59

Biochimica et Biophysica Acta, 563 ( 1 9 7 9 ) 5 9 - - 7 1 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 99461

DNA DAMAGE AND REPAIR IN RELATION TO CELL KILLING IN NEOCARZINOSTATIN-TREATED HeLa CELLS

T A K U M I H A T A Y A M A * a n d I R V I N G H. G O L D B E R G **

Department of Pharmacology, Harvard Medical School, Boston, MA 02115 (U.S.A.) ( R e c e i v e d O c t o b e r 3rd, 1 9 7 8 )

Key words: Neocarzinostatin; DNA damage; DNA repair; Cytotoxicity ; (HeLa cell)

Summary To elucidate the mechanism of the cell killing activity of neocarzinostatin on mammalian cells, the drug-induced damage of DNA and its repair were examined. Very low doses of neocarzinostatin, at which high survival of cells was observed, clearly produced single-strand breaks of DNA and decomposition of the 'DNA complex', but these damages appeared to be repaired almost completely. At higher doses of neocarzinostatin, single-strand breaks were repaired to a considerable extent while double-strand breaks seemed not to be repaired. The number of non-repairable single-strand breaks was about twice that of double-strand breaks. This implies that single-strand breaks are repaired except for those constituting double-strand breaks. Although at low levels of neocarzinostatin repair of double-strand breaks may occur, the correlation existing between the colony-forming ability of cells treated with neocarzinostatin and non-repairable DNA breakage suggests that production of a small number of critical non-repairable double-strand breaks per cell may be responsible for the cell killing activity of the drug.

Introduction The antitumor antibiotic neocarzinostatin, isolated from a filtrate of Streptomycin carzinostaticus [1], is an acidic single-chain polypeptide of molecular weight 10 700, which contains two reduction-resistant disulfide bonds [2]. Low levels of this antibiotic selectively inhibit DNA synthesis in sensitive

* Present Address: D e p a r t m e n t o f Biochemistry, Osaka City University Medical School, Osaka 545, Japan. ** To whom reprint r e q u e s t s s h o u l d be addressed.

60 bacterial and mammalian cells [3--6], induce degradation of existing DNA in bacteria [3,7] and produce breaks in DNA in mammalian cells [5,6,8--11], as well as in vitro, especially in the presence of a mercaptan [8,11]. Also, neocarzinostatin induces unscheduled DNA synthesis in lymphocytes [12] and DNA repair synthesis in HeLa cells and isolated nuclei [13]. Further, low levels of neocarzinostatin cause a block in the G2 phase of the cell cycle [14,15] and chromosome aberrations in mammalian cells [15,16], and high levels of the drug inhibit the vinblastine-induced formation of microtubular paracrystals [17], and cap formation and cell spreading in mammalian cells [18]. While it is possible that there is more than one cellular site of attack by neocarzinostatin, the involvement of DNA in the cell killing activity of neocarzinostatin was suggested by the correlation between the ability of low levels of the drug to induce breakage of cellular DNA in HeLa cells and their inhibition of DNA replication and cell growth [6] and also by the evidence that caffeine, a possible inhibitor of DNA repair, potentiates the lethality of mammalian cells treated by neocarzinostatin [19]. Further, neocarzinostatin is mutagenic for E. coli and the recA system is implicated in both mutagenicity and cell killing [20]. In this paper we examine the DNA damage caused by neocarzinostatin in HeLa cells and its repair, and also discuss the possible relation to the cell killing activity of neocarzinostatin. Materials and Methods

