Int. J . Cancer: 19, 186-193 (1977)

MOLECULAR 1NTERACTIONS BETWEEN ADRTAMYCIN AND X-RAY DAMAGE IN MAMMALIAN TUMOR CELLS

John E. BYFIELD, Young C. LEE and Lorna Tu Laboratory of Nuclear Medicine and Radiobiology, Los Angeles, Calif: I. and Division of Radiation Oncology, School of Medicine, University of Calijornia, San Diego, San Diego, Calif., USA

The effect of the anthracycline antibiotic, Adria- mycin (Ad), on the sedimentation properties of pre-labelled mammalian D N A has been studied. Ad induces D N A degradation in vivo in both excision repair-competent (HeLa and Me-180) cells and in excision repair-deficient (REQ) cells. When X- irradiated cells are exposed to Ad during the period of repair of D N A single-strand breaks. small numbers of residual breaks persist following com- pletion of repair. These are attributable to those induced by Ad alone. The effects of Ad and X-rays therefore appear to be similar and additive. N o clear-cut evidence that Ad can inhibit the repair of X-ray-induced D N A single-strand breakage was found. Ad also induces the formation of D N A double-strand breaks and inhibits the repair of X-ray-induced base damage (repair replication). The induction of D N A strand breakage may be responsible for Ad cell toxicity and may contribute to i t s capacity to enhance primary X-irradiation damage when the two types of lesions co-exist.

The development of the anthracycline antibiotics has represented a major advance in cancer chemo- therapy. The historical aspects of this research have recently been described in detail (Ghione, 1975). Most initial studies with these compounds utilized the antibiotic now most commonly known as daunomycin or rubidomycin which currently has specific but limited clinical usefulness (Bernard et a[., 1969). The development of its 14-hydroxyl derivative, Adriamycin (Ad) has dramatically extended the clinical usefulness of this class of antibiotics. The initial clinical studies by Bonadonna and his colleagues have been confirmed and expanded on a world-wide basis (Bonadonna et al., 1969; Carter et al., 1975). The clinical usefulness of Ad has b-en extensively reviewed recently (Blum and Carter, 1974; Carter et al., 1975; Skovsgaard and Nissen, 1975) and it is apparent that significant clinical response rates have been shown for a wide range of tumors. We became interested in Ad when it was ob;erved that this antibiotic could enhance the X-ray killing of mammalian cells in culture (Byfield, 1973, 1974). This observation suggested that coincident use of Ad and X-ray therapy (XRT) might be useful and also indicated that normal tissue reactions seen with combined Ad/XRT regimens would be exaggerated. A number of recent publications have indicated that clinically useful enhanced radiation effects may occur when these

two modalities are combined (Watring et al., 1974; Byfield et al., 1975). Similarly, the observation that clinical “ recall ” phenomena occur indicates that latent residual toxicity from XRT can be activated by Ad (Donaldson et al., 1974; Cassady et al., 1975. Cardiotoxicity, the dose-limiting side-effect of Ad, is also exacerbated by previous cardiac irradiation (Gilladoga et al., 1975; Merrill et al., 1975).

To better understand the interactions between Ad and X-rays, we have examined the effect of Ad on DNA integrity and cell survival in tissue culture. The results presented in this and the accompanying communication indicate that Ad can induce both single and double DNA strand breaks and that this damage can interact with X-ray damage, leading to enhanced cell killing. Ad also inhibits the excision repair of X-ray damaged bases in a limited fashion. In this paper we report the results of studies on DNA integrity and the repair of radiation damage. In the accompanying communication the cellular interactions between Ad and radiation damage arz illustrated and the possible relationships between the molecular and cellular events are discussed. The biochemical effects of Ad have recently been dis- cussed in detail (DiMarco, 1975; Lenaz et al., 1974).

