the relationship between dna strand-scission and dna synthesis inhibition in hela cells treated with...

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Biochimica et Biophysica Acta, 475 (1977) 2 8 1 - - 2 9 3 © E l sev ie r /Nor th -Hol l and Biomedica l Press

BBA 9 8 8 8 7

THE RELATIONSHIP BETWEEN DNA STRAND-SCISSION AND DNA SYNTHESIS INHIBITION IN HeLa CELLS TREATED WITH NEOCARZINOSTATIN

T E R R Y A. B E E R M A N ** and I R V I N G H. G O L D B E R G *

Department of Pharmacology, Harvard Medical School, Boston, Mass. 02115 (U.S.A.)

(Rece ived A u g u s t 3rd, 1976)

Summary

Neocarzinostatin inhibits DNA synthesis in HeLa $3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37°C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0°C before washing, the subsequent incubation at 37°C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA.

Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa $3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug.

These data suggest that the drug's ability to induce breakage of cellular DNA in HeLa $3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cyto toxic and growth-inhibiting actions of neocarzinostatin.

* Address all correspondence to: Dr. I.H. Goldberg, Department of Pharmacology, Harvard Medical Schoo l , 25 Shat tuck Street, Boston, Mass. 02115, U.S.A.

** Present address: Grace Cancer Drug Center, Roswell Memorial Park Institute, Buffalo, N.Y. 14263, U.S.A.

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Introduct ion

Neocarzinostatin is an acidic, single-chain protein of molecular weight 10 700 [1] isolated from a filtrate of Streptomyces carzinostaticus [2]. The amino acid sequence has been determined and found to contain only naturally occurring amino acids [1]. Physical characterizations of neocarzinostatin show it to possess a rigid conformation containing two reduction-resistant disulfide bonds [3]. Biologic activity is resistant to most proteolytic enzymes [4].

Neocarzinostatin has been found to be active against a variety of experi- mental tumors including Ehrlich ascites tumor in mice [5], and recently has been used in the t reatment of human acute leukemia [6]. Neocarzinostatin is known to inhibit cell growth and DNA synthesis in gram positive bacteria such as Sarcina lutea [7], as well as in a variety of mammalian cell lines like HeLa $3 [8--10], L 1210 [11,12] and Burkitt lymphoma [13]. In addition to inhibiting DNA synthesis, neocarzinostatin induces the degradation of DNA to acid- soluble material in bacteria [7] and DNA strand-scission in mammalian cells [8, 9,11,12] while in vitro incubation of neocarzinostatin with purified DNA in the presence of 2-mercaptoethanol also results in single-strand scissions in the DNA [8,14].

The mechanism of action of neocarzinostatin is still unknown but it is thought to involve some form of interaction with the cellular DNA. In the intact cell system, DNA breakage may be a direct effect of the drug or an indirect one, perhaps due to a neocrazinostatin-induced endonuclease (possibly a consequence of neocarzinostatin interaction with the cell membrane}. Recent studies have also shown that neocarzinostatin blocks HeLa $3 cells at the G2 phase of the cell cycle at drug levels causing no inhibition of DNA synthesis [15].

This paper will present a detailed analysis of the interaction of neocarzinostatin with HeLa cells and their subcellular components and will further charac- terize the drug's inhibitory action on DNA synthesis and cell growth, as well as on the strand-scission of cellular DNA.

Materials and Methods

[Me-3H]Thymidine, [Me-~4C]thymidine and [3H]dTTP were obtained from New England Nuclear. All tissue culture medium and serum were from Grand Island Biological. HeLa $3 cells, stock CCL2.2, were from American Type Cul- ture Company. Highly purified neocarzinostatin was generously provided by Dr. T.S.A. Samy.

Maintenance of HeLa $3 cultures HeLa $3 spinner cultures were maintained as previously described [8]. HeLa

$3 plate cultures were maintained in Eagle's minimal essential medium with 5% fetal calf serum at 37°C under 5% CO2 and at densities between 4 • 104 cells/ ml and 6 • l 0 s cells/ml.

Measurement of growth of HeLa $3 cells HeLa $3 cells were grown on 35 mm tissue culture dishes maintained at 37°C

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in 5% CO2 and containing 2 ml of Eagle's minimal essential medium with 5% fetal calf serum. Cell suspensions (4 • 104 cells/ml) were added to tissue culture dishes and after 24 h were treated with drug. After 3 days of growth, the cell suspension density was measured by washing the plates twice with 2 ml of D~ glucose {0.14 M NaC1, 5 mM KC1, 0.17 mM Na2HPO4, 0.22 mM KH2PO4 and 0.1% glucose, pH 7.0) followed by another wash with D~ glucose containing 0.25% trypsin. After 5 min to allow for trypsin release of the cells from the dishes, samples were counted in a hemocytometer .

Measurement o f DNA synthesis in HeLa $3 cells DNA synthesis was measured in HeLa $3 cells grown in spinner culture by

pulsing 10 ml of cell suspension (3 • 10s--4 • l 0 s cells/ml) for 30 min at 37°C with [3H]thymidine (50 Ci/mmol, 0.4 pCi/ml). Samples were then centrifuged at 3000 rev./min for 5 min at 4°C and resuspended in 2 ml of phosphate-buff- ered saline (0.17 M NaC1, 0.27 mM KCI, 8.1 mM Na2HPO4, 0.21 mM KH2PO4, pH 7.2). This was followed by two additional centrifugation in which the pellets were resuspended in 2 ml of cold 0.4 M perchloric acid. The washed pel- let was taken up in 0.5 ml of 0.4 M perchloric acid and the suspension was added to 10 ml of scintillation fluid (Scintisol-Isolab Inc.}. The tubes were then rinsed with 0.5 ml of distilled water and the rinse was added to the scintillation fluid for counting.

