Resistance and cross-resistance to chromosome damage in human blood lymphocytes adapted to bleomycin

Mutation Research, 211 (1989) 1-5

1

Elsevier MTR 04707

Resistance and cross-resistance to chromosome damage in human blood lymphoeytes adapted to bleomycin V i j a y a l a x m i a n d W. B u r k a r t Radiation Biology Unit, Paul Scherrer Institute, CH-53173 Wurenlingen (Switzerland)

(Received3 June 1988) (Revision received25 August 1988) (Accepted26 August 1988)

Keywords: Adaptiveresponse; Chromosomedamage, resistance and cross-resistance; Human blood lymphocytes;Bleomycin

Summary Human peripheral blood lymphocytes cultured in the presence of low concentrations of bleomycin (BLM), 0.01-0.1 pg/ml, for 48 h and then treated with a high concentration (1.5 # g / m l ) of the same agent or with 1.5 Gy X-rays, became significantly less sensitive to the induction of chromosomal damage than those which did not receive the pre-treatment with BLM. They responded with lower frequencies of chromatid and isochromatid breaks. These results lend further support to the operation of an adaptive repair system in lymphocytes which offers resistance and cross-resistance to the induction of chromosomal damage by the same or similar DNA-damaging agents.

An extensive search for an inducible DNA repair process, the 'adaptive repair', analogous to that found in Escherichia coli (Samson and Cairns, 1977) is now underway in human peripheral blood lymphocytes. Olivieri et al. (1984) first reported that human blood lymphocytes pre-exposed to low levels of radioactive thymidine ([3H]dThd) became less susceptible to chromatid aberrations induced by a subsequent high dose of X-rays. This observation was interpreted as the result of induction of a chromosomal repair mechanism; and subsequently it was shown that this repair process was inhibited by 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase (Wiencke et al., 1986). Shadley and Wolff (1987) also observed

Correspondence: Dr. Vijayalaxmi, Radiation Biology Unit, Paul ScherrerInstitute, CH-5303Wurenlingen(Switzerland).

that an X-ray dose, as low as 0.01 Gy, can protect the lymphocytes against the chromosomal damage induced by a subsequent exposure to 1.5 Gy Xrays. A 4-h interval between the 2 doses of X-rays was found to be sufficient for the expression of the adaptive response, while shorter intervals were not. Furthermore, once induced by 0.01 Gy X-rays, the adaptive response was shown to persist for at least 3 cell cycles (Shadley et al., 1987). The dose rate and the quality of radiation seemed to have an effect on the induction of adaptation response (Wiencke et al., 1987). More recently, Sankaranarayanan et al. (1989) have confirmed the results of Olivieri et al. (1984); in their studies, lymphocytes which were chronically exposed to a low concentration of [3H]dThd or other radioisotopes responded with lower frequencies of chromatid and isochromatid breaks, when subsequently irradiated with 1.5 Gy X-rays.

0027-5107/89/$03.50 © 1989 ElsevierSciencePubfishersB.V.(BiomedicalDivision)

Bleomycin (BLM) is an antitumor antibiotic having a broad spectrum of activity against a wide variety of human malignancies (Bonadonna et al., 1972). It also shares a number of properties of ionizing radiation in terms of its effects on DNA and chromosomes (Tamura et al., 1974; Dresp et al., 1978). In view of this similarity and the above-mentioned investigations, the present study was conducted to examine whether such an adaptive response can be induced by low concentrations of BLM in human blood lymphocytes and whether such cells can be protected against chromosomal damage by subsequent exposure to a high dose of X-rays.

jected to statistical analysis. In order to obtain information on cell kinetics, 500 consecutive metaphases were examined from each culture for the incidence of first (M1), second (M2) and third or later (M3) mitoses. X-Irradiation was performed using a Philips X-ray machine, model MCN 321 under the conditions of 240 kV, 7 mA and 1 mm aluminium filter. The cultures were protected from light during irradiation and kept at a distance of 50 cm from the radiation source. The dose rate of 1.5 G y / m i n was checked using a Farmer dosemeter 2570.

