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DNA crosslinking induced by X-rays and chemical agents
343
Biochimica et Biophysica Acta, 477 (1977) 3 4 3 - - 3 5 5 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 9 8 9 7 2
DNA CROSSLINKING INDUCED BY X-RAYS AND CHEMICAL AGENTS
A L B E R T J. F O R N A C E , Jr. * and J O H N B. L I T T L E
Laboratory of Radio biology, Dept. of Physiology, Harvard University School of Public Health, Boston, Mass. 02115 (U.S.A.) (Received F e b r u a r y 2nd, i 9 7 7 )
Summary DNA corsslinking was measured in human diploid fibroblasts by the method of alkaline elution [13,14]. Following treatment of the cells by known DNA crosslinking agents (HN2, mitomycin-C), a controlled frequency of DNA strand breaks was induced by a small test dose of X-rays at 0°C. The effect of the prior treatment with a crosslinking agent was to reduce the rate of DNA elution in a dose dependent manner. When cells were X-irradiated, incubated for 2 h at 37°C in order to permit rejoining of most of the induced DNA strand breaks, and then exposed to the small test dose of X-rays at 0°C, a reduction in the rate of DNA elution occurred. The magnitude of this X-ray induced DNA crosslink effect was dependent upon the initial dose of X-rays (1--60 krad), and was increased by irradiation under hypoxic conditions. A similar level of crosslinking was present 2 h after 5 krad of X-ray as was present with a 30 min exposure to a 1 pM nitrogen mustard. Evidence is presented that crosslinking occurs immediately after X-irradiation and is repaired with incubation at 37 ° C. X-ray crosslinking was attributed to DNA-protein crosslinks since treatment of the cell lysates with proteinase removed the effect. X-ray induced DNA single strand breaks can be measured over an increased range (25 rad--10 krad) by alkaline elution when the crosslink effect is first removed by proteinase digestion.
Introduction Previous investigators have presented evidence suggesting that ionizing radiation produces DNA crosslinks; these studies have employed various methods which measure either the extractability of DNA from protein [1--4], or changes in the viscosity and sedimentation of DNA [5--10]. These methods * Present address: Dept. of Pathology, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Mass. 02115 (U.S.A.).
344 have usually required very high doses of irradiation. Recently a filter technique [11] has been described which can detect DNA-protein crosslinks produced in vitro at more biologically relevant doses. In aqueous solution, covalent bonds are produced by X-ray or ultraviolet light between purines and small molecules; these molecules may mimic functional groups present in nucleic acids and proteins [12]. It has recently been shown that the alkaline elution technique can detect both DNA crosslinks in intact mammalian cells produced by exposure to low concentrations of nitrogen mustard (HN2} [13,14] as well as the production and repair of DNA-protein crosslinks induced by low doses of ultraviolet light [13,15]. In the present investigation , DNA-protein crosslinks have been examined following relatively low doses of X-irradiation by alkaline elution and evidence is presented for their possible repair. DNA crosslinking by various chemical and physical agents has also been studied with this technique and an approach has been proposed to quantitate these crosslinks to very low doses. After the removal of X-ray induced DNA crosslinking by proteinase digestion, DNA single-strand breaks produced by X-ray have been studied over an expanded range (25 rad--10000 rad) as compared to previous results [13,14]. Materials and Methods Cells and cell labeling: The fibroblast cell strain CRL 1220 (American Type Culture Collection, Rockville, Md.) was derived from a normal human adult donor and used at passages 5 to 18 (1 : 4 split}. The cells were grown and prepared for experiments as previously described [13]; [2-J4C]thymidine was added to growing fibroblast cells for several days and then removed. On the day of the experiments, these cells were in a confluent monolayer. Exponentially growing L1210 mouse leukemia cells [16] were labeled with [3H]thymidine as previously described [ 13]. Irradiation. X-rays were delivered by a GE Maximar X-ray generator operating at 220 kV and yielding a dose rate of 80 rad/min. When cells were incubated following X-ray, they were irradiated at 37°C and incubated for the indicated time at 37°C. They were then rinsed with warm saline, incubated with trypsin at 37°C for 60 s, and suspended in cold medium buffered with 0.01 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonate). When cells were not incubated after X-ray, they were X-irradiated while suspended in cold medium buffered with 0.01 M HEPES at 0°C; unless otherwise indicated, all X-irradiations done at 0°C were in medium and employed the X-ray generator described above. At high radiation doses (5--60 krad), a cobalt gamma-ray source was used which contained approximately 3000 C of cobalt-60 and yielded a dose rate of 160 rad/s. Hypoxic irradiation was performed with a 100 kV Philips X-ray unit yielding a dose rate to the cells of 248 rad/min. The irradiation chamber was modified such that humidified N2 continuously flowed over the fibroblasts which were in a confluent monolayer in a 30 cm 2 petri dish covered with 1.5 ml of warm medium. The cells were flushed with N~ for 15 min prior to irradiation; the 02 c o n t e n t of the outflow of the irradiation chamber was determined with a Thermox-Analyzer combustion chamber (Thermo-Lab Instruments, Inc., Glenshaw,
345 Pa.) and was 10--35 ppm at the time of irradiation. Each experiment contained controls which were X-irradiated with the same apparatus but flushed with room air. The radiation effect in air determined by alkaline elution was the same with either the 100 kV X-ray generator or the cobalt gamma ray source at the same dose. Chemical agents. HN2, nitrogen mustard (NSC 762); HN1, 2-chloro-N,Ndimethylethylamine hydrochloride (NSC 1917); and mitomycin-C (NSC 26980) were kindly supplied by the Drug Development Branch, Division of Cancer Treatment, National Cancer Institute; these agents were stored at --120°C. HN2 and HN1 were dissolved in 0.01 M HC1; mitomycin-C was dissolved in 30% methanol; these stock solutions were stored at --15°C. When fibroblasts were treated with these agents, the stock solution was added directly to the cells and medium such that the final concentration of solvent was 0.2%. At the end of this treatment, the medium was removed, the monolayer was rinsed four times with warm saline, and the cells exposed to 0.25% trypsin for 60 s, then suspended in cold medium buffered with 0.01 M HEPES buffer. Alkaline elution. The procedure used [13,15] is a modification of that described by Kohn et al. [17]. Cells were filtered onto a polyvinylchloride filter, lysed with 2 M NaC1, 0.02 M Na3 EDTA/0.2% Sarkosyl (pH 10.2), washed with 0.02 M Na3 EDTA (pH 10.3), and then eluted with 0.10 M tetrapropylammonium hydroxide/0.02 M EDTA (acid form, pH 12.2) at a pump speed of 0.04 ml/min. Eluted fractions were collected and assayed for radioactivity as previously described [ 13]. In order to provide for an internal standard, 3H-labeled L1210 cells which had received 150 rad at 0°C were included in each assay. The fraction of sample DNA which is retained on the filter when 50% of the internal standard DNA has eluted has been termed the "relative r e t e n t i o n " [131. Proteinase-K treatment. The proteinase treatment employed has been described previously [13]. Cells were lysed on the filter for alkaline elution and, after the pH 10.2 lysis solution had run through, the filters were washed with 0.01 M Tris/0.01 M EDTA/0.01 M NaC1/0.5% sodium dodecyl sulfate (pH 8.0); 0.5 mg/ml proteinase-K (E. Merck, Darmstadt, G.F.R.) in the same buffer was then added for 30 min at 23°C. When proteinase-K was omitted, incubation was in the same buffer. The solution was then allowed to drip out and alkaline elution was carried out at pH 12.2 [ 13]. Results
Evidence has been presented that the alkaline elution technique can quantitatively measure DNA single-strand breaks produced by low doses of X-ray [13--15]. At X-ray doses of 400 rad or less, elution as measured by relative retention (see Materials and Methods) decreased approximately according to a first-order relation of the X-ray dose; the Do dose was approximately 220 rad. In each elution an internal reference, L1210 cells, which had been X-irradiated with 150 rad at 0 ° C, was included. In order to improve quantification, elution of sample DNA was plotted against the elution of the internal reference DNA; there was little change in the shape of the elution curves since the internal DNA eluted in a nearly linear fashion. This method of plotting is demonstrated in
346
Fig. 1A (upper abscissa) in terms of the fraction of internal standard DNA retained on the filter. In order to simplify this method of plotting, and since the internal reference DNA elutes at a constant rate on a logarithmic plot, hours of elution (corrected) is defined such that 50% of the internal reference cell DNA had eluted off the filter in 12 h (lower abscissa Fig. 1A). The relative retention is defined as the fraction of sample DNA retained on the filter after 12 h of elution (corrected). Cells were first treated with a known corsslinking agent. To elicit the DNA crosslinking effect, they were subsequently irradiated with a small dose of X-ray. The effect of a crosslinking agent is to decrease the apparent level of strand breakage induced by X-ray [13,18]. In Fig. 1A, fibroblasts were treated with varying doses of nitrogen mustard (HN2) and then X-irradiated with 400 rad. Repair of X-ray induced DNA single-strand breaks was prevented by maintaining the cells at 0°C during and following X-irradiation. There is a dose dependent increase in the fraction of DNA retained on the filter with increasing concentration of HN2. In Fig. 1B, HN1 (a monofunctional analogue of HN2) had no crosslinking effect, i.e. the effect of the single-strand breaks induced by HN1 damage to the cell DNA was additive with the effect produced by X-ray. At a less toxic concentration of HN1, 5 • 10 -6 M for 30 min, there was also no crosslink effect (data not shown). After cells have been treated with a crosslinking agent and DNA single-strand
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HOURS OF ELUTION ( c o r r e c t e d ] Effect of nitrogen lnustard and half-mustard, (2-chloro-N-dimethylethylaminc) on X-ray sensitivity. Confluent c u l t u r e s w e r e t r e a t e d w i t h H N 2 , f I N 1 or s o l v e n t f o r 3 0 rain at 3 7 ° C ; t h e c e l l s w e r e then suspended in cold medium, X-irradiated at 0°C where indicated (X), and analyzed by alkaline elut i o n . A . E f f e c t o f v a r y i n g d o s e s o f H N 2 ( c o h e n . in t o o l ) ; w i t h o u t X - r a y ( X ) all t t N 2 - t r e a t e d samples had approximately t h e s a m e e l u t i o n p r o f i l e ( l a b e l e d " H N 2 " ) . B. E f f e c t o f h a l f - m u s t a r d , H N I . Fig. 1.
