150-170 Gy/min; Atomic Energy of Canada Limited, Ottawa, Canada), re-

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spectively, and then frozen immediately or in- cubated for given times under normal culture con- ditions. Irradiations were carried out in buffer (5 ml phosphate-buffered saline per 9 cm plastic dish) in equilibrium with air.

Corresponding experimental and control cul- tures cultures were fast-frozen on dry-ice and stored at - 20°C prior to colysis and DNA extrac- tion. DNA single-strand breaks (including alkali- labile lesions) and extract-susceptible sites were estimated by velocity sedimentation in alkaline sucrose gradients of DNA samples that had been incubated in the absence or presence of a protein extract of Micrococcus luteus cells. In any given experiment, the incidence of a particular class of DNA damage for a single DNA sample was calculated from at least two separate determina- tions.

(b) Repair replication. This technique has been described previously [3] but was modified such that thymidine-deficient growth medium was used. Furthermore, no hydroxyurea was employed to reduce de novo DNA synthesis so that the effect of aphidicolin on this process could be measured in parallel.

In brief, the technique measures the incorpora-

tion of [methyl-3H]thymidine into damaged cellu- lar DNA during the course of post-treatment in- cubation; the labelled thymidine is present at high specific activity in culture medium containing flu- orodeoxyuridine, an inhibitor of endogenous thymidine synthesis. Late logarithmic-phase cul- tures (approx. 2. l 0 6 cells per 15 cm plastic dish) were exposed to oxic y-radiation in a Gamma-cell 220 unit (5.66 G y . s- l; Atomic Energy of Canada Limited). Cultures were then incubated for the indicated times. De novo DNA synthesis and re- pair replication of DNA were distinguished, using a BrdUrd DNA-density label followed by separa- tion of the relevant DNA species by isopycnic centrifugation in neutral ethidium bromide-sodium iodide gradients. D N A associated with repair rep- lication was rebanded to ensure minimal con- tamination with DNA undergoing de novo DNA synthesis.

R e s u l t s

(A) Effects of aphidicolin on DNA metabolism in normal cells

(1) De novo DNA synthesis. Fig. 1 indicates that aphidicolin is a rapid and potent inhibitor of

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Fig. 1. Aphidicolin-induced inhibition of de novo DNA synthesis in normal human cells. Each point represents the mean of at least two determinations (S.E. ~< 10%). Symbols: O, r-q, r, and v represent 0.02, 0.2, 2 and 5/Lg aphidicolin/ml, respectively.

Fig.2. Levels of "t-ray (500 Gy)-induced D N A damage remaining in normal cells following a 2 h post-irradiation incubation period. Symbols represent cells exposed to 3,-radiation and aphidicolin (O, o) or aphidicolin alone (,x, • ) ((3, zx represents DNA single-strand breaks in alkali, O, • represent extract-sensitive sites) (per 107 Da). ©, initial yield = 1.90 (90% repair), o, initial yield = 1.33 (50% repair).

DNA synthesis in normal human fibroblasts. At 0.02-5.0 /~g aphidicolin/ml the maximal level of inhibition is achieved within l0 rain of treatment, and a concentration of 0.2 /~g/ml is sufficient to inhibit [3H]thymidine incorporation by approx. 90% of control. At 5/~g/ml, inhibition is effec- tively complete, since the 2-5% residual level of DNA synthesis can be attributed to aphidicolin-re- sistant mitochondrial DNA synthesis involving DNA polymerase y. Our findings are consistent with a previous report [21] demonstrating the aphidicolin concentration-dependency for the in vivo inhibition of DNA synthesis in HeLa cells.

(2) Repair of y-ray-induced radioproducts in DNA. We have examined the effect of aphidicolin on the repair of two classes of oxic y-ray-induced (50 Gy) radioproducts in cellular DNA: (i) single- strand breaks (including alkali-labile lesions) and (ii) radiogenic base and sugar damage detected as DNA sites sensitive to strand-incising activities present in a crude protein extract of M. luteus cells.

Fig. 2 shows the effects of various aphidicolin concentrations on the residual levels of DNA damage in normal cells after a 2 h period of post- irradiation incubation period. Controls (Fig. 2) in- dicate that a 2 h aphidicolin treatment alone, does not induce single-strand breaks or enzyme-sensi- tive lesions at a level greater than 1.1 per 10 9 Da. The residual levels of enzyme-sensitive lesions and single-strand breaks are not significantly modified by the presence of aphidicolin (0.02-5/~g/ml).