Similar experiments were done with HeLa cells synchronized at the G1/S transition by t reatment with hydroxyurea [16].

Alkaline sucrose gradient analysis o f strand-scission o f cellular HeLa $3 DNA by neocarzinostatin

Cell suspensions (2 • 10s--3 • 10 s cells/ml) were prelabelled for 16--18 h with [~4C]thymidine (50 mCi/mmol, 0.04 pCi/ml) and the DNA was analyzed on alkaline sucrose gradients as previously described [8], except that 0.3% Sarko- syl was used in the alkaline lysis solution. Samples were centrifuged at 20°C in a Beckman SW 50.1 rotor for 40 min at 40 000 rev./min. Sedimentation was always from left to right in all figures. When the time course of DNA-cutting by neocarzinostatin was measured, cells were labelled with higher levels of [14C]- thymidine (50 mCi/mmol, 0.5 pCi/ml) and after the incubation with drug, 100 ~l was placed directly on the alkaline sucrose gradient.

Isolation o f HeLa $3 cell lysate, cytoplasmic fraction and nuclei The preparations of HeLa cell lysate, cytoplasmic fraction and nuclei from

cells suspended in buffer A (10 mM Tris, pH 7.8, 1 mM EDTA, 4 mM MgC12 and 6 mM 2-mercaptoethanol) using a Dounce glass homogenizer were essen- tially as described by Hershey et al. [16]. The mixture of nuclei and cytoplasmic material is designated as the cell lysate. The cell lysate was spun at 100 000 × g at 2 ° C for 1 h and the recovered supernate is designated as the cytoplasmic fraction.

Measurement o f DNA synthesis in HeLa $3 nuclei and cell lysate preparations Nuclei suspensions (0.5 ml) prepared as described above were added to 0.4

ml of buffer A and 0.2 ml of a standard reaction mixture which consisted of

284

300 mM NaC1, 100 mM HEPES, pH 7.65, 20 mM MgC12, 15 mM ATP and 0.3 mM each of dATP, dCTP and dGTP, 0.1 mM dTTP and 0.5 pCi [3H]dTTP (50 Ci/mmol) [16]. After incubation at 37°C for 45 min, 2 ml of cold 0.4 M perchloric acid was added to the samples for measurement of DNA synthesis as described by Hershey et al. [16].

In experiments involving use of the cytoplasmic fraction 0.4 ml of the supernate obtained from a 100 000 × g centrifugation of a cell lysate (2 • 107--3 • 107 ceUs/ml) replaced buffer A in the nuclear DNA-synthesizing system.

In experiments involving use of the cell lysate, cell suspensions (2 • 107- 7 • 10 T cells/ml) were disrupted as described above and 0.5 ml of the lysate was added to buffer A and the standard reaction mixture.

Analysis of strand-scissions in D N A from nuclei isolated from HeLa $3 cells HeLa cells prelabelled with [~4C]thymidine were divided into two equal

parts and from one nuclei were isolated as described above, while with the other, buffer A was replaced by NaC1/EDTA (0.075 M NaC1, 0.024 M EDTA, pH 7.5) buffer in the procedure for isolating nuclei. After t reatment with drug, 100 pl of nuclear suspension (1 • 107--2 • 10 ~ nuclei/ml) were analyzed by sedi- mentat ion at 50 000 rev./min for 30 min on alkaline sucrose gradients.

Results

Inhibit ion o f D N A synthesis in HeLa cells grown in culture The effect of neocarzinostatin on DNA synthesis was examined in a suspen-

sion culture of HeLa $3 cells (Fig. 1). Drug at 0.5 pg/ml gave significant inhibition after only 1 h and near maximal levels of inhibition were reached by 2 h. The rapidity of onset of inhibition of DNA synthesis was dependent upon the amount of neocarzinostatin present. At the lowest drug concentration (0.1 ~g/ml) giving significant inhibition the rate of DNA synthesis eventually returned to that of untreated cells. In other experiments, inhibition of DNA synthesis by neocarzinostatin (5 tlg/ml) was found as early as at 5 min of incubation.

Strand-scission o f cellular D N A as a function o f drug concentration The breakdown of cellular DNA by neocarzinostatin was examined over a

wide range of drug concentrations. Breakdown of DNA by drug is followed by the appearance of DNA on the alkaline sucrose gradient, while control cellular DNA because of its very large size is pelleted to the bot tom of the tube (Fig. 2). Using an internal marker of superhelical PM2 DNA (gift from Dr. Jacob Lebowitz), the s value of the DNA from cells treated at 5.0 pg/ml of neocarzinostatin was estimated to be between 70 and 90 compared to control DNA which sediments at over 200 S. Between drug concentrations of 5.0 and 25 ug/ ml there is still a shift to smaller-sized DNA although the s value is changed little. This is expected from the equation of Abelson and Thomas [17] relating the s value to DNA size. When the gradients were spun for longer time periods to separate the peaks further, there was a slight shift to smaller DNA for cells treated with 100 t~g/ml of drug compared to those treated at 25 ug/ml (data not presented). Using drug levels of 100 pg/ml for 2 h instead of 30 min pro-