347 breaks have been produced by X-ray, the elution profile of the DNA is characteristically non-linear as shown in Fig. 1A. There is an early rapid elution which appears to be X-ray dependent, and then a late slow elution which is crosslink dependent. An estimate of the a m o u n t of DNA crosslinking can be derived if one examines the a m o u n t of DNA left on the filter during this late phase. The relative retention (the fraction of DNA left on the filter after 12 h of elution (corrected)) has been chosen for this purpose. The increase in elution with X-ray at this time point, designated "relative elution," is shown in Fig. 2A. Again, the first-order relation of elution to X-ray dose is demonstrated. After treatment of the cells with a crosslinking agent, HN2, the X-ray sensitivity is decreased, but the relative elution still increases in a linear manner with increasing X-ray dose. The effect of a crosslinking agent, therefore, is to decrease the slope of the DNA X-ray sensitivity by a constant factor. If the relative elution of non-crosslinked DNA (mean) at a specific X-ray dose is divided by the relative elution of crosslinked DNA at the same X-ray dose, then this ratio will be the factor by
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Fig. 2. E f f e c t of crosslinking agent~ o n X-ray s e n s i t i v i t y . " R e l a t i v e e l u t i o n " r e p r e s e n t s the increase in e l u t i o n of D N A a f t e r X-ray a n d is d e f i n e d as: (log o f relative r e t e n t i o n of f i b r o b l a s t D N A w i t h o u t X - r a y ) - - (log of relative r e t e n t i o n of f i b r o b l a s t D N A f o l l o w i n g X-ray at 0 ° C ) ; e, X-ray alone, ± S.E.; o, f o l l o w i n g 30 rain t r e a t m e n t w i t h H N 2 , 2.5 • 10 -6 M for 3 0 rain. B. Crosslink f a c t o r is d e f i n e d as: ( m e a n value of t h e relative e l u t i o n a t a g i v e n X-ray d o s e ) - (relative e l u t i o n o f f i b r o b l a s t D N A a f t e r t r e a t m e n t of t h e cells w i t h a crosslinking a g e n t f o l o w e d b y t h e s a m e X-ray d o s e ) . T r e a t m e n t w i t h H N 2 or m i t o m y c i n - C (MMC) was f o r 3 0 rain as d e s c r i b e d in Fig. 3; X-ray dose w a s 4 0 0 t a d . F o l l o w i n g u l t r a v i o l e t ( U V ) light t r e a t m e n t ( d a t a f r o m Ref. 13, cells w e r e C R L 1 1 1 9 n o r m a l h u m a n f i b r o b l a s t s f r o m A T C C ) , cells w e r e i n c u b a t e d for 30 rain a t 37~C, s u s p e n d e d , a n d X - i r r a d i a t e d at 0 ° C w i t h 4 4 0 t a d . F o l l o w i n g t r e a t m e n t w i t h u l t r a v i o l e t light, t h e crosslink f a c t o r was d e r i v e d b y dividing t h e relative e l u t i o n at 4 4 0 t a d ( t a k e n f r o m Fig. 2a) by the relative e l u t i o n o b t a i n e d f r o m [ 13] a t v a r i o u s d o s e s o f u l t r a v i o l e t light. U n t r e a t e d s a m p l e (no ultra° v i o l e t ) fell w i t h i n 1 S.D. o f e x p e c t e d v a l u e , 1.0.
348 which the X-ray sensitivity, as determined by alkaline elution, has been decreased and will be independent of the X-ray dose (200--400 rad) used to elicit the crosslink effect. This ratio, designated "crosslink factor", is plotted in Fig. 2B. The "crosslink f a c t o r " value is 1.0 in untreated samples and increases with increasing doses of HN2. As can be seen in Fig. 2B, known crosslinking agents, HN2, mitomycin-C, and ultraviolet light, produce a dose-dependent increase in value of the crosslink factor in an approximately linear manner. If one assumes that the a m o u n t of DNA crosslinking is directly proportional to the dose of crosslinking agent [18] at the doses we have used, then the crosslink factor appears empirically to be directly proportional to the a m o u n t of DNA crosslinking. The detection of DNA crosslinking induced by X-irradiation is complicated by the large number of DNA single-strand breaks produced. In Fig. 1A, the shape of the elution profile after 400 rad alone suggests possible DNA crosslinking; namely, the elution profile is concave upwards with a decreasing elution rate when 15--25% of the DNA is left on the filter. The interpretation after higher doses of X-ray is further obscured since most of the DNA elutes rapidly off the filter. Since most X-ray induced DNA single-strand breaks are rapidly rejoined [19,20], the crosslinking effect caused by X-rays is more easily demonstrated following post-X-irradiation incubation. In Fig. 3, fibroblasts were X-irradiated (5 krad) and incubated at 37°C for 2 h; most of the DNA single-strand breaks have been rejoined by this time and about 60% of the DNA is retained on the filter after 12 h of elution (corrected). When these cells were subsequently irradiated with a small dose of X-rays (300 rad) at 0°C, the increase in elution was less than in cells given 300 rad alone (no prior gammairradiation). This is demonstrated in Fig. 3 by the solid arrow (for crosslinked DNA) as compared to the dashed (for non-crosslinked DNA). In fact, more
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349 DNA is retained on the filter at 12 h from the sample which received the prior gamma irradiation plus the 300 rad dose than the sample which received 300 rad alone. In Fig. 4, the DNA crosslinking present 2 h after irradiation with varying doses of ionizing (cobalt-60) gamma-irradiation demonstrated. DNA crosslinking, as measured by the crosslink factor, was detected after exposure to 1 krad, and increased progressively in a near linear fashion with increasing radiation doses up to 60 krad. Thus, 2 h after irradiation of cells there was a distinct DNA crosslinking effect as measured by alkaline elution, and this crosslinking increased in a dose dependent manner similar to the crosslinking produced by known DNA crosslinking agents. In Fig. 5 a representative experiment is presented which demonstrates the effect of varying the incubation time after 5 krad. Under aerobic conditions {lower curve), there appears to be a rapid decrease in the amount of DNA crosslinking, as measured by the crosslink factor, which occurs with 2--4 h of postirradiation incubation; this is followed by a slower decrease at later times. The kinetics of this recovery from crosslink damage is obscured, however, as 7 determinations (closed circles, Fig. 5) done in separate experiments after 2 h of incubation showed significant variability. In all experiments, however, the crosslink factor after longer incubation times was always less than any of the determinations done at 2 h. Under hypoxic conditions obtained with the 02
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X-RAY (KRAD. 2 hr INCUBATION) Fig. 4. Crosslink factor after X-ray, • e, c e l l s w e r e t r e a t e d as d e s c r i b e d in p r e v i o u s l e g e n d w i t h v a r y i n g d o s e s o f X - r a y . C r o s s l i n k f a c t o r is t h c s a m e as d e f i n e d i n F i g . 2; o . . . . . . o, e f f e c t o f H N 2 , t a k e n f r o m F i g . 2 . C r o s s l i n k f a c t o r w a s d e t e r m i n e d b y 3 0 0 rad (a) o r 4 0 0 rad ( B ) at O~C. I n F i g . 4 A , t h e c r o s s l i n k e f f e c t is s h o w n 2 h a f t e r 1 - - 5 k r a d ; t h e e f f e c t P r o d u c e d b y H N 2 is i n c l u d e d f o r c o m p a r i s o n . I n F i g . 4 B , t h e r a n g e o f X - r a y d o s e s u s e d w a s e x p a n d e d t o 5 - - 6 0 k r a d , as c o m p a r e d t o t h e s a m e d o s e s o f H N 2 as u s e d in F i g . 4 A .