[Me-14C]thymidine (spec. act., 49.2 Ci/mol) and [Me-3H]thymidine (spec. act., 52.1 Ci/mmol) were purchased from New England Nuclear. CsC1 (for density gradient centrifugation) was from Harshaw Chemical Co. Pronase {free of nuclease) was from Calbiochem. HeLa $3 cells from American Type Culture collection were maintained in Eagle's minimal essential medium for suspension culture supplemented with 10% fetal calf serum (spinner medium) in a spinner flask. All tissue culture medium and serum were purchased from Grand Island Biological. Labeling o f Hela cells and neocarzinostatin treatment. HeLa cells (2 • 10 s cells/ml) were labeled in spinner medium with 0.1 pCi/ml of [14C]thymidine for 16--24 h at 37°C. The cells (4" 10S/ml) were washed once with fresh medium, resuspended in the same amount of medium and treated with various concentrations of neocarzinostatin for 1 h at 37°C. After treatment with drug, the cells were washed twice with medium, once with phosphate buffered saline (8 g NaC1/0.2 g KC1/1.15 g Na2HPO4/and 0.2 g KH2PO4/1) and finally suspended in the saline medium. In the DNA repair experiments, after washing with the spinner medium two times, the ceils were reincubated at 37°C. At appropriate times, 2-ml aliquots of cell suspension were withdrawn, washed and suspended in the saline medium. These cell suspensions were used for gradient analysis. Sucrose density gradient centrifugation. Three kinds of sucrose density gradients were employed: (1) Alkaline sucrose gradient. The gradient consisting of 30 ml of 5--20% sucrose (w/v) in 0.3 N NaOH, 0.3 M NaC1 and 0.01 M EDTA was overlayered with 1 ml of the 'alkali-dodecyl sulphate' lysis solution (0.3 N NaOH, 0.3 M

61 NaC1, 0.03 M EDTA and 0.5% sodium dodecyl sulphate) [21]. The cell suspension (5 • 104 cells in 0.1 ml) was lysed in the lysis solution at r o o m temperature for 2 h in the dark and then centrifuged at 20°C at 18 000 rev./min for 3 h using a Beckman SW 25.1 rotor. (2) Neutral sucrose gradient (A). The gradient consisting o f 30 ml of 5--20% sucrose (w/v) in 0.1 M NaC1, 0.01 M EDTA and 0.01 M Tris-HC1 (pH 7.5) was overlayered with 1 ml of the lysis solution (0.01 M Tris-HCl (pH 8.0), 0.01 M EDTA, 2% sodium dodecyl sulphate and 1.5 mg/ml of pronase which was preincubated for 2 h at 37°C) [22]. The cell suspension (5 • 104 cells in 0.1 ml) was lysed on the gradient at 37°C for 30 min, and at r o o m temperature for 20 min in the dark successively, and t h e n centrifuged at 20°C at 18 000 rev./min for 4.5 h using a Beckman SW 25.1 rotor. (3) Neutral sucrose gradient with shelf (B). The gradient consisting of 4.4 ml of 5--20% sucrose (w/v) in 2 M NaC1 and 0.01 M sodium citrate (pH 9) over 0.5 ml of a shelf of 60% sucrose saturated with CsC1 was overlayered with 0.3 ml of the lysis solution (0.2% sarkosyl/0.08% sodium deoxycholate/2 M NaC1/0.02 M EDTA and 0.01 M sodium citrate, pH 9) [23]. The cell suspension (2.5 • 104 cells in 0.05 ml) was lysed on the gradient for 30 min to 2 h at r o o m temperature in the dark, and then centrifuged at 20°C at 20 000 rev./min for 1 h using a Beckman SW 50.1 rotor. After centrifugation, 24 drop fractions (SW 25.1) or 6 drop fractions (SW 50.1) were collected from the bottom; 0.02 mg o f calf t h y m u s DNA was added to each tube as carrier. Then cold 10% trichloroacetic acid was added to a final concentration of 5%. The tubes were kept for 1 h on ice, and the contents were filtered on glass filters (GF/C Wattman), dried and counted in a toluene based scintillator using the Packard Tri-Carb liquid scintillation spectrometer. The sedimentation coefficient, S2o,w, of each fraction was calibrated [24] using ['4]thymidine or 32P-labeled T4 phage DNA, 71.1 S and 62 S in alkaline and neutral sucrose gradient, respectively [25,26]. The corresponding molecular weight (Mi) of each fraction was calculated using Studier's equations (alkaline DNA: S2o.w = 0.0528 M°'4°°, native DNA: s20,w = 0.0882 M °'346) [27]. To express the size of DNA, the n u m b e r average molecular weight (Mn) was calculated according to the formula [28] :