MATERIAL AND METHODS

Cell cultivation HeLa cells were originally obtained from the

American Type Culture Collection (CCL 2). The second cell line used was the rat excision repair- deficient REQ line which was probably derived from the Walker carcinoma-sarcoma line (Byfield et al., 1976a). The murine L-1210 leukemia cells used in these studies were originally obtained from the Southern Research Institute. Me-180 cells were kindly obtained from Dr. J. Fogh. Our methods for maintaining the L-1210 line have been described (Lee et al., 1974a). HeLa, REQ, and Me-180 cells were maintained in the log phase of cell growth by serial passage (using 0.25 % trypsin) in McCoy’s 5A modified medium (Associated Biomedic Systems, Buffalo, New York) supplemented with 20% fetal

Received : September 16, 1976.

ADRIAMYCIN A N D X-RAY DAMAGE I N D N A 187

calf serum. All stock cultures were maintained in Falcon plastic large T-flasks, either adherent to the plastic (HeLa and REQ) or in non-shaking sus- pension cultures (L-1210), placed in a tissue culture incubator at 37" C perfused with a water-saturated 95% air, 5 % C02 mixture.

DNA single-strand break rejoining

Two methods for the evaluation of the induction and repair of DNA single-strand breaks were utilized. In the first procedure (termed Method I) the cells were pre-labelled with tritiated thymidine ("-TdR, 5 Ci/mmole, 0.25 pCi/ml) for one doubling period to yield tritiated DNA. The growth medium containing the labelled 3H-TdR was then removed and the cultures washed twice in ice-cold Gey's buffered salt solution. The cultures were then irradiated under Gey's solution at ice bath tem- peratures. To evaluate the induction of DNA single-strand breaks or the repair of X-ray-induced DNA single-strand breaks, one of two procedures was followed. Either the cells were either directly evaluated using the alkaline sucrose gradient pro- cedure (to be described) or else the buffered salt solution was removed, pre-warmed growth medium added, and the cells were allowed to incubate under standard tissue culture conditions for variable (repair) periods. To determine the extent of DNA single-strand break induction, samples were tryp- sinized and the cells collected by centrifugation followed by resuspension in physiological saline (4 x lo6 cells/ml) at 0" C. One-tenth ml of each cell suspension was then loaded on the top of 0.2 ml of lysing medium (0.5 M NaOH and 0.1 M EDTA) which had been pre-layered over 4.0 ml of a 520% sucrose gradient made up in 0.1 M NaOH, 0.9 M NaCl, and 0.01 M EDTA. The gradients were maintained at room temperature for 2 h and then centrifuged in a Beckman SW 50.1 rotor at 20,000 rpm for 75 min at 10" C. Each gradient was then collected from the bottom by needle puncture and counted by liquid scintillation counting as previously described (Lee and Byfield, 1976). This method derives directly from the classical alkaline sucrose gradient for studying the X-ray induction of single- strand breaks in mammalian cells (Lett et al., 1967).

We also evaluated the induction of DNA strand breaks using the alternative procedure first described by McBurney et al., (1972). This procedure is more sensitive for studying the induction of DNA strand breakage but the DNA being observed consists of both single-sttanded lengths and some double- stranded regions which are incompletely denatured (McBurney et al., 1972; Simpson et al., 1974). In this procedure (termed Method 11), the cells were labelled as previously described under Method L Following trypsinization and washing, the cells

were suspended in ice-cold Gey's salt solution (lo6 cells/ml) and 0.1 ml of cell suspension (lo5 cells) was then carefully placed on top of 2 ml of 2% sucrose (in water) which had been previously pre- layered over a 31 ml 10-30% linear alkaline sucrose density gradient made up in 0.3 M NaOH, 0.5 M NaCl, and 0.01 M EDTA at 0" C. Each tube also contained a 3.0 ml cushion of 70% sucrose made up in the same reagents. Cell lysis and DNA release was then obtained by storing each gradient in the dark for 16 h at 4" C prior to centrifugation in a Beckman rotor SW 27 for 3 h at 20,000 rpm and 4" C. The gradients were then collected as described above and the radioactivity of each fraction was determined by liquid scintillation counting (Bollum, 1959).