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F i g . 5. R e p a i r o f t h e c r o s s l i n k i n g e f f e c t o f X - r a y . C e i l s w e r e e x p o s e d t o 5 k r a d a n d i n c u b a t e d a t 3 7 ° C f o r v a r y i n g p e r i o d s o f t i m e ; o, c e l l s i r r a d i a t e d i n r o u t i n e m e d i u m ( a i r ) ; /,, c e l l s i r r a d i a t e d i n m e d i u m w h i c h h a d b e e n f l u s h e d w i t h N 2 ; '~, c e l l s i r r a d i a t e d i n m e d i u m c o n t a i n i n g 5 m M N a 2 S 2 0 4 a n d t h e n i n c u b a t e d i n routine medium. The crosslink factor was determined following a test dose of 300 rad (air) at 0°C.
scavenger sodium hydrosulfite, Na2S2O4 (upper curve, Fig. 5), there was also an apparent early rapid recovery (from 1 to 2 h) and a slow late recovery from crosslink damage. These findings indicate that the DNA crosslinking detected after X-ray under aerobic or hypoxic conditions by alkaline elution decreases (repair) from 1--2 to 12--13 h after irradiation. The effect of hypoxia on DNA crosslink production after X-ray is also demonstrated in Fig. 5. The hypoxic effect was produced by X-irradiation of cells in N2 or in the presence of 5 mM Na2S~O4; Na2S204 is an 02 scavenger which has been shown to have an oxygen enhancement ratio for survival of 2.5 as determined by colony-forming ability after X-irradiation (Ritter, M.A., unpublished results). 2 h after X-ray exposure, the crosslink factor is always greater with hypoxic X-irradiation (5 determinations) than with aerobic X-irradiation (7 determinations). Since the crosslink factor with no DNA crosslinking is 1.0, the mean crosslink factor for 5 krad with a 2 h post-X-ray incubation is 48% greater with hypoxic irradiation than with aerobic irradiation. A similar effect with hypoxic irradiation was found after 1 krad with a 2 h incubation (data not shown). To determine whether protein is involved in the X-ray-induced crosslinks, the effect of proteolytic t r e a t m e n t on elution was studied. The cell lysates were treated with proteinase-K prior to elution; this reduced the a m o u n t of protein on the filter from approx. 5% to 0.5% [13]. The proteinase-K effect on the control and internal reference cells was to slightly increase the elution (approx. 5%); when plotted such t h a t 50% of the internal reference DNA eluted in 12 h, the proteinase effect on the control cell DNA was very slight (Fig. 6A). The effect on cells which had received 300 rad alone was also slight initially, but at longer elution times the elution rate was somewhat increased with proteinase suggesting a slight protein-mediated crosslink effect.
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Fig. 7. E f f e c t o f p r o t e i n a s e - K o n e l u t i o n o f X - i r r a d i a t e d DNJk. F i b r o b l a s t s w e r e X - i r r a d i a t e d a t O°C a n d a n a l y z e d b y alkaline e l u t i o n m o d i f i e d t o i n c l u d e t r e a t m e n t o f t h e cell l y s a t e s w i t h p r o t e i n a s e - K as in Fig. 6. A. R a p i d e l u t i o n a f t e r 0.5 t o 1 0 k r a d ( c o b a l t ) , e l u t i o n o f D N A is p l o t t e d against t h e e l u t i o n of the r e f e r e n c e cells; t i m e of e l u t i o n w a s c o r r e c t e d s u c h t h a t 10% of the r e f e r e n c e cell D N A h a d e l u t e d in 45 rain; 0.3 m l f m i n , p u m p s p e e d ; f o u r 6-rnin a n d six 12-rain f r a c t i o n s w e r e c o l l e c t e d ; o . . . . . -~, 5 k r a d irrad i a t i o n b u t p r o t e i n a s e - K o m i t t e d . B. E l u t i o n a f t c r 2 5 - - 5 0 0 r a d ( 0 . 0 4 m l / m i n , p u m p s p e e d ) f o l l o w i n g proteinase t r e a t m e n t of cell lysates.
352 When the cells were irradiated with 5 krad and incubated for 2 h, then X-irradiated with 300 rad at 0°C (Fig. 6B), the proteinase-K treatment produced a marked increase in elution. The crosslink effect, namely, the decrease in elution produced by the prior X-irradiation, is abolished. If DNA crosslinks are indeed produced by X-ray, then DNA single-strand breaks should be more easy to quantify, particularly at higher doses of X-ray, if these crosslinks are first removed by proteinase treatment. Such experiments were performed utilizing doses of X-ray from 25 rad to 10 krad (Fig. 7); in Fig. 7A the pump speed was increased to 0.3 ml/min; in Fig. 7B the pump speed was 0.04 ml/min. As can be seen in Fig. 7, there was a dose dependent increase in the elution of DNA with increasing X-ray dose. The slowing of the elution rate, when 15-25% of the DNA is retained on the filter, is not seen in cells irradiated with 250 and 500 rad with proteinase digestion of the lysates. Following higher doses of X-rays, there was also a near linear elution of 90% of the DNA. In Fig. 7A, the internal reference elutes faster with the higher pump speed (0.3 ml/ min) but, as can be seen with the 500 rad dose, the fraction of DNA retained on the filter versus the fraction of the internal standard DNA retained is approximately the same with either pump speed. Without the proteinase digestion with 5 krad of X-ray, DNA elutes in a slower non-linear manner characteristic of crosslinked DNA. This indicates that DNA-protein crosslinks are present immediately after X-ray and require no incubation at 37 ° C. Discussion When DNA single-strand breaks are produced by a small test dose of X-rays, the increased elution of DNA from the filter is diminished if the cells have previously been treated with a crosslinking agent [13,14]. Owing to the large number of single-strand breaks present immediately after X-irradiation, the DNA crosslinking produced by ionizing radiation itself is more easily demonstrated with this approach after most of these strand breaks have been rejoined. At this time (2 h), a dose dependent increase in DNA crosslinking is induced by radiation doses of 1--60 krad. This crosslinking, as seen by alkaline elution, is qualitatively similar to that seen after ultraviolet irradiation [13], namely, it is proteinase sensitive and resistant to salt (2 M NaC1), detergent, (SDS, Sarkosyl), and alkali. Immediately after X-irradiation, a proteinase e f f e c t o n elution is seen with doses as low as 300 rad; this indicates that at least some of the DNA crosslinking seen after X-irradiation does n o t require incubatiqn and that following doses less than 1 krad, the crosslinking may have been repaired by 2 h of incubation. At doses greater than 1 krad, this crosslinking decreases with further incubation, also suggestive of repair. Previous studies of X-ray induced DNA' crosslinking have usually been with higher doses [1,3,5--9] and repair has not been studied. Other investigators [5--7,11] have noted an increased yield of DNA crosslinks with hypoxic X-irradiation. This appears to be the case with our results, but different rates of repair of crosslinking in the first 2 h after irradiation cannot be excluded. The DNA crosslinking agent, HN2, is at least ten times more toxic to the cell than its monofunctional analogue HN1 [21]. This suggests that linkages between a DNA strand and adjacent marcromolecules are of greater conse-
353 quence to the cell than monofunctional reactions of similar small molecules with DNA. Significant DNA crosslinking, as determined by alkaline elution, is seen with known DNA crosslinking agents at biologically relevant doses in mammalian cells. The crosslink factor at the approximate D~0 for cell lethality following exposure to various agents is: 4.2 for HN2 (3 pM, 30 min) [21] ; 3.1 for mitomycin-C (2.5 pM, 30 min) [22 and Nagasawa, H., unpublished results]; 1.8 for ultraviolet (10 J • m-:, 30 m i n ) [13 and Little, J.B., unpublished results]. The D~0 for X-ray, however, is approx. 350 tad in human diploid fibroblasts [23], while DNA crosslinking comparable to that induced by the above agents occurs only after exposure to much higher doses. 