Mnwi/Mi (wi is the radioactivity of the fraction of molecular weight Mi.) The number of strand breaks per cell (N) was calculated, based on 10 -11 g of DNA per cell as follows: N = \M-~2tMn'--1) 6"03 " 1023 1 M "n10-' l (Mnl and Mn2 are the n u m b e r average molecular weights of the control and neocarzinostatin-treated cells, respectively.) [ 29 ]. CsCl density gradient centrifugation. 4.5 ml of CsC1 solution (p = 1.7) buffered with 0.01 M Tris-HC1 (pH 8.0), was overlayered with 0.3 ml of the lysis solution, 0.05 ml of cell suspension as described under (3) above, and

62 mineral oil. Centrifugation was at 20°C at 49 000 rev./min for 45 h using a Beckman SW 50.1 rotor. The trichloroacetic acid-insoluble radioactivity o f each fraction was analyzed as described above, except that the specific density (p) of each fraction was determined before adding carrier by measurement of its refraction index using a refractometer. All sedimentation patterns are shown from right to left in the figures. Colony formation. HeLa cell suspensions ( 4 . l 0 s cells/ml) treated with various concentrations of neocarzinostatin were washed twice with the spinner medium, and resuspended in Eagle's minimal essential medium for plate culture with 10% fetal calf serum. 200 or 400 cells were seeded in 35 mm petri dishes and incubated at 37°C in 5% CO2-air for 9--10 days. The colonies formed on plates were stained by methylene blue and counted. Plating efficiency of control cells was 60--70%. DNA synthetic activity of HeLa cells. HeLa cells treated with neocarzinostatin and washed twice with medium were resuspended in the spinner medium ( 4 . l 0 s cells/ml) and incubated at 37°C in a spinner flask. At the appropriate time, 1-ml aliquots of cell suspension were withdrawn and 1 ml of fresh spinner medium containing 2 ~Ci o f [3H]thymidine was added before incubation at 37°C for 30 min. After incubation, the cells were washed once with phosphate buffered saline, resuspended in 1 ml of the saline medium, and then quickly frozen in liquid N2. After completion of the experiment, the cell suspensions were thawed and 2 ml of cold 10% trichloroacetic acid was added. Acid-precipitable material was collected on a GF/C glass filter and radioactivity was counted as described above. Results

DNA damage caused by neocarzinostatin in HeLa cells Single-strand breaks of DNA. Cells exposed to various doses of neocarzinostatin were analyzed by alkaline sucrose gradient (Fig. 1). Under the cell lysis and centrifugation conditions employed, the DNA o f control cells sedimented as a broad band with the main peak at 205 S (fraction numbers 14--16) in addition to more rapidly sedimenting material (Fig. 1). At the neocarzinostatin concentrations of 0.01--0.025 pg/ml, only DNA of the 205 S size was produced under this condition; the 205 S material increases at the expense o f the more rapidly sedimenting material. Neocarzinostatin at 0.1 ~g/ml produced n o t only more 205 S DNA b u t also induced the single-strand scission of this size DNA, as represented by the small, more slowly sedimenting shoulder o f the main peak in the gradient pattern. Neocarzinostatin concentrations of 0.5 ~g/ml and above showed clearly the dose~lependent, single-strand cleavage o f the DNA. Double-strand breaks of DNA. Neutral sucrose gradients (A) were performed to detect double-strand scission of DNA (Fig. 2). Although sedimenting DNA was assumed to be essentially free of contamination b y other cell components when cells were lysed with 2% sodium dodecyl sulphate and pronase [22], the DNA of cells either untreated or treated with low levels o f neocarzinostatin sedimented anomalously. Under the conditions used here, control D N A sedimented to the b o t t o m of the tube {over 200 S), while neocarzinostatin at