(3) y-ray-induced repair synthesis. The isopycnic gradient technique (see Materials and Methods) permitted the parallel determination of repair and de novo DNA synthesis in cells exposed to the

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TABLE II

EFFECT OF APHIDICOLIN ON DE NOVO DNA SYNTHESIS IN NORMAL CELLS

[3H]thymidine uptake was assayed in parallel with the repair study shown in Table I. ),-irradiation alone reduced DNA synthesis to 32% of untreated control. Errors represent range for two determinations.

Condition [3 H]thymidine incorporation (% untreated control) in the presence of aphidicolin

( # g / m l

0.02 0.2 2 5

Unirradiated control 98_+ 4 41_+3 20_+3 20_+ 1

Irradiated (500 Gy) 83_+ 15 48_+ 1 33_+2 15+2

same levels of y-radiation and aphidicolin de- scribed in the previous section (A2). Table I shows that the expression of y-ray-induced repair synthe- sis is not modified by aphidicolin at concentra- tions up to 0.2 /~g/ml. However, at 2.0 and 5.0 /xg/ml there is a significant increase in repair synthesis levels (by a factor of 1.6-1.9-fold). In keeping with the results of the other two repair assays (see Results Section A2), the enhancement effect can not be attributed to aphidicolin-induced damage since the antibiotic alone does not induce repair synthesis.

De novo DNA synthesis is inhibited (Table II) with a similar aphidicolin concentration depend- ency in both irradiated and control cultures. Al- though we cannot correlate the effects of aphi- dicolin on repair synthesis with the inhibition of de novo DNA synthesis, the repair enhancement

TABLE I

EFFECT OF APH1DICOLIN ON y-RAY-INDUCED DNA REPAIR REPLICATION IN NORMAL CELLS

[ 3H]thymidine was measured after a 2 h incubation. Errors represent range for two determinations.

Condition [3H]thymidine incorporation ( d p m / ~ g DNA) in the presence of aphidicolin

(~g /ml )

0 0.02 0.2 2 5

Unirradiatedcontrol 62_+ 8 51_+ 3 45_+ 7 36+11 28_+ 8 Irradiated (500 Gy) 418 _+ 28 482 + 25 469 _+ 23 669 _+ 35 785 _+ 52

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observed may result from the inhibition of repli- cons to the extent that de novo DNA synthesis is biophysically identified as repair synthesis.

(B) Inhibition and recovery of de novo DNA synthe- s& in A T cells following y-irradiation of aphidicofin t r e a t m e n t

Since de novo DNA synthesis in AT cells is resistant to the inhibitory action of irradiation, it was necessary to determine whether a similar phe- nomenon occurs in response to aphidicolin treat- ment. Thus, we have compared the inhibition and recovery of de novo DNA synthesis in cells ex- posed to either y-radiation (10 Gy, Fig. 3) or aphidicolin (0.02 and 0.2 ~g/ml , Fig. 4; 0.2 ~tg/ml, Fig. 5).

In keeping with a previous report [27], de novo D N A synthesis in y-irradiated normal cells dem- onstrates an inhibition phase (approx. 52% of con- trol synthesis) followed by a recovery phase ini- tiated approx. 1-2 h following treatment (Fig. 3). Both ataxia telangiectasia strains show resistance to inhibition (although synthesis levels decline slowly to approx. 80% of control over a 4 h period). Preliminary experiments (data not shown) estab-

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Fig. 3. T-ray (10 Gy)-induced inhibition and recovery of de novo DNA synthesis in normal (GM 38, ©) and AT cells (AT3BI, A; AT5BI, •). Each point represents the mean for at least three determinations (S.E. ~< 10%).

lished that 10 Gy is an optimal dose for dis- tinguishing between inhibition and recovery phe- nomena in normal and AT cells, there being less distinction between normal and AT5BI cells at doses of 20- 500 Gy. Fur thermore , the []4C]thymidine prelabel (see Materials and Meth- ods) does not affect DNA synthesis responses,

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TIME AFTER TREATMENT (h)

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Fig. 4. Aphidicolin-induced inhibition of de novo DNA synthesis in normal (GM 38, O, I ) and AT cells (AT3BI, zx, A; AT5BI, 13, i ) .

Each point represents the mean of two determinations (S.E. ~< 10%).

Fig. 5. Recovery of de novo D N A synthesis in normal (GM 38, O) and AT cells (AT3BI, A; AT5BI, O) following exposure to 0.2 #g aphidicol in /ml for 30 min. Each point represents the mean of two determinations (S.E. ~< 15%).

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APHIDICOLIN CONC. ( N g / m l )

Fig. 6. Toxicity of aphidicolin towards normal human cells for a 24 h exposure period. Results from a single experiment (S.E. ~< 15%).

since the parallel determination of DNA synthesis levels by specific activity measurements in non-prelabelled cells yields identical results.