Materials and methods

The results are presented in Table 1. The overall response of the lymphocytes from the 2 blood samples studied is similar although the absolute values are slightly different. As can be seen, the frequencies of chromatid and isochromatid breaks in lymphocytes adapted to increasing concentrations of BLM increase in a dose-dependent manner and the frequency at 0.01 # g / m l of BLM is not significantly different from the controls. The challenge dose of 1.5 # g / m l BLM alone, given 6 h before fixing the cells, induces 63-70% breaks. When the lymphocytes adapted to BLM are subsequently treated with the challenge dose of BLM, the yield of chromatid and isochromatid breaks is lower than the sum of the numbers of breaks induced by the adaptive and challenge doses given separately. The reduction observed with the adaptive doses of 0.01 and 0.05/~g/rnl BLM is 54-61% while that with 0.1 # g / m l dose is much less, 16-23%. The challenge dose of 1.5 G y X-rays alone, given to the lymphocytes 6 h before fixation, induces 30-35% breaks. When the ceils adapted to BLM are subsequently treated with 1.5 G y X-rays, the yield of chromatid and isochromatid breaks is lower than the sum of the effects induced by the 2 treatments separately. As in the earlier case, the effect observed with the adaptive doses of 0.01 and 0.05 # g / m l BLM is more (52-67% reduction) while that with 0.1 # g / m l dose is not as high (28-32%). The results on cell cycle kinetics are given in Table 2. The proportion of lymphocytes in their first division is slightly elevated in cultures treated

Heparinized blood samples were collected from 2 healthy, non-smoking females, aged between 30 and 50 years. From each sample, separate cultures were set up using 1 ml blood in 10 ml RPMI 1640 medium containing 16% fetal calf serum, 1% PHA (Gibco), 1% glutamine, 10 U / m l penicillin, 10 # g / m l streptomycin and 25 ttM bromodeoxyuridine. They were kept in the dark at 37 ° C in a 5% carbon dioxide incubator. 4 h later, the adaptive doses of BLM (Lundbeck, Denmark, freshly prepared in sterile distilled water) were added to give final concentrations of 0.01, 0.05 and 0.1 #g/ml and the cultures returned to the incubator. At 48 h, the cells were exposed to a challenge dose of 1.5 /gg/ml BLM or 1.5 G y X-rays. The incubation continued for a further period of 6 h. For the last 2 h, the cells were also treated with 1 p g / m l colcemid (Gibco). Lymphocytes were collected, treated with 75 m M potassium chloride for 7 min and then fixed in 3 : 1 methanol : acetic acid mixture. Fixed cells were dropped onto clean slides, air-dried and stained with the standard fluorescence-plus-Giemsa technique. Coded slides were analyzed for chromosome damage. From each culture, 200 cells in first division, as defined by the absence of harlequin staining, were examined. Gaps and achromatic lesions less than the width of a chromatid were not included in the scoring. Very few chromatid exchanges were observed and recorded but not included in the analysis. Dicentric chromosomes were not found. The data on chromatid and isochromatid breaks were sub-

Results and discussion

TABLE 1 THE EFFECT OF ADAPTIVE TREATMENT WITH VARYING CONCENTRATIONS OF BLEOMYCIN (BLM) ON THE FREQUENCY OF CHROMOSOMAL ABERRATIONS INDUCED BY SUBSEQUENT CHALLENGE WITH EITHER 1.5 #g/ml BLM OR 1.5 Gy X-RAYS, IN HUMAN BLOOD LYMPHOCYTES Adaptive treatment (BLM, #g/ml)