DNA double-strand breaks

The method used to evaluate the induction of DNA double-strand breaks was that originated by Lehmann and Ormerod (1970). In this procedure, exponentially growing cells were labelled as for studies involving single-strand DNA breaks. Fol- lowing removal of the growth medium containing 3H-TdR, pre-warmed medium containing various Ad concentrations was added and the cells incubated as described in the figure legends. After Ad exposure, the cells were washed, harvested by trypsinization and resuspended in ice-cold 0.15 M NaCI. They were then carefully loaded on the top of a 2.0 ml layer of lysing solution (0.2 % sodium dodecyl sarkosinate, 0.08% sodium deoxycholate, 2 M NaCl, 0.01 M sodium citrate, and 0.02 M EDTA, PH 9.0) that had itself been pre-layered on a 31.0 ml, 520% linear sucrose gradient made up in 2.0 M NaCl and 0.01 M

sodium citrate, PH 9 to 9.5. After each gradient had been loaded with lo6 cells, the gradient surfaces were fan-blown gently for 200 seconds and then stored at room temperature for 2 h prior to centri- fugation. They were then centrifuged at 6,500 rpm for 16 h prior to collection and scintillation counting as described for alkaline sucrose gradients.

Repair replication studies

A previous communication has outlined our procedure for quantitatively evaluating the kinetics of X-ray-induced excision repair in mouse L-1210 cells (Lee et al., 1974a). In this procedure, nomal DNA synthesis is suppressed by preexposing the cells for 1 h to 7.0 pg/ml cytosine arabinoside followed by heavy gamma radiation in suspension culture (100 krads). 3H-TdR is then added and the kinetics of incorporation measured by taking duplicate samples which are processed by the direct filter paper assay method (Byfield and Scherbaum, 1966). Ad has previously been shown to inhibit X-ray-induced excision repair in L-1210 cells.

188 BYFIELD ET AL.

0 25 50 75 I00 NORMALIZED FRACTION NUMBER

FIGURE 1 Effect of Ad on DNA strand length in REQ cells.

REQ cells were pre-labelled with $H-TdR, washed and exposed to various Ad concentrations for 2 h followed by alkaline sucrose gradient sedimentation using Method I. Ad concentrations (in ,ug/ml) are shown in each panel (C - control lacking Ad).

Gamma radiation technique A 4,000 curie cobalt-60 radiation source made up

of eight individual sources in circular array was used for these experiments. In each case, the radia- tion was performed at room temperature in air. The dose rate for doses below 2 krads was approxi- mately 75 rads/min. The high dose exposures required for the induction of excision repair used dose rates of approximately 8,000 rads/minute. Absorbed radiation doses were determined by thermo-luminescent dosimetry. Each culture was rotated 180" after one-half of the radiation was given and the dose received is assumed to be rela- tively homogeneous.

RESULTS

Effect of Ad on the induction of DNA single-strand breaks

When exponentially growing REQ cells which had been pre-labelled with 3H-TdR are exposed to Ad for 2 h and then assayed for DNA strand length integrity using the classical 520% linear alkaline sucrose gradient (Method I), there is a dose-depen- dent reduction in the extent to which DNA will sediment towards the bottom of the gradient. Low levels of drug show relatively little effect while levels approaching 1.0 pg/ml or more show progressive reduction in the median sedimentation point and progressive broadening of the sedimentation peak. These changes are consistent with the induction of DNA single-strand breaks (Fig. 1).

Since the rat REQ line has been shown to be deficient in both X-ray (Byfield et al., 1967a) and ultra-violet (Byfield, 1975) -induced excision repair replication, it seemed possible that the results obtained in Figure 1 stemmed from a property unique to excision repair-deficient cells. Accordingly, we extended our observations to evaluate the effect of Ad on DNA strand integrity in two human cell lines. The cell lines used were HeLa cells (Fig. 2), derived from an adenocarcinoma of the cervix (Jones et al., 1971) and the Me-180 line (Fig. 3) which was derived from a squamous-cell carcinoma of the cervix (Sykes et al., 1970). The more sensitive assay for DNA strand integrity (Method 11) showed that both cell lines also demonstrated a concen- tration-dependent reduction in the sedimentation of pre-labelled DNA when exposed to Ad in tissue culture (Figs. 2 and 3). While the results are quali- tatively similar to those obtained using REQ cells (Fig. 1) both HeLa and Me-180 cells appeared somewhat more resistant to this phenomenon in comparison to the rat line.

