Diacridines: Bifunctional intercalators II. The biological effects of putrescine, spermidine and spermine diacridines on HeLa cells and on the L-1210 and P-388 leukemia cells

290

Biochimica et Biophysica Acta, 418 (1976) 290--299

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98503 DIACRIDINES: BIFUNCTIONAL INTERCALATORS II. THE BIOLOGICAL EFFECTS OF PUTRESCINE, SPERMIDINE AND SPERMINE DIACRIDINES ON HeLa CELLS AND ON THE L-1210 AND P-388 LEUKEMIA CELLS

E.S. CANELLAKIS and R.A. BELLANTONE Department of Pharmacology, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.)

(Received June 30th, 1975)

Summary The effect of putrescine, spermidine and spermine diacridines on the growth of HeLa cells and of P-388 and L-1210 leukemia cells has been evaluated and compared to t h a t of the parent compound, 9-aminoacridine. The diacridines are more effective growth inhibitors than 9-aminoacridine. The primary site of action appears to be the inhibition of RNA synthesis.

Introduction We have attempted to emphasize the intercalative ability of acridines by synthesizing double intercalators, i.e., two acridines connected by a homologous series of diamines (from 2-carbon to 18-carbon diamines) as well as by the naturally occurring polyamines, putrescine, spermidine and spermine; the latter polyamines form the compounds, putrescine diacridine, spermidine diacridine and spermine diacridine respectively [1]. The basic ideas behind this project have been presented [1 ]. In this paper we present some general biological effects of these double intercalators on HeLa cells and compare them to those of the parent c o m p o u n d 9-aminoacridine. The evidence presented indicates the much greater effectiveness of the double acridine intercalators as inhibitors of cell growth. The possible sites of action of these compounds are evaluated. We also present a study of the effect of spermine diacridine on the leukemic cells P-388 and L-1210. These cells were chosen because they grow in mice when injected intraperitoneally as well as in cell culture; consequently, we could study the effects of spermine diacridine on these cells both in cell culture

291 as well as in the whole animal. In addition, they require horse serum, which does not contain polyamine oxidase rather than bovine serum which has a high level of polyamine oxidase activity [2]. The low level of polyamine oxidase activity minimizes the formation of dialdehydes from spermine and spermidine which are inhibitory to cell growth; this property of horse serum permitted us to perform the spermine and spermidine reversal experiments. Materials and Methods The synthesis and purification of the double intercalators has been described [1]. 9-Aminoacridine was purchased from Aldrich. All growth media were obtained from GIBCO, Grand Island, N.Y. The radioactive tracers used were [Me-l 4C]thymidine (51.5 Ci/mol), L-[1-14C]leucine (53.5 Ci/mol) and [2~' 4C]uridine (56.7 Ci/mol) from New England Nuclear. We wish to thank Dr. Lon Hodge for his generous supply of the HeLa cell line used in these experiments. Stock HeLa cell cultures were maintained in spinner culture using Joklikmodified minimal essential medium supplemented with 2 mM L-glutamine and 10% fetal calf serum. Log phase cells (2--4 × l 0 s per ml) were exposed to the appropriate inhibitors at the indicated concentrations in spinner culture. After defined times of incubation, the cells were removed, washed three times with complete medium, resuspended and pipetted into culture tubes; the tubes were incubated on a Wedco rotating wheel at a rate of 2 rotations/min. All above operations were performed at 37°C. Control cell growth rates do not differ between the wheel cultures and spinner flasks. At intervals, triplicate samples were removed for counting. Soft agar cloning was carried o u t using Joklikmodified minimal essential medium supplemented with 2 mM L-glutamine and 15% fetal calf serum according to the method of Chu and Fischer [3]; the visible clones were counted after 20 days and were found to be the same at 30 days. Cell number was continuously monitored and the cultures diluted whenever cell populations reached 4--6 × l 0 s cells per ml; in non-dividing cells, the medium was replenished when the pH, assayed by the color change of the phenol red indicator, dropped to below 7.2--7.1. In the short term experiments in which incorporation of radioactivity was measured, the cells were grown as monolayer cultures in 35-mm plastic petri dishes (approx. 106 cells/dish) in Dulbecco's medium supplemented with 10% fetal calf serum. Cells incubated at 37°C in a 5% CO2 atmosphere were completely attached to the plastic within 1 h. The experiments were initiated by decanting the supernatant fluid and replacing it with prewarmed ( 3 7 ° C ) m e dium containing the appropriate inhibitors for one hour. Following t w o washes with complete medium, incorporation studies were performed at prescribed time intervals. Rates of RNA, DNA and protein synthesis were measured by determining the incorporation of [ ' 4 C/uridine, (1 pCi/ml), [ 14 C / t h y m i d i n e (1 pCi/ml) and [ ' 4 C ] leucine (1 #Ci/ml) into the acid-insoluble fraction during a 30-min pulse. Unlabeled thymidine (5 #g/ml) and uridine (2 pg/ml) were added to the medium to minimize the depletion of radio-activity. At the end of the incorporation period, the medium was decanted and the dishes washed in cold 0.9% saline, 70% ethanol, acetic acid/ethanol (3 : 1, v/v) (5 min each) and