2 h after exposure to 5 krad, for example, the crosslink factor was 2.0. X-ray-induced DNA crosslinking is increased under hypoxic irradiation while cell survival is increased. These facts suggest that other types of damage are probably also responsible for the lethal effects of X-irradiation. The effect of X-ray induced DNA crosslinking on cell 1.ethality, carcinogenesis, and aging remains to be determined. The~exact mechanism by which DNA-protein crosslinks influence the alkaline,elution of DNA, is n o t known. HN2 [24] and mitomycin-C [25] are known DNA-DNA crosslinking agents which would be expected to affect an assay invQlving alkaline denaturation [13,14,18,24]. HN2, a highly reactive alkylating agent, may also produce DNA-protein crosslinks; DNA extractability from protein is decreased following treatment with HN2 [26--28]. However, DNA extractability is also decreased by monofunctional agents and various metabolic inhibitors at toxic doses [ 28]. With the alkaline elution technique, no crosslink effect is seen with HN1 (this paper) at doses as toxic or more toxic to the cell than HN2 [21] and no crosslink effect was seen with toxic doses of bleomycin [29]. These findings indicate that DNA crosslinking as detected by alkaline elution is more specific for damage produced by polyfunctional (crosslinking) agents as compared to other DNA extractability assays. Although a mathematical model representing the effect of DNA crosslinking on alkaline elution has n o t been presented, owing to the fact that the nature of the crosslinks is not well defined, several observations and speculations can be made. First, the rate of elution of DNA from the filter approximates a first order function of the number of single-strand breaks produced by X-rays at low doses where the crosslink effect of X-rays is slight [13--15 and this paper]. Empirically, the value "crosslink factor" appears to be approximately directly proportional to the dose of the crosslinking agent used. In Fig. 1A, the DNA from cells which had received X-ray alone rapidly eluted off the filter, while crosslinked DNA produced by HN2 eluted off the filter very slowly after an initial rapid phase. This slow late elution profile is nearly linear with a small negative slope. If one assumes that the crosslinked DNA is represented by the a m o u n t of DNA in this slowly eluting component, then the value "crosslink factor" is derived from an approximation of this c o m p o n e n t taken at 12 h of elution; a more accurate estimate of the amount of DNA in this slowly eluting crosslinked phase would b e to extrapolate this line back to the ordinate (e.g., 5 pM HN2, 82% of the DNA in crosslinked phase at ordinate). If one assumes that one crosslink per DNA single strand renders that DNA strand crosslinked, then the Do for crosslinked DNA (that is, the dose yielding an average of one
354
crosslink per DNA strand, 37% rapidly eluting phase, 63% slowly eluting crosslinked phase) would be approximately 1 pM HN2, 30 min treatment time. As the test X-ray dose in the experiment in Fig. 1A was 400 rad, the average DNA strand size would be 1.1 • 109 daltons, assuming an efficiency for DNA singlestrand breakage of 2.3 • 10 -~2 dalton per rad [30] ; 30 min treatment with I pM HN2 would produce one crosslink per 109 daltons of DNA. This compares with 2 DNA-DNA crosslinks per 109 dalton of DNA for a 1 h treatment with 1 pM HN2 as determined by Jolley and Ormerod [18]. If one further assfimes that the crosslink factor is proportional to the number of DNA crosslinks irrespective of the type of crosslink, then 2 h after exposure to 5 krads there is one DNA crosslink per 109 dalton of DNA or approx. 3100 DNA crosslinks per cell. Finally, Kohn et al. [14] have shown t h a t at pH 11.9 or greater DNA elutes according to a first order function of the number of X-ray-induced strand breaks (when the pH is lowered only DNA strands of a certain size or less will elute). Previously, this technique was useful for the study of strand breaks produced by doses of less than 1 krad of X-ray; at doses as low as 300--400 tad, DNA crosslinking produces significant slowing of the late phase of elution. Following proteinase treatment, DNA single-strand breaks can be studied relatively free of the crosslink effect caused by X-ray such that higher doses of X-ray (up to 10 krad) can be used. When studying DNA single strand size by alkaline sucrose sedimentation, the DNA must first be freed from a " c o m p l e x " by long lysis in alkali and other treatments. Crosslinking agents delay the release of freely sedimenting DNA from this complex [18]. It would seem logical to expect t h a t X-ray crosslinks may have the same effect:. This would argue for a proteinase digestion [14,31] of cell lysates prior t o sedimentaticn in order to minimize the DNA shear and alkaline degradation induced by other methods of freeing DNA from this complex. Acknowledgements We thank Mr. Michael Allocca and Mrs. Dorothy Foscaldo for expert assistance. This work was supported by training grant CA-09078 and research grants CA-11751 and ES-00002 from the U.S. National Institutes of Health. References 1 2 3 4 5 6 7 8 9 10 ll 12 13 14 15 16
A l e x a n d e r , P. a n d S t a c e y , K . A . ( 1 9 5 9 ) N a t u r e 1 8 4 , 9 5 8 - - 9 6 0 S m i t h , K.C. ( 1 9 6 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3, 1 5 7 - - 1 6 3 F e l d b e r g , R.S. a n d G r o s s m a n , L. ( 1 9 7 6 ) B i o c h e m . 15, 2 4 0 2 - - 2 4 0 8 Mce, L.K. a n d A d e l s t e i n , S.J. ( 1 9 7 6 ) R a d i a t . Res. 6 7 , 6 3 5 . ( A b s t r . ) L e t t , J . T . , S t a e e y , K.A. a n d A l e x a n d e r , P. ( 1 9 6 1 ) R a d i a t . Res. 1 4 , 3 4 9 - - 3 6 2 L e t t , 3.T. a n d A l e x a n d e r , P. ( 1 9 6 1 ) R a d i a t . Res. 15, 1 5 9 - - 1 7 3 l l a g e n , U. a n d Wellstein, H. ( 1 9 6 5 ) S t r a h l e n t h e r . 1 2 8 , 5 6 5 - - 5 7 1 C o q u e r e l l e , T., B o h n e , L., H a g e n , U. a n d M e r k w i t z , J. ( 1 9 6 9 ) N a t u r f o r s ~ h . 2 4 b , 8 8 5 - - 8 8 8 B o h n e , L., C o q u e r e l l e , T. a n d H a g e n , U. ( 1 9 7 0 ) I n t . J. R a d i a t . Biol. 17, 2 0 5 - - 2 1 5 Krasin, F., P e r s o n , S., L e y , R.D. a n d t t u t e h i n s o n , F. ( 1 9 7 6 ) J. Mol. Biol. 1 0 1 , 1 9 7 - - 2 0 9 M i n s k y , B.D. a n d B r a u n , A. ( 1 9 7 7 ) R a d i a t . Res. ( s u b m i t t e d ) L e o n o v , D. a n d Elad, D. ( 1 9 7 4 ) J. Org. C h e m . 3 9 , 1 4 7 0 - - 1 4 7 3 F o r n a c e , Jr., A.J. a n d K o h n , K.W. ( 1 9 7 6 ) B i o e h i m . B i o p h y s . A c t a 4 3 5 , 9 5 - - 1 0 3 K o h n , K.W., E r i c k s o n , L.C., Ewig, R . A . G . a n d F r i e d m a n , C.A. ( 1 9 7 6 ) B i o c h e m i s t r y , in the press F o r n a e e , .Jr., A.J., K o h n , K.W. a n d K a n n , Jr., H.E. ( 1 9 7 6 ) P r o c . Natl. A c a d . Sci. U.S. 73, 39---43 M o o r e , G . E . , S a n d b e r g , A . A . a n d Ulrich, K. ( 1 9 6 6 ) J. Natl. C a n c e r Inst. 3 6 , 4 0 5 - - 4 2 1
355 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Kohn, K.W., Friedman, C.A. Ewig, R.A.G. and Igbal, Z.M. (1974) Biochemistry 13, 4134--4139 Joney, G.M. and Ormerod, M.G. (1973) Biochim. Biophys. Acta 3 0 8 , 2 4 2 - - 2 5 1 Lett, J.T., CaldweU, I., Dean, C.J. and Alexander, P. (1967) Nature 2 1 4 , 7 9 0 - - 7 9 2 Kann, Jr., H.E., Kohn, K.W. and Lyles, J.M. (1974) Cancer Res. 34, 398--402 Wheeler, G.P., Bowdon, B.J., Adamson, D.J. and Vail, M.H. (1970) Cancer Res. 30, 100--111 Djordjevic, B. and Kim, J.H. (1968) J. Cell. Biol. 3 8 , 4 7 7 - - 4 8 2 Weichselbaum, R.R., Epstein, J. and Little, J.B. (1976) Radiology 1 2 1 , 4 7 9 - - 4 8 2 Kohn, K.W. and Spears, C.L. (1967) Biochim. Biophys. Acta 145, 734--741 lyer, V.N. and Szybalski, W. (1963) Microbiol. 50, 355--362 Steele, W.J. (1962) Proc. Amer. Assoc. Cancer Res. 3 , 3 6 4 . (Abstr.) Golder, R.H., Martin-Guzman, G., Jones, J., Goldstein, N.O., Rotenberg, S. and R u t m a n , R.J. (1964) Cancer Res. 2 4 , 9 6 4 - - 9 6 8 Grunicke, H., Bock, K.W., Becher, H., Gang, V., Schnierda, J. and PuschendorI, B. (1973) Cancer Res. 33, 1048--1053 lqbal, Z.M., Kohn, K.W., Ewig, R.A.G. and Fornace, Jr., A.J. (1976) Cancer Res. 36, 3834--3838 Lehman, A.R. and Ormerod, M.G. (1970) Biochim. Biophys. Acta 204, 128--143 Dingman, C.W. and Kakunaga, T. (1976) Int. J. Radiat. Res. 30, 55--66
Biochimica et Biophysica Acta, 477 (1977) 3 4 3 - - 3 5 5 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 9 8 9 7 2
DNA CROSSLINKING INDUCED BY X-RAYS AND CHEMICAL AGENTS
A L B E R T J. F O R N A C E , Jr. * and J O H N B. L I T T L E
Laboratory of Radio biology, Dept. of Physiology, Harvard University School of Public Health, Boston, Mass. 02115 (U.S.A.) (Received F e b r u a r y 2nd, i 9 7 7 )
Summary DNA corsslinking was measured in human diploid fibroblasts by the method of alkaline elution [13,14]. Following treatment of the cells by known DNA crosslinking agents (HN2, mitomycin-C), a controlled frequency of DNA strand breaks was induced by a small test dose of X-rays at 0°C. The effect of the prior treatment with a crosslinking agent was to reduce the rate of DNA elution in a dose dependent manner. When cells were X-irradiated, incubated for 2 h at 37°C in order to permit rejoining of most of the induced DNA strand breaks, and then exposed to the small test dose of X-rays at 0°C, a reduction in the rate of DNA elution occurred. The magnitude of this X-ray induced DNA crosslink effect was dependent upon the initial dose of X-rays (1--60 krad), and was increased by irradiation under hypoxic conditions. A similar level of crosslinking was present 2 h after 5 krad of X-ray as was present with a 30 min exposure to a 1 pM nitrogen mustard. Evidence is presented that crosslinking occurs immediately after X-irradiation and is repaired with incubation at 37 ° C. X-ray crosslinking was attributed to DNA-protein crosslinks since treatment of the cell lysates with proteinase removed the effect. X-ray induced DNA single strand breaks can be measured over an increased range (25 rad--10 krad) by alkaline elution when the crosslink effect is first removed by proteinase digestion.
