Synergistic effect of CCNU and bleomycin on human lymphocytes exposed at late G1 and G2 states of the cell cycle

Mutation Research, 260 (1991) 265-269 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100105D

265

MUTGEN 01670

Synergistic effect of C C N U and bleomycin on human lymphocytes exposed at late G 1 and a 2 s t a t e s of the cell cycle Radosveta P. Koldamova 1 and Ilia M. Lefterov 2 I Department of Chemistry and Biochemistry and 2 Center for Genetic Counselling, Higher Medical Institute, 6003 Stara Zagora (Bulgaria)

(Received 29 November 1990) (Accepted 22 January 1991)

Kevwords: Chloroethylnitrosourea; Bleomycin; Human tymphocytes; Synergistic effect; Chromosome aberrations

Summary The combined action of the antitumor antibiotic bleomycin and chloroethylnitrosourea (CCNU) was studied in human lymphocytes in vitro. All the experiments were carried out with 20 ~ g / m l bleomycin for a given treatment time. By adding 0.7 and 3.5 # g / m l CCNU at late G1-S phase we have demonstrated a considerable increase in both percent of aberrant cells and production of dicentrics and rings (5-fold, p < 0.001). At late S-G 2 the combined treatment led to a significant enhancement of breaks per cell ( p < 0.0001) and cells with more than 12 aberrations. A possible explanation could be the known repair-inhibitory potential of CCNU, but its pure clastogenic action still has to be considered. The results presented here point out the need for seeking chemotherapeutic regimens with reduced concentrations of the drugs in combination.

DNA-repair inhibitors are of considerable importance in anticarcinogenesis (i.e., cancer prevention) and may improve cancer therapy as well (Boothman et al., 1988). Drugs which inhibit repair greatly increase the lethality of primary antineoplastic agent to cells. Chemotherapy would be improved only if the combined drugs increase the therapeutic index relative to the primary drug. The concentration of the drugs in the combination could be reduced to keep within the acceptable toxicity range. Correspondence: Dr. Radosveta Koldamova, M.D., Section of Biochemistry, Department of Chemistry and Biochemistry, Higher Medical Institute, 11 Armeiska Street, 6003 Stara Zagora (Bulgaria).

The chloroethylnitrosoureas are compounds with clinically useful antitumor activity. They decompose under physiological conditions to yield an alkylating moiety capable of interacting with nucleic acids and proteins and a carbamoylating moiety which interacts with proteins (Erickson et al., 1978; Sariban et al., 1987). Alkylation has been accepted as the principal mechanism of the antitumor activity. The carbamoylation is the basis for many of the biochemical actions that have been observed with nitrosoureas including inhibition of macromolecular synthesis (Kann et al., 1974), inhibition of RNA processing (Kann et al., 1988) and inhibition of DNA repair (Kann, 1981). C C N U (1-(2-chloroethyl)-3-cyclohexylnitrosourea) has a broad spectrum of antitumor activ-

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ity and is clinically employed for the treatment of various malignancies. It inhibits the repair of D N A damaged by ionizing radiation as estimated by the frequency of strand breaks in cells exposed to radiation (Erickson et al., 1978). C C N U acts as a radiation synergist, enhancing the cytotoxic effects of ionizing radiation by reducing the mean lethal dose for cells in culture (Kann, 1981). The effect of C C N U on human chromosomes was studied in vitro (Best and McKenzie, 1988a) and in vivo (Vyas et al., 1988). An increased spontaneous basal level of SCE in vitro (Best and McKenzie, 1988b) and in vivo (Vyas et al., 1988) and also increased chromosome aberrations after exposure in vivo (Vyas et al., 1988) have been reported. Bleomycin (BLM) is an antitumor drug that has been shown to induce chromosome aberrations similar to those induced by X-irradiation (Chatterjee and Raman, 1988). No data are available on the effect of C C N U on the clastogenic action of radiation and BLM. Therefore in the present study, we have tested C C N U as a synergist on chromosome damage produced by BLM in human lymphocytes in the late G~ and G~ stages of the cell cycle. The result of this investigation shows that C C N U significantly enhances the frequency of BLM-induced chromosome aberrations over the additive effect of both agents. Materials and methods Chernicals