Eflect of Ad on the repair of X-ray-induced DNA single-strand breaks

Since the classical demonstration of DNA strand breaks was made following the exposure of mamma- lian cells to X-rays (Lett et a/., 1967) and because Ad and its derivatives appear to induce DNA single-strand breaks, it was of interest to determine to what extent exposure of X-irradiated cells to Ad during the period of repair of DNA single-strand breaks influenced strand break rejoining. Since the REQ cell line appeared most sensitive to the

10 4 HeLa no Ad R 5 - -

B N

I HeLa I 10 Ad, 50pg/ml 5u

I FRACTION

B

FIGURE 2 Ad induction of DNA single strand breaks in vitro in

HeLa cells. Log phase HeLa cells were pre-labelled with 3H-TdR and then exposed to various concentrations of Ad for 60 min. DNA sedimentation was then studied by sedimentation method 11. Sedimentation from right to left. T =top, B = bottom of gradient.

ADRIAMYCIN AND X-RAY DAMAGE I N DNA 189

Me 180 no Ad 10

i

T FRACTION

FIGURE 3 Ad induction of DNA single-strand breaks in Me-180

cells. Same conditions and methods as indicated in Figure 2.

induction of DNA single-strand breaks by Ad alone, this line was used for these studies. The conventional (Method I) alkaline sucrose gradient procedure was used, with the results shown in Figure 4. In the upper panel of Figure 4 the DNA sedimentation pattern obtained immediately following 500 rads X-rays is shown, while the lowest panel in Figure 4 shows the DNA sedimentation pattern obtained when the cells were incubated for a 60-min repair period in the absence of Ad. It can be seen that the repaired DNA sediments in a similar way to DNA not subjected to either X-rays or Ad (Fig. 1, bottom panel), indicating that the cells have substantially repaired the X-ray-induced DNA strand breaks. If various concentrations of Ad are present during the intervening repair period (middle panels, Fig. 4) there is a reduced capacity for the DNA to revert to DNA strand lengths approaching control (non- irradiated) values. However, based on the results obtained with Ad alone (Fig. 1) it appears that the small reductions in sedimentation obtained under these circumstances are probably attributable to the induction of Ad-dependent DNA single-strand breaks. Thus, it seems unlikely that Ad exerts a major effect on the repair of X-ray-induced DNA single-strand breaks.

Effect of Ad on the induction of DNA double-strand breaks

To examine the effects of Ad on the induction of a presumptively lethal DNA strand break lesion, we employed the neutral sucrose gradient method devised by Lehmann and Ormerod (1970) for the study of X-ray-induced DNA double-strand breaks. Again, REQ cells were used to evaluate this pheno- menon (Fig. 5) . Preliminary experiments failed to

indicate any DNA double-strand breaks demon- strable by this method for short-term incubations. However, when the incubation period was extended to one approaching the cell cycle duration, a con- centration-dependent induction of DNA double- strand breaks was found (Fig. 5).

E$ect of Ad on repair replication kinetics in L-1210 cells

The preceding experiments indicated that Ad could induce DNA strand breakage and lead to both DNA single- and double-strand breaks. Previous experiments (Lee et al., 1974a) have suggested that Ad can partially inhibit X-ray- induced excision repair in mouse L-1210 cells. Using the method we previously reported, which is a sensitive assay for the kinetics and extent of X-ray-induced excision repair, we monitored the effect of various Ad concentrations on repair replication in heavily irradiated mouse L-1210 leukemia cells. The results (Fig. 6, left panel) confirm that there is a concentration-dependent inhibition of excision repair when Ad is present in the medium during the repair period. The results suggest that the plateau level of excision repair found is also an inverse function of Ad concentration.

500 rods '

60 rnin

x

5 6- ZO;;d= " 4- Ad,0.4pg/rnl

2 -

6 - 500 rods ' 60 rnin No Ad -

2

0 25 50 75 100 NORMALIZED FRACTION NUMBER

FIGURE 4 Effect of Ad on the"repair of X-ray-induced DNA

single strand breaks. Re-labelled (SH-TdR) REQ cells were analyzed for the effect of various Ad concentrations on the repair of X-ray-induced DNA single-strand breaks. In all cases where Ad was present the antibiotic was added 60 min prior to the radiation exposure. In each case the radiation repair period was 60 rnin (at 37" C). Upper panel, 500 rads X-ray, no repair, no Ad. Lower panel, 500 rads X-ray, 60 rnin repair period, no Ad. Middle panels, Ad added (levels indicated in each panel) 60 rnin prior to X-ray and present during the 60-min repair period. Sedimentation from right to left. Alkaline sucrose gradient Method I was used.