Introduction Previous investigators have presented evidence suggesting that ionizing radiation produces DNA crosslinks; these studies have employed various methods which measure either the extractability of DNA from protein [1--4], or changes in the viscosity and sedimentation of DNA [5--10]. These methods * Present address: Dept. of Pathology, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Mass. 02115 (U.S.A.).
344 have usually required very high doses of irradiation. Recently a filter technique [11] has been described which can detect DNA-protein crosslinks produced in vitro at more biologically relevant doses. In aqueous solution, covalent bonds are produced by X-ray or ultraviolet light between purines and small molecules; these molecules may mimic functional groups present in nucleic acids and proteins [12]. It has recently been shown that the alkaline elution technique can detect both DNA crosslinks in intact mammalian cells produced by exposure to low concentrations of nitrogen mustard (HN2} [13,14] as well as the production and repair of DNA-protein crosslinks induced by low doses of ultraviolet light [13,15]. In the present investigation , DNA-protein crosslinks have been examined following relatively low doses of X-irradiation by alkaline elution and evidence is presented for their possible repair. DNA crosslinking by various chemical and physical agents has also been studied with this technique and an approach has been proposed to quantitate these crosslinks to very low doses. After the removal of X-ray induced DNA crosslinking by proteinase digestion, DNA single-strand breaks produced by X-ray have been studied over an expanded range (25 rad--10000 rad) as compared to previous results [13,14]. Materials and Methods Cells and cell labeling: The fibroblast cell strain CRL 1220 (American Type Culture Collection, Rockville, Md.) was derived from a normal human adult donor and used at passages 5 to 18 (1 : 4 split}. The cells were grown and prepared for experiments as previously described [13]; [2-J4C]thymidine was added to growing fibroblast cells for several days and then removed. On the day of the experiments, these cells were in a confluent monolayer. Exponentially growing L1210 mouse leukemia cells [16] were labeled with [3H]thymidine as previously described [ 13]. Irradiation. X-rays were delivered by a GE Maximar X-ray generator operating at 220 kV and yielding a dose rate of 80 rad/min. When cells were incubated following X-ray, they were irradiated at 37°C and incubated for the indicated time at 37°C. They were then rinsed with warm saline, incubated with trypsin at 37°C for 60 s, and suspended in cold medium buffered with 0.01 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonate). When cells were not incubated after X-ray, they were X-irradiated while suspended in cold medium buffered with 0.01 M HEPES at 0°C; unless otherwise indicated, all X-irradiations done at 0°C were in medium and employed the X-ray generator described above. At high radiation doses (5--60 krad), a cobalt gamma-ray source was used which contained approximately 3000 C of cobalt-60 and yielded a dose rate of 160 rad/s. Hypoxic irradiation was performed with a 100 kV Philips X-ray unit yielding a dose rate to the cells of 248 rad/min. The irradiation chamber was modified such that humidified N2 continuously flowed over the fibroblasts which were in a confluent monolayer in a 30 cm 2 petri dish covered with 1.5 ml of warm medium. The cells were flushed with N~ for 15 min prior to irradiation; the 02 c o n t e n t of the outflow of the irradiation chamber was determined with a Thermox-Analyzer combustion chamber (Thermo-Lab Instruments, Inc., Glenshaw,
345 Pa.) and was 10--35 ppm at the time of irradiation. Each experiment contained controls which were X-irradiated with the same apparatus but flushed with room air. The radiation effect in air determined by alkaline elution was the same with either the 100 kV X-ray generator or the cobalt gamma ray source at the same dose. Chemical agents. HN2, nitrogen mustard (NSC 762); HN1, 2-chloro-N,Ndimethylethylamine hydrochloride (NSC 1917); and mitomycin-C (NSC 26980) were kindly supplied by the Drug Development Branch, Division of Cancer Treatment, National Cancer Institute; these agents were stored at --120°C. HN2 and HN1 were dissolved in 0.01 M HC1; mitomycin-C was dissolved in 30% methanol; these stock solutions were stored at --15°C. When fibroblasts were treated with these agents, the stock solution was added directly to the cells and medium such that the final concentration of solvent was 0.2%. At the end of this treatment, the medium was removed, the monolayer was rinsed four times with warm saline, and the cells exposed to 0.25% trypsin for 60 s, then suspended in cold medium buffered with 0.01 M HEPES buffer. Alkaline elution. The procedure used [13,15] is a modification of that described by Kohn et al. [17]. Cells were filtered onto a polyvinylchloride filter, lysed with 2 M NaC1, 0.02 M Na3 EDTA/0.2% Sarkosyl (pH 10.2), washed with 0.02 M Na3 EDTA (pH 10.3), and then eluted with 0.10 M tetrapropylammonium hydroxide/0.02 M EDTA (acid form, pH 12.2) at a pump speed of 0.04 ml/min. Eluted fractions were collected and assayed for radioactivity as previously described [ 13]. In order to provide for an internal standard, 3H-labeled L1210 cells which had received 150 rad at 0°C were included in each assay. The fraction of sample DNA which is retained on the filter when 50% of the internal standard DNA has eluted has been termed the "relative r e t e n t i o n " [131. Proteinase-K treatment. The proteinase treatment employed has been described previously [13]. Cells were lysed on the filter for alkaline elution and, after the pH 10.2 lysis solution had run through, the filters were washed with 0.01 M Tris/0.01 M EDTA/0.01 M NaC1/0.5% sodium dodecyl sulfate (pH 8.0); 0.5 mg/ml proteinase-K (E. Merck, Darmstadt, G.F.R.) in the same buffer was then added for 30 min at 23°C. When proteinase-K was omitted, incubation was in the same buffer. The solution was then allowed to drip out and alkaline elution was carried out at pH 12.2 [ 13]. Results
Evidence has been presented that the alkaline elution technique can quantitatively measure DNA single-strand breaks produced by low doses of X-ray [13--15]. At X-ray doses of 400 rad or less, elution as measured by relative retention (see Materials and Methods) decreased approximately according to a first-order relation of the X-ray dose; the Do dose was approximately 220 rad. In each elution an internal reference, L1210 cells, which had been X-irradiated with 150 rad at 0 ° C, was included. In order to improve quantification, elution of sample DNA was plotted against the elution of the internal reference DNA; there was little change in the shape of the elution curves since the internal DNA eluted in a nearly linear fashion. This method of plotting is demonstrated in
346
Fig. 1A (upper abscissa) in terms of the fraction of internal standard DNA retained on the filter. In order to simplify this method of plotting, and since the internal reference DNA elutes at a constant rate on a logarithmic plot, hours of elution (corrected) is defined such that 50% of the internal reference cell DNA had eluted off the filter in 12 h (lower abscissa Fig. 1A). The relative retention is defined as the fraction of sample DNA retained on the filter after 12 h of elution (corrected). Cells were first treated with a known corsslinking agent. To elicit the DNA crosslinking effect, they were subsequently irradiated with a small dose of X-ray. The effect of a crosslinking agent is to decrease the apparent level of strand breakage induced by X-ray [13,18]. In Fig. 1A, fibroblasts were treated with varying doses of nitrogen mustard (HN2) and then X-irradiated with 400 rad. Repair of X-ray induced DNA single-strand breaks was prevented by maintaining the cells at 0°C during and following X-irradiation. There is a dose dependent increase in the fraction of DNA retained on the filter with increasing concentration of HN2. In Fig. 1B, HN1 (a monofunctional analogue of HN2) had no crosslinking effect, i.e. the effect of the single-strand breaks induced by HN1 damage to the cell DNA was additive with the effect produced by X-ray. At a less toxic concentration of HN1, 5 • 10 -6 M for 30 min, there was also no crosslink effect (data not shown). After cells have been treated with a crosslinking agent and DNA single-strand
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HOURS OF ELUTION ( c o r r e c t e d ] Effect of nitrogen lnustard and half-mustard, (2-chloro-N-dimethylethylaminc) on X-ray sensitivity. Confluent c u l t u r e s w e r e t r e a t e d w i t h H N 2 , f I N 1 or s o l v e n t f o r 3 0 rain at 3 7 ° C ; t h e c e l l s w e r e then suspended in cold medium, X-irradiated at 0°C where indicated (X), and analyzed by alkaline elut i o n . A . E f f e c t o f v a r y i n g d o s e s o f H N 2 ( c o h e n . in t o o l ) ; w i t h o u t X - r a y ( X ) all t t N 2 - t r e a t e d samples had approximately t h e s a m e e l u t i o n p r o f i l e ( l a b e l e d " H N 2 " ) . B. E f f e c t o f h a l f - m u s t a r d , H N I . Fig. 1.