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Fig. 1. Inh ib i t ion of D N A synthes is by H e L a S 3 cells as a func t i on of t ime and neoca rz inos t a t i n concen- t r a t ion . H e L a S 3 cell suspensions (3 • 105---4 • 105 cel ls /ml) were t r ea ted wi th neoca rz inos t a t i n a t the c o n c e n t r a t i o n s and fo r the t ime s ind ica ted in Fig. 1. 10-ml samples were w i t h d r a w n and pulse- labeled wi th [ 3 H ] t h y m i d i n e for 30 rain at 37°C. The da ta are expressed as p e r c e n t inhib i t ion of DNA synthes is by c o m p a r i n g the i n c o r p o r a t i o n o f [ 3 H ] t h y m i d i n e in the d rug- t r ea ted samples wi th t ha t of the con t ro l sample ( a p p r o x i m a t e l y 5000 c p m in a 30-min pulse) . Neoca rz inos t a t i n levels were as fol lows: o - o, 0 . I /zg/ml; z~ A, 0 .5 p g / m l ; o ~, 1 .0 / zg lml ; m- m, 5.0 pg /ml ; and • - a 50 pg /ml .

Fig. 2. Strand-scission of H e L a S 3 DNA as a func t ion of neoca rz inos t a t i n c o n c e n t r a t i o n . H e L a S 3 cells were label led wi th [ 1 4 C ] t h y m i d i n e and t r ea t ed wi th neoca rz inos t a t in . Drug was added to 5 ml of cell suspens ion (4 • 105 - -5 • 10 s ce l ls /ml) and a f t e r i n c u b a t i o n for 30 rain at 37°C were washed , lysed and s e d i m e n t e d on an alkal ine sucrose gradient . A l t h o u g h in s o m e cases na t ive D N A was r eco v e red in t h e pel let , only rad ioac t iv i ty t ha t appears on the g rad ien t are sh o wn because r ecovery of the pe l le ted radio- ac t iv i ty was n o t suf f ic ient ly quan t i t a t i ve . R e c o v e r y of rad ioac t iv i ty on the grad ien t was g rea te r t han 90% w h e n no D N A was in the pel let ; while for c on t ro l cells, where near ly all the D N A pel le ted , r ecove ry var ied b e t w e e n 50 and 75%. In some cases, superhel ica l bac t e r iophage PM2 D N A was used as a sed imen- t a t ion m a r k e r . • • , con t ro l ; o o, 0 .5 /zg/ml, ~ - ' : , 2.0 p g / m l ; o ~, 5.0 #g /ml : • I , 2 5 / z g / m l ; and • . . - -A 100 .ug/ml.

duced still smaller DNA. At this high level of drug exposure there is the possibility that further fragmentation of DNA results from secondary actions of the drug on the cell. In all these experiments, however, no detectable loss of acid-precipitable radioactivity was found.

Strand-scission o f cellular D N A by neocarzinostatin as a funct ion o f t ime It is important to establish that the breakdown of cellular DNA results from

the incubation of the cell with drug and is not taking place after lysis of the cells on the gradient. Cells were incubated with neocarzinostatin over a short time course and the sedimentation profile of the DNA was examined {Fig. 3). The onset of cutting occurs quite rapidly with significant breakdown at 1 min and proceeds to near maximal breakdown by 10 min (Fig. 3). Longer incubation times of up to 2 h gave results similar to that found at 10 min.

As further evidence that extracellular cutting of DNA is not responsible for

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Fig. 3. Strand-sciss ion of H e L a S 3 D N A as a func t i on of t ime of i ncuba t ion wi th neoca rz inos t a t i n . H e L a S 3 cells label led wi th [ 1 4 C ] t h y m i d i n e were t r e a t e d wi th 5.0 p g / m l of n e o c a r z i n o s t a t i n at 37°G for the t imes ind ica ted . T he reac t ions were s t o p p e d by p l a c e m e n t of the cells o n t o alkal ine sucrose grad ien ts con t a in ing a l aye r of lysing m e d i u m , and samples were ana lyzed as descr ibed . $ e , no neoea rz ino- s ta t in ; © o, 0 t i m e ; c] •, 1 rain; ~ ~° 2.5 rain; m - - - - m , 5 rain; and A - - - - - A 10 rain.

Fig. 4. Strand-seiss ion of H e L a S 3 D N A a f t e r p r e i n c u b a t i o n of cells at 0°C or 37°C wi th neoca rz inos t a t i n . H e L a S 3 cells label led wi th [14C] t h y m i d i n e were t r e a t e d wi th neocaxz inos ta t in (5.0 p g / m l ) at e i ther 0

or 37°C for 10 min . The cells were t hen washed twice by suspens ion in p h o s p h a t e - b u f f e r e d saline (2°C) and c e n t r i f u g a t i o n at 3 0 0 0 r e v . / m i n (2°G) . T h e y were r e suspended in Eagle 's m i n i m u m essential m e d i u m at the original v o l u m e of cell suspens ion and i n c u b a t e d at 37°G for 30 rain be fo re be ing ana lyzed on alka- l ine sucrose grad ien ts as descr ibed . Cont ro l cells and u n w a s h e d , d rug - t r ea t ed cells (5 .0 # g / m l neoea rz ino - s ta t in ) were also i n c u b a t e d at 0 and 3 7 ° C for 30 rain. The 37°C co n t ro l is n o t s h o w n as it is essential ly ident ica l to the 0°C con t ro l and the 0°C, u f iwashed , d rug - t r ea t ed cell suspens ion sample is n o t s h o w n as it is essent ia l ly ident ica l to the 0°C, washed , d rug - t r ea t ed sample . • "-, con t ro l , 0°C, o o, drug, 10 rain at 37°G , washed , 30 rain at 37°C; and • "~,, drug, 10 rain at 0Oc, washed , 30 rain at 37°C.