292 TABLE I E F F E C T O F S P E R M I N E D I A C R I D I N E ON T H E S U R V I V A L O F M I C E I N O C U L A T E D W I T H P-388 O R L-1210 CELLS 24 m i c e p e r g r o u p . B D F / 1 m i c e i n o c u l a t e d i n t r a p e r i t o n e a l l y w i t h 106 P-3S8 cells or 106 L - 1 2 1 0 cells. T w e n t y - f o u r h o u r s later, a n d for a t o t a l o f 6 d a y s , 4 0 pg s p e r m i n e d i a c r i d i n e p e r m o u s e w e r e a d m i n i s t e r e d daily. A d m i n i s t r a t i o n o f larger or s m a l l e r a m o u n t s o f s p e r m i n e d i a c r i d i n e yields s h o r t e r survival values. T h e s e cells are d e r i v e d f r o m t h e i n t r a p e r i t o n e a l e x u d a t e of D B A ] 2 d o n o r m i c e . N o t e t h a t t h e a v e r a g e survival of c o n t r o l a n i m a l s is m u c h s h o r t e r t h a n t h o s e n o t e d in Fig. 4. (See Materials and M e t h o d s . )

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2% HC104 (20 min). The dishes were dipped in ethanol, dried at room temperature, the b o t t o m s were punched out and counted in Triton X-100: toluene/ POPOP (1 : 8, v/v) in a Packard scintillation counter. In the long term experiments, cells were removed from stock, inhibited or control cultures at 1 day intervals, similarly attached and pulsed as above. P-388 and L-1210 cells were obtained from Arthur D. Little Co. through the kind efforts of Dr. Florence White of the National Cancer Institute. Both cell lines were maintained in suspension culture in Fischer's medium for leukemic cells supplemented with 10% horse serum at 37°C. The experimental procedures used have been described [3]. In the experiments involving mice, 1 × 106 cells were injected intraperitoneally into BDF/1 mice. In the experiments shown in Table I, these cells were obtained from the intraperitoneal exudate of donor DBA/2 mice and they kill the mice in very short periods of time. However, when these cells are taken from cell culture, the time required to kill the mice is greatly extended (see Fig. 4). Results 1. The effect of 9-aminoacridine and o f spermine diacridine on the viability o f HeLa cells as measured by their ability to form clones in agar Fig. 1 shows t h a t when HeLa cells are exposed to as little as 1 pg spermine diacridine/ml (1.5 × 10 -6 M) for one hour, then washed several times by resuspension in growth medium and cloned in normal medium in soft agar, they provide no visible clones. In contrast, if HeLa cells are exposed to 9-aminoacridine for various periods of time up to 8 h, then similarly washed and cloned in agar, at least 80% of the HeLa cells are still capable of cloning. In detail, this experiment was performed as follows: an a m o u n t of 9aminoacridine (4.5 × 10 -6 M) was added which increased the doubling time of HeLa cells from 18.5 to 38 h. In a separate flask, an a m o u n t of spermine diacridine was added (1.5 × 10 -6 M) which produced the identical inhibition of growth rate. From the control (no inhibitor added) and the two experimental flasks, samples of cells were removed at different times. These were washed several times with normal growth medium, diluted and cloned in culture tubes in soft agar. An additional experiment, (lower curve, Fig. 1) was performed in which 9.0 × 10 -6 M of 9-aminoacridine or spermine diacridine were added to