In contrast to ionizing radiation, normal and AT cells demonstrate equivalent sensitivity to the inhibitory action of aphidicolin on de novo DNA synthesis (Fig. 4), and upon removal of the an- tibiotic, both cell types demonstrate prompt and similar patterns of recovery of synthesis.

(C) Effects of aphidicolin on survival capacity of unirradiated and irradiated cells

Fig. 6 shows the toxicity of aphidicolin (24 h exposure) towards normal cells. The antibiotic is clearly toxic (< 15% survival) at concentrations greater than 1 /~g/ml. In determining the maximal time period for aphidicolin treatment of AT cells we have selected 0.2 # g / m l as a relatively non-toxic concentration for normal cells (approx. 75% con- trol survival; Fig. 6). Further experimentation (data not shown) indicates that AT cells demon- strate normal aphidicolin-sensitivity following a 24 h exposure but show an approx. 2-fold increase

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in cell killing compared to normal cells following a 48 h exposure. AT cells also show enhanced sensi- tivity to prolonged incubation with hydroxyurea (Bech-Hansen, N.T. and Smith P.J., unpublished data).

The above results establish an aphidicolin treat- ment regimen of 0.2/~g/ml for 24 h as one capa- ble of efficient inhibition of de novo DNA synthe- sis with minimal cytotoxicity and no detectable effect on cellular repair capacity for radiogenic DNA damage.

The effect of this treatment regimen on the post- 7- irradiation survival capacity of normal and AT cells is shown in Fig. 7. Clearly aphidicolin has no significant effect on the radioresponse of nor- mal cells or the hypersensitive radioresponse of AT cells. Additional experiments (data not shown) indicate that similar results are obtained for short aphidicolin exposure periods of 4 h post-irradia- tion and for cells irradiated in suspension (aphi-

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7"-RAY DOSE (Gy) Fig. 7. Effect of aphidicolin on the y-ray survival responses of normal (GM 38, O, O) and AT cells (AT3BI, ,',, A; AT5B1, t~, I ) . Open symbols represent untreated controls and closed symbols represent cells exposed to 0.2/.tg aphidicolin/ml for a 24 h post-irradiation period. Data points for each curve have been normalized to the corresponding unirradiated control. S.E. ~< 15%.

24

dicolin having no effect on cell attachment). In conclusion, inhibition of de novo synthesis per se is not sufficient to modify the post-y-irradiation survival • capacity of either normal or AT cells.

Discussion

The main objectives of this study were 2-fold. First, to define the effect of aphidicolin on the repair of two major classes of radiogenic lesions in DNA of normal cells and we report that even at high aphidicolin concentrations (e.g., 5/~g/ml), there is no significant inhibition of repair. Second, to study the action of aphidicolin on AT cells which show resistance to the inhibitory effect of ionizing radiation on DNA synthesis (a 'resis- tance' phenotype for which we suggest the desig- nation Syn r as opposed to Syn" and Syn s indicating normal and sensitive phenotypes, respectively). Under conditions of minimal toxicity but exten- sive inhibition of de novo DNA synthesis, aphi- dicolin does not significantly modify the radiation sensitivity of normal or AT cells. Our findings are pertinent to both the role of polymerase a in DNA repair and the cellular defect in ataxia telangiec- tasia.

Aphidicolin is undoubtedly toxic to human cells under certain conditions, and cytotoxicity alone rather than specific interference with repair func- tions may explain why the antibiotic apparently inhibits ultraviolet-induced pyrimidine dimer exci- sion in HeLa cells (4/~g/ml per 24 h [18]). Our findings constitute the first report on the effects of aphidicolin on the repair of defined radiogenic DNA lesions in human cells. The results indicate that the human polymerase a is not involved in processes leading to the repair of the majority of DNA strand breaks or the disappearance of sites sensitive to incising activities present in protein extracts of M. luteus cells. The apparent enhance- ment of "r-ray-induced repair synthesis at high (2 and 5 ~g /ml ) concentrations of aphidicolin does suggest that polymerase a-associated functions may normally be associated with the regulation of the extent of repair synthesis, although the possibility that severely restricted replicon activity may be mistaken for repair synthesis cannot be ruled out.