347 breaks have been produced by X-ray, the elution profile of the DNA is characteristically non-linear as shown in Fig. 1A. There is an early rapid elution which appears to be X-ray dependent, and then a late slow elution which is crosslink dependent. An estimate of the a m o u n t of DNA crosslinking can be derived if one examines the a m o u n t of DNA left on the filter during this late phase. The relative retention (the fraction of DNA left on the filter after 12 h of elution (corrected)) has been chosen for this purpose. The increase in elution with X-ray at this time point, designated "relative elution," is shown in Fig. 2A. Again, the first-order relation of elution to X-ray dose is demonstrated. After treatment of the cells with a crosslinking agent, HN2, the X-ray sensitivity is decreased, but the relative elution still increases in a linear manner with increasing X-ray dose. The effect of a crosslinking agent, therefore, is to decrease the slope of the DNA X-ray sensitivity by a constant factor. If the relative elution of non-crosslinked DNA (mean) at a specific X-ray dose is divided by the relative elution of crosslinked DNA at the same X-ray dose, then this ratio will be the factor by
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Fig. 2. E f f e c t of crosslinking agent~ o n X-ray s e n s i t i v i t y . " R e l a t i v e e l u t i o n " r e p r e s e n t s the increase in e l u t i o n of D N A a f t e r X-ray a n d is d e f i n e d as: (log o f relative r e t e n t i o n of f i b r o b l a s t D N A w i t h o u t X - r a y ) - - (log of relative r e t e n t i o n of f i b r o b l a s t D N A f o l l o w i n g X-ray at 0 ° C ) ; e, X-ray alone, ± S.E.; o, f o l l o w i n g 30 rain t r e a t m e n t w i t h H N 2 , 2.5 • 10 -6 M for 3 0 rain. B. Crosslink f a c t o r is d e f i n e d as: ( m e a n value of t h e relative e l u t i o n a t a g i v e n X-ray d o s e ) - (relative e l u t i o n o f f i b r o b l a s t D N A a f t e r t r e a t m e n t of t h e cells w i t h a crosslinking a g e n t f o l o w e d b y t h e s a m e X-ray d o s e ) . T r e a t m e n t w i t h H N 2 or m i t o m y c i n - C (MMC) was f o r 3 0 rain as d e s c r i b e d in Fig. 3; X-ray dose w a s 4 0 0 t a d . F o l l o w i n g u l t r a v i o l e t ( U V ) light t r e a t m e n t ( d a t a f r o m Ref. 13, cells w e r e C R L 1 1 1 9 n o r m a l h u m a n f i b r o b l a s t s f r o m A T C C ) , cells w e r e i n c u b a t e d for 30 rain a t 37~C, s u s p e n d e d , a n d X - i r r a d i a t e d at 0 ° C w i t h 4 4 0 t a d . F o l l o w i n g t r e a t m e n t w i t h u l t r a v i o l e t light, t h e crosslink f a c t o r was d e r i v e d b y dividing t h e relative e l u t i o n at 4 4 0 t a d ( t a k e n f r o m Fig. 2a) by the relative e l u t i o n o b t a i n e d f r o m [ 13] a t v a r i o u s d o s e s o f u l t r a v i o l e t light. U n t r e a t e d s a m p l e (no ultra° v i o l e t ) fell w i t h i n 1 S.D. o f e x p e c t e d v a l u e , 1.0.
348 which the X-ray sensitivity, as determined by alkaline elution, has been decreased and will be independent of the X-ray dose (200--400 rad) used to elicit the crosslink effect. This ratio, designated "crosslink factor", is plotted in Fig. 2B. The "crosslink f a c t o r " value is 1.0 in untreated samples and increases with increasing doses of HN2. As can be seen in Fig. 2B, known crosslinking agents, HN2, mitomycin-C, and ultraviolet light, produce a dose-dependent increase in value of the crosslink factor in an approximately linear manner. If one assumes that the a m o u n t of DNA crosslinking is directly proportional to the dose of crosslinking agent [18] at the doses we have used, then the crosslink factor appears empirically to be directly proportional to the a m o u n t of DNA crosslinking. The detection of DNA crosslinking induced by X-irradiation is complicated by the large number of DNA single-strand breaks produced. In Fig. 1A, the shape of the elution profile after 400 rad alone suggests possible DNA crosslinking; namely, the elution profile is concave upwards with a decreasing elution rate when 15--25% of the DNA is left on the filter. The interpretation after higher doses of X-ray is further obscured since most of the DNA elutes rapidly off the filter. Since most X-ray induced DNA single-strand breaks are rapidly rejoined [19,20], the crosslinking effect caused by X-rays is more easily demonstrated following post-X-irradiation incubation. In Fig. 3, fibroblasts were X-irradiated (5 krad) and incubated at 37°C for 2 h; most of the DNA single-strand breaks have been rejoined by this time and about 60% of the DNA is retained on the filter after 12 h of elution (corrected). When these cells were subsequently irradiated with a small dose of X-rays (300 rad) at 0°C, the increase in elution was less than in cells given 300 rad alone (no prior gammairradiation). This is demonstrated in Fig. 3 by the solid arrow (for crosslinked DNA) as compared to the dashed (for non-crosslinked DNA). In fact, more
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349 DNA is retained on the filter at 12 h from the sample which received the prior gamma irradiation plus the 300 rad dose than the sample which received 300 rad alone. In Fig. 4, the DNA crosslinking present 2 h after irradiation with varying doses of ionizing (cobalt-60) gamma-irradiation demonstrated. DNA crosslinking, as measured by the crosslink factor, was detected after exposure to 1 krad, and increased progressively in a near linear fashion with increasing radiation doses up to 60 krad. Thus, 2 h after irradiation of cells there was a distinct DNA crosslinking effect as measured by alkaline elution, and this crosslinking increased in a dose dependent manner similar to the crosslinking produced by known DNA crosslinking agents. In Fig. 5 a representative experiment is presented which demonstrates the effect of varying the incubation time after 5 krad. Under aerobic conditions {lower curve), there appears to be a rapid decrease in the amount of DNA crosslinking, as measured by the crosslink factor, which occurs with 2--4 h of postirradiation incubation; this is followed by a slower decrease at later times. The kinetics of this recovery from crosslink damage is obscured, however, as 7 determinations (closed circles, Fig. 5) done in separate experiments after 2 h of incubation showed significant variability. In all experiments, however, the crosslink factor after longer incubation times was always less than any of the determinations done at 2 h. Under hypoxic conditions obtained with the 02
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scavenger sodium hydrosulfite, Na2S2O4 (upper curve, Fig. 5), there was also an apparent early rapid recovery (from 1 to 2 h) and a slow late recovery from crosslink damage. These findings indicate that the DNA crosslinking detected after X-ray under aerobic or hypoxic conditions by alkaline elution decreases (repair) from 1--2 to 12--13 h after irradiation. The effect of hypoxia on DNA crosslink production after X-ray is also demonstrated in Fig. 5. The hypoxic effect was produced by X-irradiation of cells in N2 or in the presence of 5 mM Na2S~O4; Na2S204 is an 02 scavenger which has been shown to have an oxygen enhancement ratio for survival of 2.5 as determined by colony-forming ability after X-irradiation (Ritter, M.A., unpublished results). 2 h after X-ray exposure, the crosslink factor is always greater with hypoxic X-irradiation (5 determinations) than with aerobic X-irradiation (7 determinations). Since the crosslink factor with no DNA crosslinking is 1.0, the mean crosslink factor for 5 krad with a 2 h post-X-ray incubation is 48% greater with hypoxic irradiation than with aerobic irradiation. A similar effect with hypoxic irradiation was found after 1 krad with a 2 h incubation (data not shown). To determine whether protein is involved in the X-ray-induced crosslinks, the effect of proteolytic t r e a t m e n t on elution was studied. The cell lysates were treated with proteinase-K prior to elution; this reduced the a m o u n t of protein on the filter from approx. 5% to 0.5% [13]. The proteinase-K effect on the control and internal reference cells was to slightly increase the elution (approx. 5%); when plotted such t h a t 50% of the internal reference DNA eluted in 12 h, the proteinase effect on the control cell DNA was very slight (Fig. 6A). The effect on cells which had received 300 rad alone was also slight initially, but at longer elution times the elution rate was somewhat increased with proteinase suggesting a slight protein-mediated crosslink effect.