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induce complete cessation of growth. Again, samples were removed at different times, washed and cloned in soft agar. The recovery of clones from the control was 70--80% of the total cells added. We have normalized each of these values to 100%, and then compared the number of recovered experimental clones to this figure. As can be seen from Fig. 1, after exposure of HeLa cells to 9-aminoacridine (4.5 X 10 -6 M) for 1, 3, 5, 8 and 24 h, the cloning ability of these cells was 95%, 98%, 86%, 75% and 8%, respectively. After similar exposure to 9-aminoacridine (9.0 X 10 -6 M) for the same periods of time their cloning ability was 107%, 91%, 89%, 81% and 14%, respectively. In contrast, exposure to as little as 1.5 X 10 -6 M spermine diacridine for 1 h resulted in no recoverable clones from soft agar.

2. Effect of limited exposure to low concentrations of spermine diacridine and the subsequent growth of HeLa cells in the absence of the inhibitor The results given above could be better understood when the experiment was modified as follows: the cells were exposed to the drugs as before, washed free of the drug and reintroduced into normal growth medium; the cell number was then measured over many days. We thus had a continuous measure of the ability of the HeLa cells exposed to these drugs to subsequently divide and maintain themselves in the absence of the inhibitor. Fig. 2a shows that as little as a one hour exposure of HeLa cells to spermine diacridine at 1.5 × 10 -6 M (1/g/ml) results in eventual cessation of growth after 6--8 days. This is a progressive phenomenon, and exposure of HeLa cells to the same a m o u n t of spermine diacridine for longer periods of

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Fig. 2. T h e e f f e c t of l i m i t e d e x p o s u r e of H e L a ceils t o 9 - a m i n o a c r i d i n e a n d to s p e r m i n e d i a c r i d i n e u p o n t h e s u b s e q u e n t o u t g r o w t h of H e L a cells in c u l t u r e . Fig. 2a, t h e cells w e r e e x p o s e d to 4.5 × 1 0 - 6 M 9 - a m i n o a c r i d i n e (solid s y m b o l s ) a n d 1.5 X 1 0 - 6 M s p e r m i n e d i a c r i d i n e ( h o l l o w s y m b o l s ) , a n d in Fig. 2b t h e cells w e r e e x p o s e d to 9.0 × 10 -6 M 9 - a m i n o a c r i d i n e (solid s y m b o l s ) a n d 9.0 × 1 0 - 6 M s p e r m i n e d i a e r i d i n e ( h o l l o w s y m b o l s ) , T h e o u t g r o w t h o f t h e c o n t r o l p o p u l a t i o n is r e p r e s e n t e d b y a s t r a i g h t line. H e L a cells w e r e e x p o s e d t o t h e a c r i d i n e s f o r 1, 3, a n d 5 h (in Fig. 1 H , 3 H a n d 5H). T h e cells w e r e w a s h e d t h o r o u g h l y as d e s c r i b e d in t h e t e x t a n d a l l o w e d t o g r o w in c o m p l e t e m e d i u m for p e r i o d s u p to 25 d a y s . W h e n e v e r t h e cells r e a c h e d a p o p u l a t i o n size of 4 × 105 c e l l s / m l t h e y w e r e d i l u t e d ; for cells r e m a i n i n g a t t h e p l a t e a u , t h e m e d i u m was also r e p l e n i s h e d w h e n t h e p h e n o l r e d i n d i c a t o r s h o w e d t h a t t h e p H h a d fallen. All values h a v e b e e n c o r r e c t e d f o r such d i l u t i o n s .