DNA breakdown, similar sedimentation profiles are obtained whether or not the cells are washed free of drug prior to the lysis. Also the alkalinity of the gradient (pH 12) is sufficient to inactivate all in vitro neocarzinostatin cutting activity (data not presented), though it was conceivable that neocarzinostatin that has been incubated with cells was afforded some form of protection against denaturation at the time of cell lysis. To rule out this possibility, drug was incubated with unlabelled cells prior to the addition of the [14C]thymidine-labelled cells. In these studies strand-scission of the [14C]thymidine- labelled cellular DNA was observed only when the labelled cells were incubated with drug prior to cell lysis.

Strand-scission o f HeLa $3 cellular D N A and inhibition o f D N A synthesis and cell growth by neocarzinostatin as a funct ion o f temperature

Treatment of cells with neocarzinostatin (5.0 pg/ml) at 0°C does not cause breakage of cellular DNA (Fig. 4). Cells treated with drug (5.0 ug/ml) at 0 or

12.5

37°C for 10 min and then washed free of drug before incubation at 37°C showed breakage of cellular DNA only for the cells pretreated at 37°C (Fig. 4).

Experiments were designed to determine if drug must be continuously present to cause inhibition of DNA synthesis in HeLa $3 cells. When cells were treated with drug at 37°C for relatively short time periods followed by removal of the drug, the degree of inhibition of DNA synthesis was nearly the same as when the drug was not removed (Fig. 5). Similarly it was found that once cells were exposed to drug for 10 min at 37°C, cell growth was still inhibited after three days whether or not the free drug was washed from the cells after the ini- tial 10-min incubation period (Table I). On the other hand, incubation of cells with neocarzinostatin at 0°C for 10 min followed by removal of the drug and shifting the cells to 37°C resulted in no inhibition of DNA synthesis (Fig. 5) or cell growth (Table I).

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/ Fig. 5. Inh ib i t ion of D N A synthes is in H e L a S 3 cells by p r e i n c u b a t i o n a t 0 or 37°C wi th neoca rz inos t a t in . H e L a S 3 cell suspens ion (4 • 105 - -5 • 105 eel ls /ml) were i n c u b a t e d wi th neoca rz inos t a t i n (5.0 ~g /ml ) at e i ther 0 or 37°C for 10 rain be fo re be ing washed as descr ibed in Fig. 4. Cell suspens ions were t h e n resus- p e n d e d at the i r original v o l u m e in Eagle 's m i n i m u m essential m e d i u m and b r o u g h t to 37°C. [ 3 H ] T h y m i - dine was t h e n added a t zero t ime and samples were w i t h d r a w n a t the ind ica ted t imes for m e a s u r e m e n t of D N A synthes is as desc r ibed in Materials and Methods . In add i t ion , two con t ro l samples lacking neocar - z inos ta t in were at 0°C and o the rwise t r e a t e d ident ica l ly to the d rug - t r ea t ed samples . Also o t h e r cell suspens ions were i n c u b a t e d at 37°C wi th neoca rz inos t a t i n (5 .0 /~g /ml ) and were n o t washed . • e , con- t rol at 37°C; • s c on t ro l a t O°C; a ~ , d rug a t 37°C; o o drttg, I 0 rain a t 37°C, washed , i n c u b a t e d at 370C; and o D, d rug , 10 rain a t OOC, washed , i n cu b a t ed at 37°C.

Fig. 6. Effec ts of neoca rz inos t a t i n t r e a t m e n t of H e L a S 3 cells on the i n c o r p o r a t i o n of d T T P in to the D N A of isola ted nucle i and cell lysates . H e L a S 3 cells s y n c h r o n i z e d a t the G 1/S t r ans i t ion b y a m e t h o p t e r i n - adenos ine t r e a t m e n t [16 ] and t r ea t ed as descr ibed in Materials and Methods were given n e o c a r z i n o s t a t i n (5.0 ~tg/ml) I h p r io r to release f r o m the b lock . 2 h a f t e r release nucle i (A) or cell lysate (B) were isola ted and the i n c o r p o r a t i o n of [ 3 H ] d T T P in to D N A was m e a s u r e d a t the ind ica ted t imes as descr ibed in Mate- rials and Methods . In (C), 100 /~1 of ac t i va t ed s a l m o n s pe rm D N A (2 .5 m g / m l D N A t r ea t ed at 37°C for 30 m i n wi th panc rea t i c DNAase (0.1 ~g /ml ) in 0 .01 M Tris, p H 7.4 and 5 m M MgCI 2) was ad d ed to cell lysates 10 rain p r io r to in i t ia t ion of D N A synthesis , e - - -~, con t ro l ; and o • o, neoca rz inos t a t i n , 5 .0 /~g/ml .