Bleomycin (Bleocin, Nippon, Japan) was prepared at a concentration of 2 m g / m l in sterile distilled water and stored at 4 ° C for 2 weeks. 50 /~1 stock solution was added to 5 ml culture. C C N U (synthesized in this department and kindly donated by Simona Popova) was dissolved in ethanol to prepare a stock solution of 0.35 m g / m l immediately prior to use. Ethanol (Merck, F.R.G.) was added to control cultures. All cell-culture reagents except phytohemagglutinin (PHA) were from Sigma (U.S.A.). The PHA routinely used in this cytogenetic laboratory was from the Institute of Infectious and Parasitic Diseases (Sofia, Bulgaria).

Cell cultures

Venous blood from 2 healthy non-smoking donors (female 20 years, male 30 years) was used. 0.5 ml of whole blood was added to 4.5 ml of R P M I 1640 medium containing 20% newborn calf serum, 2 m M L-glutamine, 50 /xg/ml gentamycin and 2% P H A and incubated at 37 ° C. Late G 1 treatment was carried out 18 h after PHA stimulation since initiation of S phase has been observed at 24 h in human lymphocytes (Preston et al., 1987). The cultures were exposed to BLM and C C N U simultaneously for 4 I1, which is long enough to encompass more than 2 decomposition half-times for C C N U (Sariban et al., 1984). At this treatment point the cultures were treated with 2 doses of C C N U , 0.7/xg/ml and 3.5 /~g/ml. All experiments with higher concentrations led to considerable suppression of the mitotic index. BLM and C C N U were removed by washing the cells twice in R P M I 1640 and the cultures were resuspended in fresh complete medium containing 22 /xM bromodeoxyuridine and subsequently harvested at 72 h. G~ treatment was carried out 67 h after stimulation. The cultures were exposed to BLM 20 /xg/ml and C C N U 3.5 and 7/~g/ml. For the last hour the cells were also treated with 1 /xg/ml colchicine. A 0.075 mM KCI solution was used as a hypotonic for 20 rain and the cell mass was fixed in 3 : 1 methanol: acetic acid. Slides from G~ were stained using a modified FPG method (Perry and Wolf, 1974). Coded slides were examined for chromosome damage. Two hundred metaphases were analyzed from each culture for chromosome aberrations. For G~ treatments scoring of chromosome aberrations was done strictly from first-cycle metaphases. They were scored as chromatid breaks, chromatid exchanges, deletions (terminal and interstitial), dicentrics and rings. For G~ treatments chromatid and isochromatid breaks were scored and rare chromosome exchanges were recorded, but not included in the analysis. Cells with more than 12 aberrations were recorded separately. An analysis of the number of breaks per metaphase was carried out counting 2 breaks for chromatid and chromosome exchanges and 1 break for chromatid breaks and deletions (for G 1 ). The data on the incidence of chromosome aberrations

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(observed versus expected) were subjected to statistical analysis using a 1-tailed t-test. The expected value (additive effect) was the sum of the 2 individual treatments minus control. The incidence of aberrant metaphases was evaluated by X 2.

Results and discussion The results of experiments in late G 1 are summarized in Table 1. In cells exposed to BLM alone

the frequency of chromatid breaks and deletions is higher than the frequency of exchanges (chromosome and chromatid). In the presence of C C N U alone there was a dose-dependent increase over the controls of the frequencies of aberrant metaphases, chromatid breaks and chromatid exchanges. The chromosome aberrations (deletions and exchanges) were not increased with increasing the dose. C C N U 0 . 7 / ~ g / m l induces an enhancement of