63 [ 072,1

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= NCS 0.5

x

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=

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i

'~ NCS 0.1

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o

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i 0 0

5

t0

15

20

FRACT/ON NUMBER

25

5 Pellet

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15

20

25

I 30

FRACTIONNUMBER

Fig. 1. Alkaline sucrose gradient analysis o f D N A o f H e L a cells treated w i t h n e o c a r z i n o s t a t i n (NCS). HeLa cells w e r e l a b e l e d w i t h [ 14C] t h y m i d i n e , treated w i t h n e o c a r z i n o s t a t i n , and a n a l y z e d b y alkaline sucrose gradients as d e s c r i b e d in Materials and M e t h o d s . D1nzg c o n c e n t r a t i o n s ( ~ g / m l ) are s h o w n in the figure. T h e p e a k o f m a r k e r T 4 D N A w a s in fraction n u m b e r 24.

Fig. 2. Neutral sucrose gradient analysis o f D N A o f H e L a cells treated w i t h n e o c a r z i n o s t a t i n (NCS). HeLa cells w e r e labeled w i t h [ 1 4 C ] t h y m i d i n e , treated w i t h n e o c a r z i n o s t a t i n , and a n a l y z e d b y neutral sucrose gradient ( A ) as described in Materials and M e t h o d s . Drug c o n c e n t r a t i o n s ( ~ g / m l ) are given in the figure. T h e p e a k o f m a r k e r T 4 D N A w a s in fraction n u m b e r 20.

0.5 pg/ml and 1.0 pg/ml produced DNA peaks on the gradient of 55% and 75% of the total radioactivity, respectively. At the neocarzinostatin concentrations o f 2 gg/ml and above, almost all radioactivity was recovered on the gradients and the peaks shifted to lower S values as the drug concentration was increased.

Effect on rapidly sedimenting DNA ('DNA complex ') and release of free DNA When mammalian cells were lysed under mild conditions, the DNA of untreated cells sedimented very rapidly as a sharp peak through the sucrose gradient onto the shelf [23]. Ormerod and Lehmann [23] showed that the rapidly sedimenting DNA was caused in part at least by entanglement of long DNA molecules, but the association of lipid with DNA, the low density of the DNA complex and the association o f DNA with detergent bands provided evidence for an attachment between DNA and lipid, most likely derived from nuclear membrane. The addition of pronase to the lysis solution causes the degradation of the DNA complex of the control cells so that the DNA peak is in the middle o f the gradient (fractions No. 13 and 14) and its density is 1.7 by CsC1 density gradient centrifugation (data not shown). Thus, this rapidly sedimenting DNA also seems to contain a protein component.

64 This procedure was used to analyze the DNA complex and the release of free DNA by neocarzinostatin. As shown in Fig. 3, low doses of neocarzinostatin (0.025, 0.1 ~g/ml) induced the formation of slower moving peaks which may represent some kind of degradation of the complex. Neocarzinostatin concentrations of 0.5 and 1.0 pg/ml showed not only the decomposition o f the complex, some of which sedimented on the shelf again, b u t also a small amount of free DNA on the top of the gradient. To confirm the release of free DNA from the complex, CsC1 density gradient centrifugation was performed using the same lysis conditions (Fig. 4). In this experiment, the DNA complex of control cells had a density of 1.6 and the cells treated with 0.5 gg/ml neocarzinostatin produced a complex with a density below 1.4 and a small amount of free DNA (p = 1.7). High doses of neocarzinostatin (5, 50 pg/ml) produced a large a m o u n t of free DNA (Figs. 3 and 4). The percent releases of free DNA (p = 1.7) from the DNA complex were 5, 26, 74, and 85 at neocarzinostatin concentrations of 0, 0.5, 5, and 50 ~g/ml, respectively. The densities of the complex from control and neocarzinostatin (0.5 /Jg/ml) treated cells were sometimes variable.