BYFIELD ET AL. 190

2 I

2 I

2 m b l

2 2 O I

X

2 I

2 I

T FRACTION

FIGURE 5 Induction of DNA double-strand breaks by Ad in

HeLa cells. Pre-labelled (3H-TdR) HeLa cells were analyzed for the induction of DNA double-strand breaks as described under " Methods ". In all cases the Ad was present for 16% h at 37" C. C, controls, no Ad; Ad was present in the remaining panels at the indicated concen- trations. Sedimentation from right to left. T = top, B - bottom of gradient.

5-

TIME (Min)

Thus, less total repair is found as the external Ad concentration in the medium increases. Since the cytosine arabinoside used to suppress semiconserva- tive DNA synthesis in this assay is not completely effective, there is a background level of 3H-TdR incorporation. When this amount is subtracted from the kinetic pattern it was found that there was an absolute decline in the terminal amount of incor- porated radioactivity (Fig. 6, right panel). The origins of this phenomenon are obscure but could include either DNA autolysis in the heavily radiated, drug-treated cells or, less likely, an intrinsic suscep- tibility of repaired or repairing sites to Ad-induced nuclease activity. Further experiments will be rzquired to evaluate the origins of this phenomenon.

DISCUSSION

The growing literature on Ad and its analogues has indicated a wide variety of biochemical effects. The classical experiments are those of Calendi and colleagues (1965) who showed that the parent compound, daunomycin, became strongly bound to the deoxy-nucleoproteins and DNA of living cells, leading to changes in the visible and ultra-violet absorption spectra, fluorescent emission patterns and polarographic behavior. These changes were dependent on the existence of intact helical DNA. RNA was substantially less affected. In particular, Calendi et al., showed that the combination of daunomycin with DNA led to changes in the thermal denaturation-renaturation behavior, viscosity pro- perties and sedimentation behavior, which were consistent with the stabilization of the DNA double-

TIME (Min l

FIGURE 6 Inhibition of X-ray-induced ex-

cision repair by Ad in L-1210 leukemia cells. Log phase L-1210 cells were pre-treated with 7 ,ug/ml cytosine arabinoside and then ex- posed to 100 krads gamma rays. Following irradiation, SH-TdR was added and cytosine arabinoside- resistant SH-TdR incorporation fol- lowed (Lee et d., 1974~). Solid circles, no irradiation. Open circles, 100 krads, no Ad. Cells treated with Ad (in pg/ml): closed squares, 0.4, open triangles, 2.0, open .squares, 10.0, closed triangles, 50.0. Left panel, absolute levels of 3H-TdR incorporation plotted as a function of time following irra- diation. Right panel, SH-TdR in- corporation corrected for label uptake by non-irradiated cells. Symbols in the right panel are the same except for the solid circles which represent 50 pg/ml Ad in the right panel.

ADRIAMYCIN AND X-RAY DAMAGE IN DNA 191

stranded helix leading to an increase in the melting point. Since the viscosity was increased while the sedi- mentation coefficient was decreased, they proposed that a substantial change in conformation of the DNA was induced by the presence of the antibiotic (Calendi et al., 1965). An increase in viscosity would not be consistent with strand breakage. Subsequently, Pigram et al. (1972) showed that the X-ray diffraction pattern of DNA-daunomycin complexes was consistent with an intercalation of the molecule into the DNA helix.

