Effects of arabinofuranosylcytosine on the DNA synthesis of mammalian cells treated with bleomycin

Europ. J. CancerVol. 9, pp. 25-29. Pergamon Press 1973. Printed in Great Britain

Effects of Arabinofuranosylcytosine on the DNA Synthesis of Mammalian Cells Treated with bleomycin VAGN BREMERSKOV* Department of Pathology, Finseninstitutet, Kobenhavn, Denmark A b s t r a c t - - T h e antitumor peptide-mixture bleomycin inhibits the DNA synthesis of L-929 fibroblasts in vitro. The inhibition results in a parasynchronization of the cells in G1- early S-phase. Following releasefrom bleomycin more successive waves of D NA synthesis are observed, as the cells proceed into S-phase. By treating the released cells Jbr 1 hr with ARA-C (0.05 myg/ml) a considerable reduction of the DNA synthesis of the released cells can be obtained. The combination of bleomycin treatment with short pulses of ARA-C at low concentrations is proposedfor the clinical management of bleomycin sensitive human tumors.

INTRODUCTION

L-CELLS treated with bleomycin, will, on release, exhibit synchronous waves of DNA-synthesis [1]. Also microfluorometric measurements of the nuclear Feulgen-DNA content shows a gradual normalization of the distribution of cells within the S-phase [1]. The mitotic index increases within 4 hours after release from bleomycin from 2-1 to 3.6 (normal: 4.5-5.0) and the number of clonogenic ceils, in soft agar, increases from 30% under bleomycin treatment to 70% upon release [2]. These observations m a y constitute major draw-backs to an effective chemotherapy with bleomycin. We have tried to take advantage from the said parasynchronization of the nuclear DNA-synthesis [1] by successive application of bleomycin and the S-phase "specific" drug arabinosylcytosine [3]. As will be shown, cells treated with bleomycin are highly susceptible to ARA-C.

preparation in sterile ampules from Fa. H. Lundbeck, Copenhagen. The mixture which is readily soluble in water and aqueous solutions, was dissolved in sterile serum-free medium to yield the proper concentration immediately before use. In most experiments bleomycin mixture was added to the cultures (20 ml) in a final volume of 0.5 ml serum-free medium. ARA-C (1-beta-D-arabinofuranosylcytosine, Cytosar °) was obtained as sterile standard lyophilized preparation from either Merck, Sharpe and Dohme, or from Upjohn. No difference could be found between the two preparations in parallel experiments, using DNA synthesis inhibition as parameter. All radiochemicals used in these investigations were obtained through The Radiochemical Centre, Amersham, U.K. The rest of the chemicals used were analytical grade or of the highest purity available.

MATERIAL

METHODS

Bleomycin mixture (more than 80% A2, less than 4% Bt) was obtained as a lyophilized

Throughout these experiments a line of Lcells (L-929, The American Type Tissue Collection), growing in monolayer cultures in 250 cc plastic bottles (Falcon Plastics Inc., U.S.A.), was used. The cultures were grown in a modified Eagle's M E M (4), supplemented with 4-5% heat inactivated calf serum (Flow

Accepted 8 August 1972. *Supported by grants from Daell Fonden, the Danish Anti-cancer Society and the Danish Medical Research Council. 25

Vagn Bremerskov

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Laboratories, Scotland, U.K.). The cultures were propagated by trypsinization (Trypure N O V O , 0.02% in phosphate buffer pH 7.2). Following propagation, the cultures were left undisturbed for 18-24 hr in a climate room, before being used for experiments. No medium change was performed prior to addition of" cytostatics and/or labelled precursors. TREATMENT

For the initial treatment of tissue-cultures with bleomycin we chose a concentration of 4 myg/ml which will inhibit 35-40% of the DNA synthesis of our cell line [1], when treated for 24 hr. After this period the cultures to receive ARA-C were washed 3 times with 20 ml 37°C, conditioned medium, refed and given ARA-C dissolved in 0.5 ml serum-free medium to the appropriate concentrations. At the end of ARA-C treatment the cultures were washed as above, and 3H-TdR, 1 myC/ml (5 C/mM), added and allowed to incorporate for 30 min at 37°C. The reaction was terminated by washing 3 times with 0.27 N PCA (20 ml, icecold) for 5 min. Then 5 ml 1 N PCA was added and the cells were rubbed off with a rubber policeman. The precipitated cells were hydrolysed for 20 min at 70°C followed by centrifugation for 10 min at approximately 2000 g. From the supernatant aliquods were taken for DNA estimation a.m. Burton [5] and tbr counting of the activity incorporated into acid insoluble material. This was done by mixing 500 myl of the hydrolysate with 15 ml of Bray's scintillation fluid [6] after neutralization with 5 N N a O H (100-150 myl). Counting was done in a Beckman 250S liquid scintillation system. Calculation and conversion of cpm to Pmoles (10 -12 moles) 3H-TdR incorporated per OD595 = 1 per 30 min was pertbrmed on a Wang 700 B programable calculator (Wang Laboratories Ltd., U.S.A.).