Repair of DNA damage caused by neocarzinostatin in HeLa cells Repair of single-strand breaks. The repair of single-strand breaks was analyzed as shown in Fig. 5. At neocarzinostatin concentrations of 0.01 and 0.025 gg/ml, the 205 S size DNA is converted by 5 h into more rapidly sedimenting molecules such as seen in the sedimentation pattern o f control DNA. At neocarzinostatin concentrations above 0.1 ~g/ml, rejoining of single-strand breaks in the 205 S DNA molecules was clearly observed after 2 and 5 h, even at concentrations as high as 5 pg/ml of drug. But after 23 h incubation, these

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Fig. 3. E f f e c t o f neoca~rzinostatin ( N C S ) o n the ' D N A c o m p l e x ' . H e L a cells w e r e l a b e l l e d w i t h [ 1 4 C ] t h y m i d i n e , t r e a t e d w i t h n e o c a r z i n n s t a t i n , and a n a l y z e d b y n e u t r a l s u c r o s e g r a d i e n t w i t h s h e l f (B) as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . D r u g c o n c e n t r a t i o n s (/~g/ml) a r e g i v e n in the figure.

65 (o---oI ~.

Control

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15

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FRACTION NUMBER Fig. 4. CsC1 density gradient profiles of DNA of HeLa cells treated with neocarzinostatin (NCS). [14C]Thyrnidine labeled HeLa cells were treated w i t h n e o c a ~ z i n o s t a t i n and a n a l y z e d b y CsCI d e n s i t y gradient centrifugaUon as described in Materials and M e t h o d s . Drug c o n c e n t r a t i o n s (/~g/ml) are given in the figure. o . . . . . . o, 14C-radioactivity; • "-, density of CsCI. The arrow indicates w h e r e free D N A s e d i m e n t s (p = 1.7).

peaks decreased and small DNA fragments appeared on the t o p of the gradient. The decrease in the 205 S DNA peak and the increase in the amount of degraded DNA after a period of repair vary directly with the dose of neocarzinostatin. Repair of double-strand breaks. Repair of double-strand breaks was analyzed b y neutral sucrose gradients after a 5 h incubation of the neocarzinostatintreated cells at which time m a x i m u m repair of single-strand breaks was observed. In contrast with the results on alkaline sucrose gradients, there was no obvious shift of these peaks toward the b o t t o m o f the tube at the relatively high concentrations of drug needed to produce detectable double-strand breaks (0.5--10 pg/ml) (data n o t shown). Repair of the 'DNA complex '. As shown in Fig. 6, the slower moving peaks caused b y low doses of neocarzinostatin (0.025, 0.1 ~g/ml) appeared to be repaired within 5 h. Even at a drug concentration of 0.5 ~g/ml, large amount o f the complex was reformed after 23 h. It should be noted, however, that at 0.1 gg/ml neocarzinostatin and higher increasing amounts o f degraded DNA appeared on the t o p of the gradient after a 23 h incubation.

Summary of DNA damage and repair To quantitate the DNA damage and repair, the n u m b e r of breaks per cell was determined as described in Materials and Methods. The n u m b e r of singlestrand breaks was calculated from the data of alkaline sucrose gradient (Figs. 1 and 5, and in some cases, 4.5 h centrifugation instead of 3 h was performed (data not shown)), from which the number average molecular weight of control DNA was 1 • 109. Since the recovery of DNA on the gradient at drug concentrations of 1/~g/ml and above was high, the n u m b e r of double-

66

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FRACTION NUMBER Fig. 5. Repair o f = L ~ - s t ~ m d break= caused by neoea=-'zinoststi~ (NCS). [14C]7bymidL~e pre]abeled H e L a cells t r e a t e d w i t h n e o c a r z i n o ~ t a t i n w e r e r e i n c u b a t e d a t 3 7 ° C a n d a t t h e i n d i c a t e d t i m e , t h e D N A p r o f i l e o f t h e s e cells w a s a n a l y z e d b y alkaline s ~ e r o s e g r a d i e n t as d e s c r i b e d in Materlm~ a n d M e t h o d s . o...... o, n o n e o c a ~ z i n o s t a t i n ; • "-, 0 h; • ..... s, 2 h; ¢ ~, 5 h; v . . . . . . o, 23 h.