TABLE 1 EFFECT OF CCNU AND BLM ON CHROMOSOME GI-S STAGE Treatment

Control BLM CCNU CCNU BLM+CCNU BLM+CCNU

ABERRATIONS

INDUCED

Concentration

Cells

(/Lg/ml)

scored a a b e r r a n t cells

chromatid breaks

chromatid exchanges

400 350 200 410 200 405

0.025 0.35 0.13 0.41 0.42 1.10

0.036_+0.005 0.095_+0.01 0.04 _+0.01 0.26 -+0.02 * *

20 0.7 3.5 20+0.7 20+3.5

IN HUMAN

LYMPHOCYTES

deletions

chromosome exchanges

0.16_+0.02 0.03_+0.01 0.03_+0.01 0.25-+0.03 0.31_+0.05 *

0.056_+0.01 0.01 +0.001 0.007_+0.001 0.14 -+0.015 * * * 0.30 _+0.045 * * *

Percent of A b e r r a t i o n s per cell_+SE

2.5 24.5 14.0 33.7 51.5 +++ 65.2 ++

+ 0.003 +0.05 _+0.03 _+0.04 _+0.05 -+0.07 * * *

IN LATE

Breaks per cell

0.02 0.70 0.18 0.64 1.03 2.41

a Pooled d a t a of 2 i n d e p e n d e n t experiments. * p < 0.02, * * p < 0.01, * * * p < 0.001, 1-tailed t-test. +÷ p < 0.01, +++ p < 0.001, X 2.

TABLE 2 EFFECT OF CCNU AND BLM ON CHROMOSOME Treatment

DAMAGE IN HUMAN

Concentration (~tg/ml)

Cells scored

Percent of aberrant cells

N u m b e r of chromatid and isochromatid breaks

20 3.5 7.0 20+3.5 20+7.0

200 200 200 200 200 200

3.5 27.5 15.0 27.5 86.6 +++ 89.8 +++

7 138 35 78 550 778

20 3.5 7.0 20+3.5 20+7.0

200 200 200 258 200 200

2.0 25.2 26.0 45.0 73.1 +++ 98.6 ÷++

4 86 52 233 452 1380

LYMPHOCYTES

Breaks per cell + SE

N u m b e r of chromatid exchanges

AT G 2 STAGE N u m b e r of chromosome exchanges

Percent of cells w i t h more t h a n 12 aberrations

2 2

6.33 12.00

Sample 1 Control BLM CCNU

CCNU BLM+CCNU BLM+CCNU

0.035 0.69 0.17 0.39 2.75 3.89

+ 0.01 _+0.04 +0.02 +0.05 +0.29"*** +0.15 ****

0.02 0.43 0.26 0.90 2.26 6.94

+ 0.01 + 0.06 _+0.03 ±0.06 +0.14"*** _+0.24"***

1

2 7

Sample II Control BLM CCNU CCNU BLM+CCNU BLM+CCNU

+++ p < 0.001, X 2 * * p < 0.0001 (1-tailed t-test).

* *

0.6 1 3 1

1

1 1

4.90 45.50

268 the frequency of aberrant metaphases ( p < 0.001) and chromosome exchanges ( p < 0.001) when it is added simultaneously with BLM. The frequencies of chromatid breaks, chromatid exchanges and deletions are not increased over the additive effect. C C N U 3.5/~g/ml induces significant increases in the frequency of aberrant metaphases ( p < 0.01), chromatid breaks ( p < 0.001), chromatid exchanges ( p < 0 . 0 1 ) , deletions ( p < 0 . 0 2 ) and chromosome exchanges ( p < 0.001) over the additive effect. The frequency increases by a factor of,approximately 2 for chromatid exchanges and deletions and approximately 5 for dicentrics and rings. The results of the experiments in the G 2 phase of the cell cycle are presented in Table 2. The overall response of the lymphocytes from the 2 blood samples studied is similar, although the absolute values are different. C C N U alone increases the frequencies of aberrant metaphases and chromosome breaks over the control values. When C C N U (3.5/xg/ml) is added with BLM (20 ~tg/ml) the enhancement in the incidence of chromosome damage is more than 3-fold over the expected values ( p < 0.0001) for both cultures. The enhancement from C C N U is greater at a dose of 7 . 0 / t g / m l . The effect of the combination C C N U - B L M (7.0 and 20 /~g/ml, respectively) is 3.9-fold and 6.9-fold greater than the sum of the individual treatments (blood samples I and II, respectively, p < 0.0001). The percentage of cells with > 12 aberrations is significantly elevated.