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Fig. 7. E f f e c t o f p r o t e i n a s e - K o n e l u t i o n o f X - i r r a d i a t e d DNJk. F i b r o b l a s t s w e r e X - i r r a d i a t e d a t O°C a n d a n a l y z e d b y alkaline e l u t i o n m o d i f i e d t o i n c l u d e t r e a t m e n t o f t h e cell l y s a t e s w i t h p r o t e i n a s e - K as in Fig. 6. A. R a p i d e l u t i o n a f t e r 0.5 t o 1 0 k r a d ( c o b a l t ) , e l u t i o n o f D N A is p l o t t e d against t h e e l u t i o n of the r e f e r e n c e cells; t i m e of e l u t i o n w a s c o r r e c t e d s u c h t h a t 10% of the r e f e r e n c e cell D N A h a d e l u t e d in 45 rain; 0.3 m l f m i n , p u m p s p e e d ; f o u r 6-rnin a n d six 12-rain f r a c t i o n s w e r e c o l l e c t e d ; o . . . . . -~, 5 k r a d irrad i a t i o n b u t p r o t e i n a s e - K o m i t t e d . B. E l u t i o n a f t c r 2 5 - - 5 0 0 r a d ( 0 . 0 4 m l / m i n , p u m p s p e e d ) f o l l o w i n g proteinase t r e a t m e n t of cell lysates.
352 When the cells were irradiated with 5 krad and incubated for 2 h, then X-irradiated with 300 rad at 0°C (Fig. 6B), the proteinase-K treatment produced a marked increase in elution. The crosslink effect, namely, the decrease in elution produced by the prior X-irradiation, is abolished. If DNA crosslinks are indeed produced by X-ray, then DNA single-strand breaks should be more easy to quantify, particularly at higher doses of X-ray, if these crosslinks are first removed by proteinase treatment. Such experiments were performed utilizing doses of X-ray from 25 rad to 10 krad (Fig. 7); in Fig. 7A the pump speed was increased to 0.3 ml/min; in Fig. 7B the pump speed was 0.04 ml/min. As can be seen in Fig. 7, there was a dose dependent increase in the elution of DNA with increasing X-ray dose. The slowing of the elution rate, when 15-25% of the DNA is retained on the filter, is not seen in cells irradiated with 250 and 500 rad with proteinase digestion of the lysates. Following higher doses of X-rays, there was also a near linear elution of 90% of the DNA. In Fig. 7A, the internal reference elutes faster with the higher pump speed (0.3 ml/ min) but, as can be seen with the 500 rad dose, the fraction of DNA retained on the filter versus the fraction of the internal standard DNA retained is approximately the same with either pump speed. Without the proteinase digestion with 5 krad of X-ray, DNA elutes in a slower non-linear manner characteristic of crosslinked DNA. This indicates that DNA-protein crosslinks are present immediately after X-ray and require no incubation at 37 ° C. Discussion When DNA single-strand breaks are produced by a small test dose of X-rays, the increased elution of DNA from the filter is diminished if the cells have previously been treated with a crosslinking agent [13,14]. Owing to the large number of single-strand breaks present immediately after X-irradiation, the DNA crosslinking produced by ionizing radiation itself is more easily demonstrated with this approach after most of these strand breaks have been rejoined. At this time (2 h), a dose dependent increase in DNA crosslinking is induced by radiation doses of 1--60 krad. This crosslinking, as seen by alkaline elution, is qualitatively similar to that seen after ultraviolet irradiation [13], namely, it is proteinase sensitive and resistant to salt (2 M NaC1), detergent, (SDS, Sarkosyl), and alkali. Immediately after X-irradiation, a proteinase e f f e c t o n elution is seen with doses as low as 300 rad; this indicates that at least some of the DNA crosslinking seen after X-irradiation does n o t require incubatiqn and that following doses less than 1 krad, the crosslinking may have been repaired by 2 h of incubation. At doses greater than 1 krad, this crosslinking decreases with further incubation, also suggestive of repair. Previous studies of X-ray induced DNA' crosslinking have usually been with higher doses [1,3,5--9] and repair has not been studied. Other investigators [5--7,11] have noted an increased yield of DNA crosslinks with hypoxic X-irradiation. This appears to be the case with our results, but different rates of repair of crosslinking in the first 2 h after irradiation cannot be excluded. The DNA crosslinking agent, HN2, is at least ten times more toxic to the cell than its monofunctional analogue HN1 [21]. This suggests that linkages between a DNA strand and adjacent marcromolecules are of greater conse-
353 quence to the cell than monofunctional reactions of similar small molecules with DNA. Significant DNA crosslinking, as determined by alkaline elution, is seen with known DNA crosslinking agents at biologically relevant doses in mammalian cells. The crosslink factor at the approximate D~0 for cell lethality following exposure to various agents is: 4.2 for HN2 (3 pM, 30 min) [21] ; 3.1 for mitomycin-C (2.5 pM, 30 min) [22 and Nagasawa, H., unpublished results]; 1.8 for ultraviolet (10 J • m-:, 30 m i n ) [13 and Little, J.B., unpublished results]. The D~0 for X-ray, however, is approx. 350 tad in human diploid fibroblasts [23], while DNA crosslinking comparable to that induced by the above agents occurs only after exposure to much higher doses. 2 h after exposure to 5 krad, for example, the crosslink factor was 2.0. X-ray-induced DNA crosslinking is increased under hypoxic irradiation while cell survival is increased. These facts suggest that other types of damage are probably also responsible for the lethal effects of X-irradiation. The effect of X-ray induced DNA crosslinking on cell 1.ethality, carcinogenesis, and aging remains to be determined. The~exact mechanism by which DNA-protein crosslinks influence the alkaline,elution of DNA, is n o t known. HN2 [24] and mitomycin-C [25] are known DNA-DNA crosslinking agents which would be expected to affect an assay invQlving alkaline denaturation [13,14,18,24]. HN2, a highly reactive alkylating agent, may also produce DNA-protein crosslinks; DNA extractability from protein is decreased following treatment with HN2 [26--28]. However, DNA extractability is also decreased by monofunctional agents and various metabolic inhibitors at toxic doses [ 28]. With the alkaline elution technique, no crosslink effect is seen with HN1 (this paper) at doses as toxic or more toxic to the cell than HN2 [21] and no crosslink effect was seen with toxic doses of bleomycin [29]. These findings indicate that DNA crosslinking as detected by alkaline elution is more specific for damage produced by polyfunctional (crosslinking) agents as compared to other DNA extractability assays. Although a mathematical model representing the effect of DNA crosslinking on alkaline elution has n o t been presented, owing to the fact that the nature of the