strand breaks could be determined from the data of neutral sucrose gradients (A) (Fig. 2), assuming that sedimenting DNA was practically free of contamination by other cell components [22]. The size of control DNA analyzed on neutral sucrose gradients was assumed to have a number average molecular weight of 2 . 1 0 9 , or twice that on alkaline sucrose gradients. Also Mn was directly calculated from the gradient profiles to avoid error of estimation of M, due to non-random distribution [28], as mentioned in Materials and Methods. As shown in Fig. 7, the number of single-strand breaks remaining after 5 h reincubation of the neocarzinostatin-treated ceils is about twice that of the double-strand breaks not repaired at the relatively high doses of neocazzinostatin required to detect double-strand breaks under our conditions of analysis.

Cell killing activity of neocarzinostatin To correlate the DNA damage with the cell killing activity of neocarzinostatin, the colony forming ability of the treated cells was determined. Colony

67

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NEOCARZINOSTAT/ (,ug/ml) N

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Fig. 6. R e p a i r of t h e d e c o m p o s i t i o n o f t h e ' D N A c o m p l e x ' c a u s e d b y n e o c a r z l n o s t a t i n ( N C S ) . [ 1 4 C ] T h y m i d i n e p r e l a b e l e d H e L a cells t r e a t e d w i t h neocarzinostat~-n w e r e r e i n c u b a t e d a n d a t t h e i n d i c a t e d t i m e s , t h e D N A p r o f i l e w a s a n a l y z e d b y n e u t r a l s u c r o s e g r a d i e n t s w i t h s h e l f (B). a ~, 0 h ; -~, 5h;~ ~, 23 h. Fig. 7. S u m m a r y o f D N A d a m a g e c a u s e d b y n e o c a r z i n o s t a t l n a n d its repair. T h e details are d e s c r i b e d in Results. = =, t o t a l n u m b e r o f single-strand b r e a k s p e r cell at O h; • • , t o t a l n u m b e r of d o u b l e s t r a n d b r e a k s p e r cell a t 0 h ; o p e n s y m b o l s r e p r e s e n t r e s p e c t i v e b r e a k s a f t e r 5 h r e i n c u b a t i o n . T h e regress i o n lines w e r e d e t e r m i n e d b y t h e m e t h o d of least s q u a r e s .

formation of HeLa cells treated with neocarzinostatin is shown in Fig. 8. Do, which is the dose of drug at which there is 37% survival of the cells, is determined by a line parallel to the linear portion of the curve through the 100% survival point, Dq (quasithreshold dose) which is the dose equivalent to the intercept of the extrapolation of the linear portion of the curve with a horizontal line drawn through the 100% survival point, and n (extrapolation number) which is a number equivalent to the intersection with the log survival axis, are 0.01 gg/ml, 0.004 ug/ml, and 1.5, respectively.

Recovery of DNA synthetic ability It has been suggested that the ability of neocarzinostatin to induce breakage of cellular DNA may be an essential aspect of its inhibition of DNA replication [6]. Therefore, the recovery of DNA synthesis after inhibition by neocarzinostatin was determined for comparison with cell survival (Fig. 9). At 0 time using even very low concentrations of neocarzinostatin, inhibition of DNA synthesis was observed and its extent depended on the drug dose. At these low doses of neocarzinostatin (0.01, 0.025, 0.1 ~g/ml), recovery from inhibition was observed after 2 or 5 h incubation, but after 24 h all cells treated with drug (even 0.01/~g/ml) had decreased DNA synthetic ability.