These effects were reminiscent of other inter- calating agents including actinomycin D (cf. Kersten and Kersten, 1974). Since actinomycin D is the only clinically useful drug that has been reported to interfere with the repair of sub-lethal X-ray damage (Elkind et al., 1964) it seemed likely that the mole- cular effects of the two types of agents would be similar. Our initial studies (Byfield, 1973, 1974) confirmed that toxic levels of Ad, like actinomycin D, could radiosensitize oxygenated tumor cells in culture as illustrated by a steeper slope on the radiation survival curve. This observation strongly implied that intercalating agents either affected the ability of cells to repair X-ray damage or that they initiated damage that interacted with sub-lethal X-ray damage. Initially we found that daunomycin and Ad, like actinomycin D, inhibited the excision repair of X-ray damage (Lee et al., 1974~). This is confirmed in the studies reported here (Fig. 6). However, simultaneous work in these laboratories has failed to develop any convincing evidence that excision repair contributes to recovery in a split-dose assay (Byfield et al., 19766). Therefore, radio- sensitization by this mechanism seems unlikely. Another possible mechanism might be an inhibition of DNA strand break repair. Since we had already noted clinically useful synergism between Ad and conventional X-ray treatments (Watring et al., 1974) we elected to pursue the studies of alternative molecular pathways reported here.

Of central interest is the observation that Ad can induce changes in the DNA sedimentation properties of both rodent and human tumor-cell DNA con- sistent with the induction of DNA single-strand breaks (Figs. 1-3). In some preliminary studies reported several years ago (Lee et al., 1972), we suggested that actinomycin D inhibited the repair of X-ray-induced DNA single-strand breaks and that this might account for its ability to radio- sensitize. However, it was subsequently found that actinomycin D can itself induce DNA single-strand breaks (Lee et al., 1974b; Pater and Mak, 1974). Therefore, its inhibition of repair of X-ray breaks is equivocal. Both preliminary results with murine cells (Byfield et al., 1974; Schwartz and Kanter, 1975) and those reported here for human cells

show that Ad has identical effects. This phenomenon requires intact cells since even very high concen- trations of Ad had very little effect on the sedi- mentation properties of purified DNA when analyzed by identical alkaline sucrose gradient methodology (Lee and Byfield, 1976). The current data shows that the induction of DNA degradation by Ad is independent of the alkaline sucrose gradient method used for analysis (Figs. 1-3). Since an intact excision repair system is not required (Fig. l), it is unlikely that the process originates from repair of an adduct but rather probably results from Ad-induced distortion of an otherwise normal DNA helix.

The extent of DNA breakage may vary somewhat depending on the cell line being studied (cf. Figs. 1-3). The origins of these quantitative differences in sensitivity are as yet unknown. The doubling times of REQ and HeLa cell lines are both approximately 24 h in our laboratory and the duration of exposure with respect t o the cell cycle time therefore does not appear to be responsible for the differences in Ad sensitivity. Since HeLa cells originated from an adenocarcinoma (Jones et al., 1971) while the Me-1 80 cells originated from a squamous-cell carcinoma (Sykes et al., 1970) but both arose in the same organ (cervix) it seems unlikely that histological origins are important in this pheno- menon. Since Ad is thought to be transported into cells (Meriwether and Bachur, 1972) the difference in sensitivity can perhaps be related to a reduced intracellular level in the two human cell lines. However, this remains to be rigorously documented by intracellular measurement of Ad concentrations.

Of interest is the observation that Ad does not appear to inhibit the repair of most X-ray-induced DNA single-strand breaks (Fig. 4). In analyzing the origins of X-ray-induced cell killing, there is no general agreement as to the role played by DNA single-strand breaks in the causes of cell death, particularly when drugs and X-rays are combined (Byfield, 1974). Mammalian cells are known to repair the great majority of X-ray-induced DNA single-strand breaks (Lett et al., 1967). However, since all of the currently available means for evalu- ating the repair of DNA single-strand breaks are quantitatively ambiguous (Simpson et al., 1974) it seems unlikely that alkaline sucrose gradient methodology is capable of determining whether Ad-induced enhancement of cell killing is related to an interaction between these two types of DNA strand damage.