to treatment with bleomycin for various periods are given in Fig. 1. From the histogram of the control culture (A) it is seen that the diploid (2n) value corresponds to 60-70 working units (WU), while the tetraploid value (4n) is 120 to 140 WU. Since 2n is the value of cells in G/-phase and 4n is that of G2-phases the intermediary values represent the S-phase cells. After 24 hr treatment with bleomycin the profile has changed considerably, showing a high proportion of the cells accumulated in G t and early S-phase while late S-phases and G2phases are reduced. Histogram C of Fig. 1 shows the situation 45 min after release from bleomycin. At this time a high number of S-phases are present corresponding to the CELL NUMBER

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Fig. 1. la) Relative nuclear-Feulgen-D N A content o[200 acriJtavine slai~wd cells from an untreated co~ltrol culture. (b) Relative nuclear Feulgen-DNA content ¢f 200 acriJlavine stai~ed calls j}'om a culture treated with 4 myg/ml ofbleomycin for 24 hr. (c) Relative nuclear Feulgen-DNA content of 200 cells from a culture 45 rain after release from bleoT~tycin. Ordinates : cell number. Abscissas : D N A content in arbitrary working units (HIU). 2~ : diploid D N A content. .in : letraploid D N A content.

D NA Synthesis after bleomydn and A R A - C parasynchronized population of G 1- and early S-phase cells seen in Fig. l(b). The results will be published in greater detail elsewhere.

D N A synthesis after release from bleomycin U p o n release from bleomycin, 4 myg/ml for 24 hr, equal to an inhibition in the actual experiment of 60% of the D N A synthesis (Fig. 2) the capacity of the released cultures for D N A synthesis was followed by pulse labeling with a H - T d R of the acid insoluble material. Cultures labeled from time: 0 after release, to time 60 min after release, were labeled for 5 min, while later cultures were labeled for 30 min. After counting all activities were corrected to incorporation per 30 min. This was found justified since a preliminary experiment had shown a linear correlation between 3 H - T d R incorporation and time in bleomycin-treated cells for up to 45 min. ~ / / / / / / / / / / / / / / / / V / / / . : ~///U/2",/~y////////~.~.,. .~.

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From Fig. 2 it appears that the release from bleomycin is followed by more successive waves of D N A synthesis, one from 30 to 60 min, the next from 150 to 300 min and a third between 360 and 390 min. The nature of the D N A synthesized in the different periods will be the subject of a subsequent paper. Effect of ARA-C on the D N A synthesis of bleomycin-treated and released cells. In this experiment one set of cultures received bleomycin 4 myg/ml for 24 hr, while another set had 0.5 ml serum-free medium. After 24 hr the medium was changed on all cultures (4 cultures/experimental point) and ARA-C was applied as indicated in Figs. 3 and 4. These figures thus illustrate the combined effects o f a pre-treatment with bleomycin and a post-release treatment with ARA-C, versus the effects of ARA-C alone. It appears that ARA-C is able to abolish the increase in D N A synthesis of released cells which would be expected from the results given in Fig. 2. Furthermore, the parallel decline of the D N A synthesis of pretreated and released cells and that of cells treated with ARA-C alone, indicate that the effects of ARA-C are additive to those of bleomycin. These effects are relatively more marked for smaller concentrations of ARA-C and tbr shorter treatments.

Effect of ARA-C on the D N A synthesis of released cells Figure 5 illustrates the time-course of D N A synthesis in L-cells released from bleomycin but subjected to continued treatment with ARA-C, 0-05 myg/ml from time: 0-60 min after release. The pattern shows some similarity to that of released cells without subsequent ARA-C treatment, although the extent to which D N A is synthesized is considerably smaller. However, even the combination of ARA-C to bleomycin will not abolish the synthesis of some D N A after release from ARA-C. DISCUSSION

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Fig. 2. D N A synthesis of L-ceU cultures after release from bleomycin. Cultures were incubated for 24 hr with 4 myg bleomycin/ml medium. Then the cultures were washed 3 times with conditioned medium and refed with 20 ml of conditioned modified M E M containing 4-5% heat inactivated calf serum. At the indicated times 4 released, 2 untreated and 2 unreleased cultures were labeled with 3H-TdR (1 myC/ml, 5C/mM) as described under methods. The values given for released cultures are the mean of the 4 cultures corresponding to each point. Ordinate: Pmoles (10- az moles) of 3H-TdR incorporated per mmymoles TdR per 30 rain. Abscissa : Time.