It is interesting to note that high aphidicolin concentrations (17/~g/ml) alone, or following X-

irradiation of human G O lymphocytes, can en- hance the frequencies of chromatid-type aberra- tions - a cytogenetic feature observed in X-irradi- ated AT cells [25]. The cytogenetic effects of aphidicolin may relate to the high concentrations of antibiotic employed, since in the present experi- ments normal cells were not sensitized to y-radia- tion by lower levels of aphidicolin. However, severe restriction of polymerase a activity appears to permit the expression of chromosome anomalies in terms of inherent (spontaneous) instability and radiation-induced aberrations, although the lethal consequences of such pathways could not be fol- lowed due to the cytotoxicity of the aphidicolin treatment levels required. It would be of interest to determine whether aphidicolin enhances chro- matid-type aberrations in irradiated AT cells, since one might expect that if some facet of the poly- merase a system involved in chromosome stability is already defective, then AT cells should be rela- tively refractory to the action of aphidicolin.

We have previously reported [18] that there is heterogeneity, possibly associated with DNA re- pair capacity, in the patterns of DNA synthesis in irradiated AT cells. Complementation studies [28] have also characterized heterogeneity in the Syn r phenotype. Our previous experiments were per- formed with low doses of y-radiation and rela- tively long pulse label periods - a protocol which may tend to obscure the Syn r phenotype [27]. In the present study we have adopted a short pulse regimen and used two AT cell strains which have previously been shown to display the Syn r pheno- type [9]. Our results confirm those of Painter and Young [9,27].

A striking feature of AT cell strains is their consistent hypersensitivity to ionizing radiation [1 ]. It has been suggested [9,8,13,27] that in irradiated AT cells, the Syn r phenotype may be a factor in (i) increased cell killing, (ii) reduced mitotic delay and (iii) increased frequencies of chromosomal

• aberrations. Thus, the Syn r phenotype could per- mit DNA replication to occur on damaged tem- plates and allow cells to progress prematurely from G 2 phase into mitosis before pre-aberrational damage is completely repaired.

Such a simple temporal model described above does not appear to be consistent with either the present data or with previous observations. Firstly,

no extensive enhancement of recovery of irradia- ted AT cells is provided by the blocking of de novo DNA synthesis by aphidicolin (see Results section) or by holding cells in plateau phase cul- ture [29]. It should be noted that the experiments described in the present paper were also per- formed with hydroxyurea as the blocking agent (1-10 mM concentration range) with results simi- lar to those described for aphidicolin (Smith, P.J., Paterson, M.C. and Bech-Hansen, N.T., unpub- lished data). Aphidicolin and contact inhibition- prevention of DNA synthesis differ in their in- fluence on the post-irradiation survival capacity of normal cells, suggesting that only cells in G o are capable of demonstrating recovery from poten- tially lethal damage. Secondly, autoradiographic studies (data not shown) indicate that in the cur- rent survival experiments the S phase fraction of the asynchronous population, at the time of treat- ment, ranged from 25-30% for normal and AT cells. Thus, the majority of cells are not candidates for enhanced lethality by the Syn r phenotype, un- less potentially lethal damage is relatively long lived. Thirdly, it has been observed that radiogenic chromosome abnormalities occur with enhanced frequencies in AT cells compared with normal cells, when irradiated during interphase [25,30,31]. Collectively, the evidence strongly suggests that a temporal model for the expression of radiosensitiv- ity in ataxia telangiectasia is unlikely.

Radiation and aphidicolin-induced inhibition of DNA synthesis differ greatly in the processes in- volved at the chromatin level. Radiogenic damage (e.g., direct DNA strand breaks or repair-related enzymatic incision events) is thought to give rise to relaxation of regions of DNA thus inhibiting repli- con activity [32]. On the other hand, aphidicolin affects the polymerase a, and chromatin structure presumably remains relatively undisturbed. A chromatin-associated abnormality in ataxia telan- giectasia (e.g., reduced accessibility of repair en- zymes to DNA) would be expressed irrespective of the presence or absence of aphidicolin and during all phases of the cell cycle. The latter situation could explain both the cytogenetic features of irradiated AT cells and the radiation Syn r pheno- type.

To conclude, we regard the radiation Syn r phe- notype as a reflection of a heterogeneous defect in

25

DNA repair expressed at the enzyme, cofactor or substrate (chromatin) level. This defect would re- sult in a reduced number of repair events, in AT cells, contributing to premature initiation of DNA synthesis [ 11].

Clearly, ataxia telangiectasia offers a unique cellular system in which to study the interrelation- ship between the mechanisms for DNA repair and biological responses to carcinogens in terms of chromosome aberrations and lethality. The use of specific inhibitors, such as aphidicolin, should fur- ther prove to be useful in identifying the role played by DNA replication.

Acknowledgements

This work was partially supported by U.S. NCI Contract NO1-CP-81002 with the Clinical Epide- miology Branch, NCI, Bethesda, MD. We are grateful for the expert technical assistance of De- borah Adams and P.A. Knight.

References

1 Paterson, M.C. and Smith, P.J. (1979) Annu. Rev. Genet. 13, 291-318

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