On the other hand, it is generally agreed that DNA double-strand breaks are probably lethal events (Setlow and Setlow, 1972; Chadwick and Leenhouts, 1973). It was thus of interest to note that prolonged exposure of tumor cells to relatively low concentrations of Ad leads to the induction of

192 BYFIELD ET AL.

DNA double-strand breaks (Fig. 5) . .If Ad-induced DNA double-strand breaks are lethal, then it is apparent that cell mortality based on this lesion might be involved in the killing of growing cells by Ad. These results may be compared to those of Hittelman and Rao (1975), who used the process of premature chromosome condensation to analyze the effect of Ad on cellular DNA. They showed that, while Ad did not inhibit the progression of G1 cells into S phase, it led to a substantial inhibition of progression of S phase cells into G2. Prolonged treatments at relatively low Ad concentrations produced almost complete elimination of normal G2 chromosomes from the culture. Of interest was their observation that a variety of chromosome gaps, breaks, and exchanges were demonstrable in Ad- treated G2 cells and that this response was dose- dependent. The degradation of DNA demonstrated in the experiments reported here would suggest that the chromosome damage found by Hittelman and Rao in G2 cells may originate from in vivo degra- dation of DNA due to nuclease activity in Ad- treated cells. Their data also suggested that some degree of repair was possible in cells treated with low levels of Ad. We have not been able to demon- strate the repair of Ad-induced DNA single-strand breaks. However, it is probably not possible to remove the Ad from the cultures to such a degree that continued breakage does not occur, obscuring low levels of repair.

It is also of interest to consider these results in relationship to the observations that Ad can increase the transformation rate of rat cells in tissue culture

(Price et al., 1975), and can also increase the mutation frequency in bacteria (Pani et al., 1975). Our exper- iments suggest that rat cells are quite sensitive to the induction of DNA strand breakage by Ad and such breakage could be related to its capacity to transform such cells in tissue culture. Since both Ad and daunomycin have now been demonstrated to inhibit excision repair, it may be speculated that such interference may perhaps lead to alternative repair pathways associated with an increased rate of mutagenesis. It is well established that exposure of non-virus-producing cell lines carrying viral information to pyrimidine analogues can induce virus production (Lowy et al., 1971). Since such analogues also render cells highly susceptible to DNA strand breakage (cf. Setlow and Setlow, 1972), a relationship between Ad-induced cell transformation and DNA strand breakage and repair may exist.

The correlation between these molecular studies and the interaction between Ad and X-rays at the level of cell survival are discussed in the accompany- ing communication (Byfield et al., 19766).

ACKNOWLEDGEMENTS

This work was supported by Division of Cancer Treatment contract NO1-CM-43791 and in part by USPHS grant CA-12691 from the National Cancer Institute, NIH, US Department of Health, Education and Welfare. A portion of it was performed in facilities supported by funds from the US Environ- mental Protection Agency.

INTERACTIONS MOL~CULAIRES ENTRE LES LESIONS CAUSEES PAR L’ADRIAMYCINE ET PAR LES RAYONS x DANS LES CELLULES DE TUMEURS DE MAMMIF~RES

Les auteurs ont 6tudiQ I’effet d’un antibiotique de la catdgorie des anthracyclines, I’Adriamycine (Ad), sur les propriites de sedimentation de I’ADN de mammifhres prdmarqud. L’Ad induit une degradation de I’ADN in vivo dans les cellules competentes (HeLa e t Me-180) et deficientes (REQ) pour la rdparation des lesions. Lorsque les cellules irradiees sont exposees h I’Ad pendant la periode de rdparation des rup- tures d’un brin d’ADN, un petit nombre de ruptures residuelles subsistent aprhs la riparation. Elles sont imputables A I’action de I’Ad. Les effets de I’Ad et des rayons X semblent donc analogues et cumulatifs. On n’a pas pu demontrer nettement que I’Ad inhibe la reparation des ruptures d’un brin d’ADN induites par les rayons X. L’Ad entrahe aussi la formation de ruptures des doubles brins d’ADN et inhibe la rdpara- tion des lesions des bases induites par les rayons X (replication de la rdparation). Le fait que I’Ad provoque des cassures des brins d’ADN peut expliquer sa toxicit6 pour les cellules et sa capacite d’activer les lesions primaires dues aux rayons X lorsque les deux types de ldoions coexistent.

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ADRIAMYCIN AND X-RAY DAMAGE IN D N A 193

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