During the last few years the present laboratory has been investigating the mechanism of action of bleomycin. As a means of getting insight into the intracellular processes influenced by bleomycin a number of release experiments have been pertbrmed with special reference to D N A synthesis regulation. As shown in Fig. 2 the release from bleomycin is followed by more successive waves of DNA synthesis. This observation may explain some of the difficulties experienced in the clinical

28

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Figs. 3 - 4 . Combined effect of 24 hr treatment with bleomycin (4 myg/ml) and A R A - C for various periods compared with treatment with either bleomycin or A R A - C alone. O - © : Incorporation of 3H- TdR into acid insoluble material of cells treated with A R A C without pretreatment with bleomycin. Z \ - - - - - - Z \ : Incorporation of 3H- TdR into acid insoluble material of cells pretreated with bleomycin (4 myg/ml, 24 hr) followed by A R A - C for various periods. Abscissas: Concentration of A R A - C (logarithmic scale). Ordinates: 1 0 - 12 moles 3H-TdR x 0D595- 1 x time-

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Fig. 5. Effect of A R A - C (0.05 myg/ml, 1 hr) on the D N A synthesis of cells pretreated with bleomycin (4 myg/ml, 24 hr) compared to the D N A synthesis of cells released from bleomycin treatment. 2s------ZX : For details see legend to Fig. 2. x------x: 3H-TdR incorporation into acid insoluble material of cultures treated with bleomycin, released (see methods), treated with A R A - C for 1 hr and released once more. Controls: Control values for D N A synthesis indicated by horizontal lines : at 0-08 :for cells treated with A R A - C only; at 2-0 :for cells treated with bleomycin only; at 4.25 : for untreated controls. Abscissa : time after release from bleomycin. Ordinate: 3H- TdR incorporation ( 10 - 12 moles x mmymoles T d R - 1 x time- ~).

D NA Synthesis after bleomycin and A R A - C treatment of malignant neoplasms with bleomycin. We have tried to take advantage of the observation that bleomycin causes some degree of parasynchronisation of the cell population (Fig. 1) by adding ARA-C, a potent S-phase inhibitor [3], to the cells after release from the block caused by bleomycin. As shown in Fig. 5 this treatment reduces quite considerably the

capacity of the cells for D N A synthesis. With the reservations necessitated in any conclusion from in vitro experiments the present observations seem to suggest that a combination of bleomycin with short pulses of A R A - C in small concentrations may be of use in the clinical therapy.

REFERENCES

1. 2. 3. 4. 5. 6. 7.

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V. BREMERSKOV,DNA synthesis during the life cycle of L-cells: Morphological, quantitative histochemical and biochemical investigations with bleomycin. (In preparation). V. BREMERSKOV,Cytokinetic studies with bleomycin. (In preparation). F . L . GRAHAMand G. F. WHITMORE,The effect of 1-beta-D-arabinofuranosylcytosine on growth, viability, and DNA synthesis of mouse L-cells. Cancer Res. 30, 2627 (1970). C. MITTERMAYER,P. KADENand W. SANDRITTER,Untersuchungen an teilungssynchronen L-Zellen: I. Optimale Wachstumsbedingungen und Charakterisierung des synchronen Zellsystems. Histochemie. 12, 67 (1968). K. BURTON,The relation between the synthesis of deoxyribonucleic acid and the synthesis of protein in the multiplication of bacteriophage T2. Biochem. or. 61, 473 (1955). G.A. BRAY,A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Biochem. 1, 279 (1960). V. BREMERSKOV,P. KADENand C. MITTERMAYER,DNA synthesis during the life cycle of L-cells: Morphological, histochemical and biochemical investigations with arabinosylcytosine and thioarabinosylcytosine. Europ. J. Cancer 6, 379 (1970). N. BOHMand E. SPRENGER,Fluorescence cytophotometry: a valuable method for the quantitative determination of nuclear Feulgen-DNA. Histochemie. 16, 100 (1968). ABBREVIATIONS

G 1: presynthetic phase S: synthetic phase G2: postsynthetic phase MEM: minimum essential medium TdR : thymidine PCA: perchloric acid