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T A B L E I

E F F E C T S O F T E M P E R A T U R E O N N E O C A R Z I N O S T A T I N - I N H I B I T I O N O F H e L a S 3 C E L L G R O W T H

H e L a S 3 cells on p la tes were m a i n t a i n e d and g r o w t h m e a s u r e d as desc r ibed in Mater ia ls and Me thods .

Cells (8 • 104 ce l l s ]ml) were i n c u b a t e d w i t h or w i t h o u t n e o c a r z i n o s t a t i n (1 .0 p g / m l ) a t e i t he r 37 or 0°C . Where i n d i c a t e d , the p la tes were w a s h e d w i t h 3 r inses of 2 ml of p h o s p h a t e - b u f f e r e d sal ine fo l lowed by

the a d d i t i o n o f f resh Eag le ' s m i n i m u m essent ia l m e d i u m and i n c u b a t i o n was c o n t i n u e d at 3 7 ° C fo r 72 h.

T r e a t m e n t I n c u b a t i o n c o n d i t i o n s Cell c o u n t a t 72 h

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Effect o f neocarzinostatin on DNA synthesis in nuclei To determine whether the inhibition of thymidine incorporation into DNA

occurred before or after formation of the nucleoside triphosphate, the effect of neocarzinostatin on the incorporation of nucleoside triphosphates into DNA by isolated nuclei was examined. When nuclei are isolated from HeLa S3 cells that have been treated with drug, there is a strong inhibition of the rate of DNA synthesis (Fig. 6A). Also the level of inhibition, is dependent upon the length of time the cells were treated with drug prior to nuclei isolation (data not shown).

Similarly, hypotonical ly swelled cells and lysed cell preparations can be used to measure nucleoside tr iphosphate incorporation into DNA. As with isolated nuclei, those preparations derived from neocarzinostatin-treated cells were defi- cient in DNA synthesis (Fig. 6B shows the data for the lysate system only). In the lysate system the addition of activated DNA {calf thymus DNA nicked by pancreatic DNAase) stimulates DNA synthesis presumably due to the presence of an extranuclear form of the DNA polymerizing enzyme [18]. As shown in Fig. 6C, lysates from drug-treated and untreated cells to which activated DNA was added exhibit virtually the same amount of stimulation indicating that the drug does not affect the enzymes needed for replication as they appear fully functional in the presence of exogenous DNA template. Rather, the template DNA of the treated cell appears to be defective.

Hershey et al. [16] have shown that it is possible to isolate from the cell homogenate a fraction designated as the cytoplasmic fraction which contains the various components , including DNA polymerases, involved in replication, that when added to nuclei is able to stimulate DNA synthesis. Isolated cytoplasmic fraction from drug-treated and untreated cells were compared for their abilities to stimulate DNA synthesis in nuclei from untreated cells (Fig. 7A). It is clear that the cytoplasmic fraction from drug-treated cells has not been affected. In Fig. 7B it is shown that the cytoplasmic fraction produces the expected stimulation in control nuclei bu t has no affect on nuclei from drug- treated cells.

All the experiments presented in this section, concern the effects of drug on

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I I I ,1 2~) 25 20 40 60 20 40 60 S IO

T I M E (rain) FRACTION NUMBER Fig. 7. I n t e r a c t i o n of n e o c a r z i n o s t a t i n wi th the c y t o p l a s m i c f rac t ion of H e L a S 3 cells. A. Th e cy toplas - mic f rac t ion was isola ted f r o m s y n c h r o n i z e d H e L a S 3 cell suspens ions (1 • 107- -2 • 107 cel ls /ml) t ha t were i n c u b a t e d wi th or w i t h o u t neoca rz inos t a t i n (5 #g/re.l) 1 h be fo re release as desc r ibed in Materials and Methods . Nuclei i so la ted f r o m c on t ro l or ne oc a rz inos t a t i n - t r e a t ed cells were i n c u b a t e d wi th 4 0 0 #l of b u f f e r A and also nuc le i i sola ted f r o m con t ro l cells were i n c u b a t e d wi th 4 0 0 #1 of c y t o p l a s m i c frac- t ion der ived f r o m con t ro l or d rug - t r ea t ed cells. The i n c o r p o r a t i o n of [ 3 H ] d T T P in to D N A was m e a s u r e d as descr ibed in Materials and Methods at the ind ica ted t imes . • $, con t ro l nucle i + b u f f e r A; o o, drug-nucle i + b u f f e r A; ~ ~, c on t ro l nucle i + co n t ro l cy to p l a smic f rac t ion ; and [] . . . . . . a , con t ro l nucle i + d rug -cy top l a smic f rac t ion . B. Cy top l a s mic f rac t ion and nucle i we re p r e p a r e d as in (A) . Nuclei f r o m con t ro l and ne oc a rz inos t a t i n - t r e a t e d cells were i n c u b a t e d wi th 4 0 0 #I of b u f f e r A or 4 0 0 #I of the c y t o p l a s m i c f rac t ion f r o m c on t ro l cells. The inco113oration in to D N A was m e a s u r e d as in (A). • : , con t ro l nucle i + b u f f e r A; ~ e, con t ro l nuc le i + co n t ro l c y t o p l a s m i c f rac t ion ; o o, d rug-nucle i + b u f f e r A; and D . . . . . . ~, d rug-nucle i + c on t ro l c y t o p l a s m i c f rac t ion .