time (or to larger amounts of spermine diacridine for the same periods of time, Fig. 2b) further limits their total growth and results in an earlier plateau of the curve. Consequently, the reason t h a t no clones could be seen in the cells exposed to spermine diacridine was t h a t these cells could not go through enough divisions to provide a visible clone. A clone of HeLa cells visible to the naked eye requires approx. 500 cells (Dr. G.A. Fischer, personal communication); this would require approximately 9 divisions. As can be seen, the cells exposed to spermine diacridine will only go through a limited number of doublings; these vary from approx. 0 to 3 (Figs. 2a and 2b). In contrast, exposure of HeLa cells to 9-aminoacridine for the same periods of time and under the identical conditions results in slower rates of growth which are subsequently maintained from the 22 day duration of the experiment but not in elimination of the growth of the cells; this therefore permits the observation of clones in agar. It is particularly interesting to note t h a t in the presence of spermine diacridine the number of HeLa cells reaches a plateau level and remains at this plateau for m a n y days. These cells are not interrupted at any unique state of cell division insofar as can be ascertained through the phase contrast microscope. We may, therefore, deduce t h a t this is an all or none phenomenon, because if a small percent of the cells were unaffected, t h e y would have continued growing and would have eventually outgrown the non-dividing cells. These results therefore explain the apparent contradiction in the previous experiment.

295

3. Inhibition of RNA, DNA and protein synthesis by 9-aminoacridine or spermine diacridine The experiment was performed in two sets. One measures these parameters over a short term (5 h) immediately following exposure to 9-aminoacridine or spermine diacridine as compared to controls (Fig. 3b). The other was performed over a long term (16 days) to establish any changes that may occur in these parameters during the period of observation (Fig. 3c). At the same time the cell count of the three cell populations was monitored (Fig. 3a) so that the •

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effects on RNA, DNA and protein synthesis could be related to the growth of the cells. Fig. 3a shows the characteristic continuous increase in cell number of the control population, the increased generation time of cells exposed to 9-aminoacridine, and the establishment of a numerically constant cell population for the cells exposed to spermine diacridine. Fig. 3b shows that immediately after exposure to 9-aminoacridine or to spermine diacridine and subsequent to their resuspension in fresh medium, R N A synthesis is immediately inhibited. Protein and DNA synthesis are inhibited later; most experiments have shown a one hour delay before protein synthesis was inhibited. Quantitatively there does not appear to be any great difference in action of these two c o m p o u n d s over a 5-hour period. However, where these measurements are extended over a long term, discrete qualitative and quantitative differences appear (Fig. 3c). Note that in b o t h cases a maximum of inhibition occurs at 24 h for RNA, DNA and protein synthesis. However, in the cells exposed to 9-aminoacridine which show a continuous increase in cell number, the inhibition of macromolecular synthesis is reversed after 24 h with values slowly approaching normal. In contrast, the cells exposed to spermine diacridine which do not increase appreciably in number over the long term, continue to synthesize RNA, DNA and protein at the low, inhibited 24-h level; they also show a tendency for a continuing decreased synthetic ability. These results are in agreement with the inability of cells exposed to spermine diacridine to proceed through an increase in cell number while the cells exposed to 9-aminoacridine can continue increasing in number, b u t in the latter

297 case their newly acquired generation time is dependent upon the extent of their prior exposure to 9-aminoacridine (Fig. 2a and 2b).