crosslinks is not well defined, several observations and speculations can be made. First, the rate of elution of DNA from the filter approximates a first order function of the number of single-strand breaks produced by X-rays at low doses where the crosslink effect of X-rays is slight [13--15 and this paper]. Empirically, the value "crosslink factor" appears to be approximately directly proportional to the dose of the crosslinking agent used. In Fig. 1A, the DNA from cells which had received X-ray alone rapidly eluted off the filter, while crosslinked DNA produced by HN2 eluted off the filter very slowly after an initial rapid phase. This slow late elution profile is nearly linear with a small negative slope. If one assumes that the crosslinked DNA is represented by the a m o u n t of DNA in this slowly eluting component, then the value "crosslink factor" is derived from an approximation of this c o m p o n e n t taken at 12 h of elution; a more accurate estimate of the amount of DNA in this slowly eluting crosslinked phase would b e to extrapolate this line back to the ordinate (e.g., 5 pM HN2, 82% of the DNA in crosslinked phase at ordinate). If one assumes that one crosslink per DNA single strand renders that DNA strand crosslinked, then the Do for crosslinked DNA (that is, the dose yielding an average of one
354
crosslink per DNA strand, 37% rapidly eluting phase, 63% slowly eluting crosslinked phase) would be approximately 1 pM HN2, 30 min treatment time. As the test X-ray dose in the experiment in Fig. 1A was 400 rad, the average DNA strand size would be 1.1 • 109 daltons, assuming an efficiency for DNA singlestrand breakage of 2.3 • 10 -~2 dalton per rad [30] ; 30 min treatment with I pM HN2 would produce one crosslink per 109 daltons of DNA. This compares with 2 DNA-DNA crosslinks per 109 dalton of DNA for a 1 h treatment with 1 pM HN2 as determined by Jolley and Ormerod [18]. If one further assfimes that the crosslink factor is proportional to the number of DNA crosslinks irrespective of the type of crosslink, then 2 h after exposure to 5 krads there is one DNA crosslink per 109 dalton of DNA or approx. 3100 DNA crosslinks per cell. Finally, Kohn et al. [14] have shown t h a t at pH 11.9 or greater DNA elutes according to a first order function of the number of X-ray-induced strand breaks (when the pH is lowered only DNA strands of a certain size or less will elute). Previously, this technique was useful for the study of strand breaks produced by doses of less than 1 krad of X-ray; at doses as low as 300--400 tad, DNA crosslinking produces significant slowing of the late phase of elution. Following proteinase treatment, DNA single-strand breaks can be studied relatively free of the crosslink effect caused by X-ray such that higher doses of X-ray (up to 10 krad) can be used. When studying DNA single strand size by alkaline sucrose sedimentation, the DNA must first be freed from a " c o m p l e x " by long lysis in alkali and other treatments. Crosslinking agents delay the release of freely sedimenting DNA from this complex [18]. It would seem logical to expect t h a t X-ray crosslinks may have the same effect:. This would argue for a proteinase digestion [14,31] of cell lysates prior t o sedimentaticn in order to minimize the DNA shear and alkaline degradation induced by other methods of freeing DNA from this complex. Acknowledgements We thank Mr. Michael Allocca and Mrs. Dorothy Foscaldo for expert assistance. This work was supported by training grant CA-09078 and research grants CA-11751 and ES-00002 from the U.S. National Institutes of Health. References 1 2 3 4 5 6 7 8 9 10 ll 12 13 14 15 16
A l e x a n d e r , P. a n d S t a c e y , K . A . ( 1 9 5 9 ) N a t u r e 1 8 4 , 9 5 8 - - 9 6 0 S m i t h , K.C. ( 1 9 6 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3, 1 5 7 - - 1 6 3 F e l d b e r g , R.S. a n d G r o s s m a n , L. ( 1 9 7 6 ) B i o c h e m . 15, 2 4 0 2 - - 2 4 0 8 Mce, L.K. a n d A d e l s t e i n , S.J. ( 1 9 7 6 ) R a d i a t . Res. 6 7 , 6 3 5 . ( A b s t r . ) L e t t , J . T . , S t a e e y , K.A. a n d A l e x a n d e r , P. ( 1 9 6 1 ) R a d i a t . Res. 1 4 , 3 4 9 - - 3 6 2 L e t t , 3.T. a n d A l e x a n d e r , P. ( 1 9 6 1 ) R a d i a t . Res. 15, 1 5 9 - - 1 7 3 l l a g e n , U. a n d Wellstein, H. ( 1 9 6 5 ) S t r a h l e n t h e r . 1 2 8 , 5 6 5 - - 5 7 1 C o q u e r e l l e , T., B o h n e , L., H a g e n , U. a n d M e r k w i t z , J. ( 1 9 6 9 ) N a t u r f o r s ~ h . 2 4 b , 8 8 5 - - 8 8 8 B o h n e , L., C o q u e r e l l e , T. a n d H a g e n , U. ( 1 9 7 0 ) I n t . J. R a d i a t . Biol. 17, 2 0 5 - - 2 1 5 Krasin, F., P e r s o n , S., L e y , R.D. a n d t t u t e h i n s o n , F. ( 1 9 7 6 ) J. Mol. Biol. 1 0 1 , 1 9 7 - - 2 0 9 M i n s k y , B.D. a n d B r a u n , A. ( 1 9 7 7 ) R a d i a t . Res. ( s u b m i t t e d ) L e o n o v , D. a n d Elad, D. ( 1 9 7 4 ) J. Org. C h e m . 3 9 , 1 4 7 0 - - 1 4 7 3 F o r n a c e , Jr., A.J. a n d K o h n , K.W. ( 1 9 7 6 ) B i o e h i m . B i o p h y s . A c t a 4 3 5 , 9 5 - - 1 0 3 K o h n , K.W., E r i c k s o n , L.C., Ewig, R . A . G . a n d F r i e d m a n , C.A. ( 1 9 7 6 ) B i o c h e m i s t r y , in the press F o r n a e e , .Jr., A.J., K o h n , K.W. a n d K a n n , Jr., H.E. ( 1 9 7 6 ) P r o c . Natl. A c a d . Sci. U.S. 73, 39---43 M o o r e , G . E . , S a n d b e r g , A . A . a n d Ulrich, K. ( 1 9 6 6 ) J. Natl. C a n c e r Inst. 3 6 , 4 0 5 - - 4 2 1
355 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Kohn, K.W., Friedman, C.A. Ewig, R.A.G. and Igbal, Z.M. (1974) Biochemistry 13, 4134--4139 Joney, G.M. and Ormerod, M.G. (1973) Biochim. Biophys. Acta 3 0 8 , 2 4 2 - - 2 5 1 Lett, J.T., CaldweU, I., Dean, C.J. and Alexander, P. (1967) Nature 2 1 4 , 7 9 0 - - 7 9 2 Kann, Jr., H.E., Kohn, K.W. and Lyles, J.M. (1974) Cancer Res. 34, 398--402 Wheeler, G.P., Bowdon, B.J., Adamson, D.J. and Vail, M.H. (1970) Cancer Res. 30, 100--111 Djordjevic, B. and Kim, J.H. (1968) J. Cell. Biol. 3 8 , 4 7 7 - - 4 8 2 Weichselbaum, R.R., Epstein, J. and Little, J.B. (1976) Radiology 1 2 1 , 4 7 9 - - 4 8 2 Kohn, K.W. and Spears, C.L. (1967) Biochim. Biophys. Acta 145, 734--741 lyer, V.N. and Szybalski, W. (1963) Microbiol. 50, 355--362 Steele, W.J. (1962) Proc. Amer. Assoc. Cancer Res. 3 , 3 6 4 . (Abstr.) Golder, R.H., Martin-Guzman, G., Jones, J., Goldstein, N.O., Rotenberg, S. and R u t m a n , R.J. (1964) Cancer Res. 2 4 , 9 6 4 - - 9 6 8 Grunicke, H., Bock, K.W., Becher, H., Gang, V., Schnierda, J. and PuschendorI, B. (1973) Cancer Res. 33, 1048--1053 lqbal, Z.M., Kohn, K.W., Ewig, R.A.G. and Fornace, Jr., A.J. (1976) Cancer Res. 36, 3834--3838 Lehman, A.R. and Ormerod, M.G. (1970) Biochim. Biophys. Acta 204, 128--143 Dingman, C.W. and Kakunaga, T. (1976) Int. J. Radiat. Res. 30, 55--66