Table 3 shows the distribution of chromosome breaks per metaphase at the G 2 stage for BLM and C C N U treatments alone and in combination. The analysis shows clearly that the combination B L M - C C N U leads to an increase in the number of metaphases with more than 3 breaks per cell. The frequency of aberrant metaphases is increased 1.7-fold. Chloroethylnitrosoureas contain 2 separate structure groups - alkylating and carbamoylating. Alkylation at guanine 0 6 can form interstrand crosslinks (Kohn, 1977) and alkylation at other sites can cause monoadducts. Alkylation at various D N A sites may be repaired by nucleotide excision repair which proceeds through the transient formation of single-strand breaks (SSB). Carbamoylation produces a variety of biochemical effects including inhibition of a strand-rejoining step in DNA-excision repair (Fornace et al., 1978), as well as a rejoining of X-ray-induced breaks (Kann et al., 1980). According to Kann et al. (1981) repair-inhibiting nitrosoureas are a new class of radiation synergists, whose mechanism of action apparently differs from that of other previously studied radiation synergists. Bleomycin is a radiomimetic antibiotic and the aberrations produced by BLM (40 ~tg/ml) and X-rays (2 Gy) are quantitatively and qualitatively similar (Chatterjee and Raman, 1988). The results presented in this study show that C C N U and BLM have synergistic action on the induction of chromosome aberrations in human

TABLE 3 POOLED DATA FROM SAMPLES I AND II ON THE DISTRIBUTION OF CHROMOSOME BREAKS PER CELL INDUCED BY BLM AND CCNU AT THE G2 STAGE IN HUMAN LYMPHOCYTES Treatment (~g/ml)

Percent of aberrant metaphases

Percentage of metaphases with the aberration distribution 1/M

2/M

3/M

4/M

Control BLM (20) CCNU (3.5) CCNU (7.0) BLM (20)+CCNU (3.5) BLM (20)+ CNU (7.0)

2.7 26.3 20.5 26.5 79.5 94.0

100 63.1 79.7 38.9 26.6 11.3

23.1 16.5 21.6 19.8 9.5

11.6 3.0 7.2 17.1 12.4

4.2 2.5 9.5 12.8

5/M

> 5/M 7.4

0.8 9.0 8.0

0.8 17.9 46.0

269 l y m p h o c y t e s . T h e s y n e r g i s t i c e f f e c t is e v i d e n t in b o t h G 1 a n d G 2 stages, h o w e v e r G2 s h o w e d m o r e e n h a n c e m e n t o f b r e a k s p e r cell. T h i s m a y b e bec a u s e of t h e l o n g e r t i m e for r e p a i r in l a t e Gx c o m p a r e d to G 2. The CCNU-mediated p o t e n t i a t i o n was m o r e e f f e c t i v e in t h e p r o d u c t i o n o f d i c e n t r i c s a n d rings (5-fold) t h a n c h r o m a t i d b r e a k s , c h r o m a t i d exc h a n g e s a n d d e l e t i o n s in G 1. T h i s is o f i n t e r e s t , b e c a u s e of t h e l o w p e r c e n t o f d i c e n t r i c s a n d rings p r o d u c e d b y C C N U in this s t a g e (0.7%). T h e s y n e r g i s t i c a c t i o n of C C N U a n d B L M in t h e p r o c e s s of c h r o m o s o m e a b e r r a t i o n i n d u c t i o n r e p o r t e d h e r e m i g h t b e d u e to i n h i b i t i o n o f D N A r e p a i r r a t h e r t h a n to the c l a s t o g e n i c i t y o f C C N U .

Acknowledgements W e w i s h to t h a n k D r . D o n k a B e n o v a f o r e n c o u r a g e m e n t d u r i n g the c o u r s e o f this s t u d y a n d D a n i e l a I v a n o v a f o r t e c h n i c a l s u p p o r t in p r e p a r ing the manuscript.

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