68 i

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Fig. 8. C o l o n y f o r m i n g ability o f H e L a cells t r e a t e d w i t h n e o c a r z i n o s t a t i n . C o l o n y f o r m a t i o n w a s m e a s u r e d as d e s c r i b e d in Materials and M e t h o d s . C l o s e d circles and bars r e p r e s e n t e d m e a n s and standard errors o f the m e a n , r e s p e c t i v e l y . A t a drug c o n c e n t r a t i o n o f 0 . 1 ~ g / m l , n o c o l o n i e s w e r e f o u n d . Fig. 9. R e c o v e r y o f D N A s y n t h e t i c ability o f H e L a cells t r e a t e d w i t h n e o c a r z i n o s t a t i n ( N C S ) . D N A synthesis w a s m e a s u r e d as d e s c r i b e d in Materials and M e t h o d s . Cells w e r e t r e a t e d w i t h the i n d i c a t e d c o n c e n ° t r a t i o n o f drug f o r 1 h prior t o d e t e r m i n a t i o n o f D N A s y n t h e t i c ability at t h e i n d i c a t e d t i m e s o f incubat i o n in t h e a b s e n c e o f drug. T h e D N A s y n t h e t i c abilities o f c o n t r o l cells are e x p r e s s e d as 1 0 0 % at the indicated times.

Discussion Alkaline and neutral sucrose gradient centrifugation are usually used for analyzing strand breaks in D N A , but in the case of cellular D N A , the results depend critically on the cell lysis conditions, rotor speed, and cell number loaded [ 2 2 , 2 9 - - 3 2 ] . While chromosomal D N A is generally thought to exist as a single, continuous, double-stranded molecule [ 3 3 - - 3 5 ] , upon prolonged alkaline lysis o f cells the sequential appearance o f smaller D N A o f 208 S and then 165 S has been observed [30,31]. However, evidence has been provided that D N A o f these sizes and larger, as found under alkaline lysis conditions, result from incomplete denaturation or aggregation of the D N A [36,37]. In an effort to avoid artifacts o f these sorts, we have used the 'alkali-dodecyl sulphate' lysis method by which D N A from rat liver nuclei was obtained as large, single-stranded molecules (mol. wt. > 1.0 • 109) [21]. Under these conditions the D N A from control HeLa cells sedimented as a broad band with a main peak at 2 0 5 S and u p o n treatment with as little as 0.01 ug/ml (10 -9 M) neocarzinostatin the D N A sedimenting at 2 0 5 S increased at the expense o f the faster sedimenting material. These changes, sensitive to very low drug levels, likely result from single-strand breaks in the DNA. Since neocarzinostatin at concentrations of 0.1 ~g/ml and above clearly cleave the 205 S D N A in a dosedependent manner, the~e latter results can be used to determine the number of single-strand breaks before and after a period o f repair. The single-strand breaks produced by higher levels of neocarzinostatin were