Fig. 8. E f fec t of n e o c a r z i n o s t a t i n on the s t rand-scission of D N A in isolated H e L a S 3 nuclei . Nuclei we re i so la ted f r o m a s y n c h r o n o u s H e L a S 3 cells p re labe l led w i th [ 14C] t h y m i d i n e as desc r ibed in Materials and Methods . Nuclei i sola ted in b u f f e r A or in the NaC1-EDTA b u f f e r were i n c u b a t e d for 30 min at 37°C wi th or w i t h o u t neoca rz inos t a t i n (5 .0 p g / m l ) a nd were t h e n sub jec ted to alkal ine sucrose s e d i m e n t a t i o n analysis as descr ibed in Materials a nd Methods . • ±, con t ro l , b u f f e r A; ~ ~, d rug - t r ea t ed , b u f f e r A; • • , con t ro l , Na CI -EDTA buf fe r ; and o o, d rug - t r ea t ed , NaCI -EDTA buf fe r .

HeLa $3 cell components isolated from cells pretreated with neocarzinostatin. On the other hand, when isolated nuclei were incubated with neocarzinostatin there was no inhibition of DNA synthesis (Table II). It was thus important to examine the effects of neocarzinostatin on the DNA of isolated nuclei. When cells are treated with drug and their nuclei are then isolated, as expected, the sedimentation profile of the DNA shows strand-scissions on alkaline sucrose gradients in comparison with DNA from control cells (data not shown). Using the same technique the sedimentation profiles of DNA from nuclei that were incubated with drug subsequent to isolation were determined (Fig. 8). When the nuclei were processed in a buffer containing Mg 2÷ and 2-mercaptoethanol, the DNA from both the control and drug-treated nuclei sedimented together, peaking at fraction No. 12. On the other hand, when nuclei were isolated in an

2 9 0

T A B L E I I

E F F E C T O F N E O C A R Z I N O S T A T I N O N D N A S Y N T H E S I S B Y I S O L A T E D N U C L E I

As i n d i c a t e d , n u c l e i w e r e i s o l a t e d f r o m H c L a S 3 cel l s u s p e n s i o n s (4 • 1 0 S - - 5 • l 0 s c e l l s / m l ) w h i c h h a d

b e e n 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 ( 5 . 0 t t g / m l ) 1 h p r i o r to r e l e a s e f r o m t h e a m e t h o p t e r i n - a d e n o s i n e

b l o c k a n d t h e i n c o r p o r a t i o n o f [ 3 H ] d T T P i n t o D N A w a s m e a s u r e d 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 .

A l s o , n u c l e i w e r e i s o l a t e d in a s i m i l a r m a n n e r b u t n e o c a r z i n o s t a t i n w a s n o t a d d e d u n t i l a f t e r i s o l a t i o n .

N u c l e i s u s p e n s i o n s , w h i c h w e r e c o n c e n t r a t e d to t h e e q u i v a l e n t o f 1 - 107 c e l l s / m l , w e r e e x p o s e d t o n e o - c a r z i n o s t a t i n ( 5 . 0 t t g / m l ) b a s e d o n t h e o r i g i n a l v o l u m e o f cel l s u s p e n s i o n , i .e . t h e s a m e a m o u n t o f d r u g

as t h e n u c l e i f r o m d r u g - t r e a t e d ce l ls r e c e i v e d . A f t e r i n c u b a t i o n w i t h d r u g f o r 1 h a t 3 7 ° C , [ 3 H ] d T T P

i n c o r p o r a t i o n i n t o D N A f o r 4 5 m i n w a s d e t e r m i n e d 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 T i m e o f a d d i t i o n [ 3 H] T h y m i d i n e P e r c e n t n e o c a r z i n o s t a t i n i n c o r p o r a t i o n i n h i b i t i o n

( p g / m l ) ( c P m )

0 P r i o r t o n u c l e i i s o l a t i o n 2 1 1 3 - -

5 .0 P r i o r t o n u c l e i i s o l a t i o n 8 7 1 60

0 A f t e r n u c l e i i s o l a t i o n 2 0 5 4 - -

5 .0 A f t e r n u c l e i i s o l a t i o n 2 0 2 1 2

EDTA buffer to avoid activation of nucleases, control DNA was larger than that isolated in the Mg 2+ and 2-mercaptoethanol containing buffer and now the addition of drug does produce a smaller DNA (Fig. 8).

Discussion

Our results on the pattern of inhibition of DNA synthesis in neocarzinostatin- treated HeLa $3 cells are in essential agreement with a similar s tudy done by Homma et al. [10]. Also, in other experiments cells synchronized at the G1/S transition of the cell cycle [16] and then given neocarzinostatin showed inhibition of DNA synthesis that was independent of whether the drug was present before, during or after releasing cells to enter S phase. This result speaks against the existence of a substance required for DNA synthesis other than DNA, whose formation occurs outside the S phase and is sensitive to neocarzinostatin, and is compatible with DNA itself being the target of the drug.