4. Intraperitoneal growth of P-388 and L-1210 cells which have been inhibited by prior exposure to spermine diacridine I>-388 and L-1210 cells were exposed to spermine diacridine washed and resuspended in normal growth medium. Such cells will cease to increase in number, reach a plateau and eventually decrease in number. The resultant growth curve is represented in Fig. 4. At various time intervals 1 X 10 6 cells from this inhibited culture were removed and administered intraperitoneally to mice. The survival times of the mice were noted. These numbers are presented on the curves corresponding to Fig. 4. It was also noted that these cells did increase in number intraperitoneally in the mice as was evident from microscopic examination and the volume of the exudate. We conclude that cells which are inhibited from subsequent growth in cell culture can grow when placed intraperitoneally in the animal. The fact that cells later along the growth curve take longer to kill the mice is not necessarily an index of the decreased viability of the cells in culture but may simply be due to a decreased ability of these cells to grow in the animal for as yet unidentified reasons; it should be noted that cells exposed to spermine diacridine and injected into mice at zero time will grow in mice and kill them at the same time as control untreated cells although these same cells will not grow when maintained in cell culture. (b)

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Fig. 5. (a) T h e r e v e r s a l b y s p e r m / d i n e a n d s p e r m i n e o f t h e g r o w t h i n h i b i t o r y e f f e c t o f s p e r m i n e d i a c r i d i n e o n P - 3 8 8 cells, r e s p e c t i v e l y . Cells w e r e e x p o s e d t o s p e r m i n e d i a c r i d i n e (1 h, 4 . 5 × 1 0 -6 M), w a s h e d , a n d r e s u s p e n d e d in n o r m a l g r o w t h m e d i u m i n t h e p r e s e n c e o r a b s e n c e o f 2 5 ~ g / m l o f s p e r m i d i n e o r s p e r m i n e . C o n t r o l cells ( I o), c o n t r o l cells p l u s s p e r m i d i n e ( a a ) , cells e x p o s e d t o s p e r m i d i n e d i a c r i d i n e , t h e n g r o w n in t h e p r e s e n c e o f s p e r m i d i n e o r s p e r m i n e ( s e), cells e x p o s e d tO s p e r m i n e d i a c r i d i n e and g r o w n in t h e a b s e n c e o f s p e r m / d i n e o r s p e r m i n e (o o). T h e r e s u l t s o b t a i n e d w i t h s p e r m i d i n e o r s p e r m i n e a r e i n d i s t i n g u i s h a b l e . S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h L - 1 2 1 0 cells. (b) T h e r e v e r s a l b y s p e r m / d i n e o r b y s p e r m i n e o f t h e g r o w t h i n h i b i t o r y e f f e c t o f s p e r m i n e d i a e r i d i n e o n P - 3 8 8 cells respectively. Polyamines added at various times after the exposure to spermine diacridine. The experiment is i d e n t i c a l t o t h a t d e s c r i b e d in 5a, e x c e p t that s p e r m / d i n e o r s p e r m i n e ( 2 5 / m i ) w e r e a d d e d a t v a r i o u s t i m e s a f t e r e x p o s u r e t o s p e r m L n e d i a c r i d i n e (3 h 1 X 5 1 0 - 6 M). C o n t r o l (e e), spermine diacridine, n o p o l y a m i n e s (A A); s p e r m i n e d i a e r i d i n e , p o l y a m i n e s a d d e d a f t e r 1 h (o o), 2 4 h (1 1), 7 2 h (3 3), 1 2 0 h ( 5 5), a n d 1 6 8 h (7 7). T h e e f f e c t s o f s p e r m i n e a n d s p e r m i d i n e a r e i n d i s t i n g u i s h a b l e . S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h L - 1 2 1 0 cells.