69 found to be repaired (Fig. 5). Repair proceeded to a maximum between 2 and 5 h after drug removal, although at 23 h small DNA fragments were observed at the top of gradients at drug concentrations of 0.1 gg/ml and above. It is unclear whether this further degradation of DNA results from the death of a particular cell population, as reported in the case of belomycin [38], or represents the selective destruction of a unique DNA in all cells. In view of the data on colony formation (Fig. 8) and growth kinetics (not shown), it seems possible that this result may simply reflect the extent of cell death at this time point. Further, it is clear that at neocarzinostatin concentrations above 0.1 ~g/ml repair of singlestrand breaks is incomplete (Fig. 5). Thus it was of importance to determine the possible relationship between cell death and the persistence of single-strand breaks in the DNA and to establish whether these breaks could, in fact, represent unrepaired double-strand breaks. To detect double-strand breaks different conditions of neutral sucrose gradient centrifugation were surveyed. No matter what conditions were employed anomalous sedimentation behavior of the DNA of the control and low dose drug-treated cells was encountered, as previously reported in the case of X-irradiation [29,32]. Nevertheless, it seems clear that under the conditions of analysis used in this paper double-strand breaks were found at neocarzinostatin concentrations as low as 0.5 ~g/ml. These breaks appear not to be random, since discrete peaks of lower S value are formed (Fig. 2). The ratio of double-strand to single-strand breaks is very high (1 : 5). Since it has been found that in vitro neocarzinostatin preferentially attacks deoxythymidylic and deoxyadenylic acid residues, i.e. members of a base pair, in DNA [39,40], generating gaps [41] from which these bases have been eliminated [39,40], it is not surprising that the chance of producing double-strand breaks is increased over that due to the random placement of single-strand lesions [40]. Nevertheless, the ratio of double- to single-strand breaks calculated from the in vitro data [39] is about 1 : 30. It seems likely, therefore, that the high ratio found in vivo is due to the rapid repair of the single-strand breaks even during the period of drug treatment. The results reported in this paper are quite different from those of Ohtsuki et al. [10], who required very high levels of drug (50 pg/ml) and longer exposure times to detect single- or double-strand breaks. The insensitivity of DNA strand breakage under their conditions probably originates from the fact that the cellular DNA was already sheared before loading onto the gradients due to the prior mixing of the cell suspension with the lysis solution. We were not able to detect significant repair of double-strand breaks as analyzed on neutral sucrose gradient (A), as has also been reported in the case of X-irradiated cells [32,42]. As is the case for X-ray induced DNA damage [42], the non-repairable single-strand breaks can be accounted for by doublestrand breaks, since there are approximately twice as many of the former as there are of the latter at the relatively high neocarzinostatin concentrations of 0.5--10 pg/ml (Fig. 7). On the other hand, evidence for the rejoining of radiation-induced DNA double-strand breaks in eukaryotic cells has been reported [43--47]. These disparate results may originate from the different cell types, doses of X-irradiation and analysis conditions used. It is also possible that double-strand breaks produced by low concentrations of neocarzinostatin

70 (below 0.5 pg/ml) are repaired to a considerable degree and such repair may account for the regeneration of the 'DNA complex' found at these drug levels on neutral gradients. Whether the 'complex' represents a real structure in which DNA is attached to nuclear membrane [48,49] or is an artifact of the isolation conditions is unclear, but it is o f interest that the very low levels of neocarzinostatin which are found to produce single-strand breaks only also lead to degradation of the 'complex'. A similar result has been obtained with the antibiotic bleomycin [50]. It has been proposed that the major lethal event produced by X-irradiation is a double-, not a single-strand break [42,44,45,47]. Extrapolating from the n u m b e r of double-strand breaks produced at drug levels of 1--5 pg/ml we find that there are about 55--60 double-strand breaks per cell at the mean lethal dose (Do) of 0.01 pg/ml. Thus it seems possible that, even though some repair of double-strand breaks m a y occur at low doses of neocarzinostatin, a small n u m b e r of such breaks in a critical region of the genome may be the lethal event. The other main actions of neocarzinostatin on mammalian cells are inhibition of DNA synthesis and block in G2 of the cell cycle. It was found that the fragmentation of DNA preceded the inhibition of DNA synthesis and that the breakage of DNA correlated with the inhibition of DNA synthesis [6]. Similarly we observed the transient recovery of DNA synthesis at low doses o f neocarzinostatin (below 0.1 ~g/ml) at which repair o f single-strand breaks was almost complete in a 5 h reincubation (Fig. 5). There is the possibility, however, that this transient 'recovery' is due to a synchronization effect o f neocarzinostatin due to transient block of cell cycle in G2 [14,15]. As experiments showing effects of neocarzinostatin on microtubular proteins required much higher neocarzinostatin doses (5--50 /~g/ml) [17,18], they appear not to be responsible for the cell killing activity of neocarzinostatin. The precise mechanism whereby neocarzinostatin damages DNA remains to be elucidated, but recent evidence implicating free radical mechanisms has been presented [ 51,52].

Acknowledgement This work was supported by U.S. Public Health Service Research Grant G M 12573 from the National Institutesof Health.

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