Both inhibition of DNA synthesis and the strand-scission of cellular DNA occur at similar drug levels and the cutting of DNA strands is faster than the inhibition of DNA synthesis (Figs. 1 and 3). The time course of the reaction indicates that DNA fragmentation is occurring inside the cell and is not taking place after cell lysis in the lysing medium. Similarly, cell lysis induced by drug t reatment itself, with subsequent breaking of DNA, appears not to be impli- cated as determined by both microscopic observations and by DNA recovery experiments after cell washing. Further, as expected, removal of free drug prior to cell lysis does not decrease the amount of DNA scission.

In contrast with the plant alkaloid camptothecin which produces only alkali- labile breaks in DNA [19], neocarzinostatin-induced breaks are true strand- scissions as shown by the equal levels of DNA breakdown on both alkaline and formamide gradients {unpublished data). Similarly, in vitro strand-scission of superhelical viral DNA by neocarzinostatin has also been found to take place in the absence of alkali [8]. Further, the sedimentation profile of DNA from drug- treated cells on neutral gradients shows that the scissions in the DNA are single-

291

stranded in nature since there was a reduction in the amount of cut DNA on neutral gradients compared with alkaline gradients (unpublished data}. There appears to be a limit to the extent of DNA strand-scission for even at high levels of neocarzinostatin the remaining DNA is of a relatively large size and acid-soluble material is no t produced.

There is evidence that both the inhibition of DNA synthesis and the cutting of cellular DNA require some form of temperature-dependent drug binding to or penetration of the cell surface. If cells were treated with drug at 0 or 37°C for 10 min and the free drug was removed before incubation at 37 ° C, there was inhibition of DNA synthesis and nicking of cellular DNA only if pretreatment was at 37°C. Presumably, drug is not able to bind to or penetrate cells at 0°C and is removed by the washing. Similarly, cell growth inhibition also requires pretreatment at the elevated temperature. It should be noted that under these conditions of preincubation, neocarzinostatin would have almost completed its breaking of the DNA.

The data discussed so far suggest that a primary action of neocarzinostatin on HeLa $3 cells may be the cutting of cellular DNA and that this results in the observed effects on DNA synthesis and possibly even on cell growth. Our study of the consequences of neocarzinostatin on the incorporation of dTTP into DNA by nuclei or lysate derived from HeLa $3 cells supports this possibility. Lysate from drug-treated cells was inhibited in the incorporation of dTTP into DNA and this inhibition could be overcome by adding primer DNA to the system. Thus it appears that neocarzinostatin altered the activity of the DNA template rather than that of the replicating enzymes. Further evidence for such a mechanism is the lack of effect that neocarzinostatin t reatment of cells has on the activity of the isolated cytoplasmic fraction. Cytoplasmic fraction from drug-treated cells retained its ability to stimulate the incorporation of dTTP into DNA by isolated nuclei. Only when cytoplasmic fraction from untreated cells was added to nuclei from drug-treated cells was there a loss of stimulation.

It is of interest that nuclei derived from cells pretreated with neocarzinostatin show inhibition of DNA synthesis while neocarzinostatin added directly to isolated nuclei results in no such inhibition. This apparent inconsistency was resolved, at least in part, when it was found that isolating nuclei from HeLa $3 cells with a Mg 2.- and 2-mercaptoethanol-containing buffer resulted in strand- scission of cellular DNA (Fig. 8). Because nucleases from cell nuclei are susceptible to activation by Mg ~÷ [20], the buffer used in the isolation procedure likely induced breakage of the DNA by activation of endogenous nucleases. The addition of neocarzinostatin to the isolated nuclei did not result in additional strand-scissions, perhaps because only exposed regions in the chromatin are susceptible to attack by either drug or nucleases. When a buffer containing a high concentration of EDTA was used in an effort to restrict nuclease activity, the DNA was of a much larger size than that found when Mg 2÷ and 2-mercaptoethanol were present in the buffer. Neocarzinostatin was now able to degrade this larger-sized DNA but again to a limit. This rcsult is similar to that reported by Sarma et al., [21] with nuclei prepared from rat liver.

The data presented in this paper suggest that strand-scission of cellular DNA is an important aspect of the mechanism of action of neocarzinostatin. The precise means whereby this is accomplished in the intact cell and its relation-

292

ship to the in vitro DNA strand-breaking reaction remain to be elucidated. In particular, the relationship between DNA synthesis inhibition and inhibition of cell growth is not obvious since levels of neocarzinostatin lower than those necessary for DNA synthesis inhibition block cells in G: of the cell cycle [15]. While there is evidence suggesting that there is a link between DNA fragmentation and DNA replication inhibition, that between the former and cell growth requires additional documentat ion. It should be pointed out, for instance, that recent experiments (Beerman, T.A. and Goldberg, I.H., in preparation) show that DNA strand-scission can be found even at 0.03 pg/ml neocarzinostatin where cell growth is inhibited by 50%. Similarly, Tatsumi et al. [22] have reported that concentrations of neocarzinostatin as low as 0.05 pg/ml will stimulate DNA repair of cultured lymphocytes, presumably a consequence of the direct damage of the cellular DNA by the drug. The fact that very low levels of neocarzinostatin will produce DNA strand-breaks permits speculation that the insertion of a few critical breaks may determine whether or not a cell can pass into mitosis.