298 5. Is there a tissue factor which permits the spermine diacridine inhibited cells to resume growth? We sought for a tissue factor which may reverse the growth inhibition of P-388 and L-1210 cells imposed by the spermine diacridine. Extensive fractionation of the mouse exudate indicated that in all probability basic compound(s) were involved. Rather than pursue the investigation we used some of the k n o w n naturally occurring basic compounds, spermidine and spermine. As can be seen from Fig. 5a, P-388 cells whose growth has been inhibited by prior exposure to spermine diacridine will grow normally when they are subsequently exposed to spermidine or spermine. In order to establish whether leukemia cells inhibited over long periods of time will revert to normal doubling times with spermine, P-388 cells were exposed to spermine diacridine under standard conditions, resuspended in normal growth medium and were then treated with spermine at different times following their exposure to spermine diacridine. Fig. 5 shows that even as late as seven days after exposure to spermine diacridine, when the cells are well into the stationary portion of the growth curve, they resume normal growth rates immediately after addition of spermine. Consequently, we seem to be dealing with cells which have been inhibited in some critical process related to cell replication, whose inhibition spermine will reverse. Discussion The primary site of the action of these diacridine intercalators appears to be the inhibition of RNA synthesis as in the case of 9-aminoacridine. However, t h e y are more effective inhibitors of cell growth than the parent compound; this effect correlates with their better intercalative ability with DNA. We have presented results for a limited number of diacridines and for HeLa and P-388 cells only. Similar results were obtained for the other diacridines mentioned [1] and for L-1210 cells. In due time, a detailed comparative study of the effect of various diacridines so far synthesized on a variety of cell lines will be presented. We have used P-388 and L-1210 cells grown in the presence of horse serum which does n o t contain diaminooxidase [2] and therefore does n o t produce dialdehydes from added spermine or spermidine; such dialdehydes if formed are inhibitors of cell growth [4]. HeLa cells, however, grow in the presence of fetal calf serum and are n o t amenable to reversal of growth by the addition of spermine over long periods of time because the serum of ruminant origin contains high levels of diaminooxidase [5] ; the consequent formation of dialdehydes is known to be inhibitory to protein synthesis [4]. It is difficult at present to provide a complete explanation for the prolonged inhibition of cell growth following limited exposure to spermine diacridine One possibility is that the numbers of new cells and the numbers of dying cells reach a dynamic equilibrium resulting in no increase in cell number. Another is that the cells are inhibited at some crucial synthetic state and abort part of the newly synthesized DNA. Such a p h e n o m e n o n has been defined for phytohemagglutinin and antigen stimulated lymphocytes [6,7] as well as for frog auricles [8].

299

The relief of inhibition of growth by the polyamines may be due to the removal of endogenous bound diacridines. These effects can be better substantiated when radioactive diacridines become available.

Acknowledgments This research was supported in part by Contract N01-CM-12339 from the Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Department of Health, Education and Welfare and in part by a USPHS Research Career Award to E.S. Canellakis, No. 5 KO6GM 03070.

References 1 Canellakis, E.S., Shaw, Y.H., Hanners, W.E. and Schwartz, R.A. (1976) Biochim. Biophys. Acta 418, 300---314 2 Cohen, S.S. (1971) in I n t r o d u c t i o n to P o l y a m i n e s , Prentice-Hall 3 Chu, M.Y. and Fischer, G.A. (1968) Biochem. Pharmacol. 17, 753 4 Alaveon, R.A. (1970) Aeta Biochim. Biophys. 1 3 7 , 3 6 5 5 Halevy, S., Fuchs, Z. and Mager, J. (1962) Bull. Res. Coun. Israel, Sec. A. II, 52 6 Rogers, J.C., Boldt, D., Kornfeld, S., Skinner, A. and Valeri, C.R. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1685 7 0 l s e n , I. and Harris, G. (1974) I m m u n o l o g y 27, 973 8 Stroun, M. and Anker, P. (1972) Biochimie 54, 1443