Acknowledgements

The authors would like to thank Dr. T.S.A. Samy for his generous contribu- t ion of highly purified neocarzinostatin. Also the skilled technical assistance of Peggy Whitbred and Susan Curran is highly appreciated. This work was supported in part by U.S. Public Health Service Research Grant GM 12573 from the National Institutes of Health. T.A.B. was supported by an Anna Fuller Fellow- ship, a Sterling Winthrop Fellowship and a Fellowship from the National Insti- tutes of Health.

References

1 M e i e n h o f e r , J . , M a e d a , H. , Glaser , C.B. , C z o m b o s , J . a n d K u r o m i z u , K. ( 1 9 7 2 ) Sc ience 178 , 8 7 5 - - 8 7 6

2 I sh ida , N. , M i y a z a k i , K. , K u m a g a i , K. a n d R i k i m a r u , M. ( 1 9 6 5 ) J . A n t i b i o t . ( T o k y o ) Set . A 18 , 6 8 - - 7 6

3 S a m y , T .S .A . , A t r e y i , M., M a e d a , H. a n d M e i e n h o f e r , J . ( 1 9 7 4 ) B i o c h e m i s t r y 13 , 1 0 0 7 - - 1 0 1 4 4 M a e d a , H. , K u m a g a i , K . a n d I sh ida , N. ( 1 9 6 6 ) J . A n t i b i o t . ( T o k y o ) Ser . A. 19 , 2 5 3 - - 2 5 9 5 B r a d n e r , W.T. a n d H u t c h i n s o n , D.J . ( 1 9 6 6 ) Can . C h e m o t h e r . R e p . 50 , 7 9 - - 8 9 6 H i r a n k i , K. , K a n i m u r a , O. , T a k a h a s h i , I., N a g a o , T., K i t a j i m a , K. a n d I r ino , S. ( 1 9 7 3 ) Rev . Fr .

D ' H e m a t o l . T. 1 3 , 4 4 5 - - 4 5 1 7 0 n o , Y., W a t a n a b e , Y. a n d I sh ida , N. ( 1 9 6 6 ) B i o c h i m . B i o p h y s . A e t a 1 1 9 , 4 6 - - 5 8 8 B e e r m a n , T .A . a n d G o l d b e r g , I .H. ( 1 9 7 4 ) B i o c h e m . B i o p h y s . Res . C o m m u n . 59 , 1 2 5 4 - - 1 2 6 1 9 B e e r m a n , T .A . , G o l d b e r g , I .H. a n d P o o n , R . ( 1 9 7 4 ) E x c e r p t a Med ica I n t e r n a t i o n a l C o n g r e s s Ser ies

No . 3 5 3 , S u r g e r y , R a d i o t h e r a p y a n d C h e m o t h e r a p y o f Cance r , E x e e r p t a Med ica , A m s t e r d a m 5, 3 4 7 - - 3 5 2

10 H o m m a , M., K o i d a , T. , S a i t o - K o i d e , T. , K a m o , I., Se to , M., K u m a g a i , K. , a n d I sh ida , N. ( 1 9 7 0 ) Pro- gress in A n t i m i c r o b i a l a n d A n t i c a n e e r C h e m o t h e r a p y , P r o c e e d i n g s o f t he VI I n t e r n a t i o n a l C o n g r e s s o f C h e m o t h e r a p y 2 , 4 1 0 - - 4 1 5

11 T a t s u m i , K. , N a k a m u r a , T. a n d Wak i sak i , G. ( 1 9 7 4 ) G a n n 65 , 4 5 9 - - 4 6 1 1 2 S a w a d a , H. , T a t s u m i , K. , Sasada , M., S h i r a k a w a , S., N a k a m u r a , T. a n d W a k i s k a , G. ( 1 9 7 4 ) C a n c e r

Res. 34 , 3 3 4 1 - - 3 3 4 6 13 K a w a i , Y. a n d K a t o h , A. ( 1 9 7 2 ) J . Na t l . Can . Ins t . 48 . 1 5 3 5 - - 1 5 3 8 14 I sh ida , R . a n d T a k h a s h i , T. ( 1 9 7 6 ) B i o c h e m . B i o p h y s . Res . C o m m u n . 68 , 2 5 6 - - 2 6 1 1 5 E b i n a , T. , O k u t s u k i , K. , Se to , M. a n d I sh ida , N. ( 1 9 7 5 ) Eur . J . Can . 11 , 1 5 5 - - 1 5 8 16 H e r s h e y , H. , S t r i be r , S. a n d Mul ler , G .C . ( 1 9 7 3 ) Eur . J . B i o c h e m . 3 4 , 3 8 3 - - 3 9 4 17 A b e l s o n , J .N . a n d T h o m a s , J r . , C .A. ( 1 9 6 6 ) J . Mol. Biol . 18 , 2 6 2 - - 2 9 1

293

18 Friedman, D.L. and Mueller, G.L. (1968) Biochim. Biophys. Acta 161,455--468 19 Abelson, H.T. and Penman, S. (1973) Biochem. Biophys. Res. Commun. 50, 1048--1054 20 Hewish, D.R. and Burgoyne, L.A. (1978) Biochem. Biophys. Res. Commun. 52, 5 0 4 - - 5 1 0 21 Sarma0 D.S.R., Rajalakshmi, S. and Samy, T.S.A. (1976) Biochem. Pharmacol. 25, 789--792 22 Tatsumi, K., Sakane, T., Sawada, H., Shirakawa, S., Nakamura, T. and Wakisaka, G. (1975) Gann

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