Challenge treatment

Chromatid and isochromatid breaks in 200 cells

Expected

Percent decrease

144 153 171 73 82 100

58 54 23 59 52 32

128 135 153 61 68 86

61 58 16 67 53 28

Chromatid exchanges

Blood sample 1 ~

~

0.01 0.05 0.10 0.01 0.05 0.10 0.01 0.05 0.10

BLM X-rays -

BLM BLM BLM X-rays X-rays X-rays

4

141 70 7 16 34 60 * * * 71 * * * 132 * * * 30 * * * 39 * * * 68 * * *

Blood sample 2: --

--

0.01 0.05 0.10 0.01 0.05 0.10 0.01 0.05 0.10

BLM X-rays -

-

BLM BLM BLM X-rays X-rays X-rays

3

126 59 5

12 30 50 * * * 57 * * * 128 * 20 * * * 32 * * * 62 *

The treatment schedule is described in Materials and methods. Expected values are the sum of 2 individual treatments minus the control. *

p <0.01;

***

p<0.001.

w i t h the a d a p t i v e (alone), c h a ll e n g e (alone) doses of B L M a n d X - r a y s (alone), as c o m p a r e d to c o n trois, suggesting s o m e d e la y in the cell cycle p r o gression. H o w e v e r , the a d d i t i o n a l c h a l le n g e treatm e n t with B L M a n d X-rays does n o t cause f u r t h e r slow i ng of cell cycle in a d a p t e d cells, i n d i c a t i n g that the r e d u c t i o n in c h r o m o s o m e b r e a k s o b s e r v e d in a d a p t e d l y m p h o c y t e s is n o t a spurious conseq u e n c e of d i f f e r e n t i a l cell d e v e l o p m e n t rates. T h e r e are s o m e rep o r ts in the literature w h e r e the a d a p t a t i o n response was n o t o b s e r v e d in cult u r e d m a m m a l i a n cells ( F o x et al., 1982; K a r r a n et al., 1982). H o w e v e r , the available d a t a i n d i c a t e

that the doses of m u t a g e n s used for a d a p t a t i o n a n d ch al l en g e t r e a t m e n t s as well as the time interval b e t w e e n the 2 t r e a t m e n t s should be w i t h i n a p a r t i c u l a r r a n g e in o r d e r to o b s e r v e a p r o n o u n c e d a d a p t i v e r e s p o n s e ( S a m s o n a n d Schwartz, 1983; S h ad l ey a n d Wolff, 1987; Sh ad l ey et al., 1987). This is true o f the p r e s e n t st u d y as well: the a d a p t i v e doses o f 0.01 a n d 0.05 / ~ g / m l B L M elicited a m o r e p r o n o u n c e d a d a p t i v e r e s p o n s e t h a n the 0.1 # g / m l dose. It is i n t er est i n g to n o t e t h a t the l y m p h o c y t e s a d a p t e d to l o w c o n c e n t r a t i o n s o f B L M b e c a m e resistant to the c h r o m o s o m e d a m a g e i n d u c e d b y

TABLE 2 P R O P O R T I O N a O F H U M A N BLOOD L Y M P H O C Y T E S I N F I R S T (M1), S E C O N D (M2) A N D T H I R D OR L A T E R (M3) DIVISIONS IN C U L T U R E AT 5z; h A F T E R TREATM E N T W I T H B L E O M Y C I N (BLM) A N D X-RAYS Challenge treatment

% ceils in M1

M2

M3

Blood sample 1 0.01 0.05 0.10 0.01 0.05 0.10 0.01 0.05 0.10

BLM X-rays BLM BLM BLM X-rays X-rays X-rays

62 82 72 72 74 82 79 80 88 74 69 80

36 16 27 26 25 17 21 19 12 25 30 20

2 2 1 2 l 1 0 1 0 1 1 0

Blood sample 2 0.01 0.05 0.10 0.01 0.05 0.10 0.01 0.05 0.10

BLM X-rays BLM BLM BLM X-rays X-rays X-rays

58 80 67 79 76 79 82 79 83 78 79 75

39 18 31 19 22 21 18 21 16 21 21 22

3 2 2 2 2 0 0 0 1 1 0 3

Adaptive treatment (BLM, # g / m l )

a 500 consecutive metaphases were examined from each culture. The treatment schedule is described in Materials and methods. For challenge treatment 1.5 # g / m l BLM or 1.5 Gy X-rays was used.

the challenge dose of not only BLM but also X-rays. While this manuscript was under preparation, Wolff et al. (1988) reported that human blood lymphocytes adapted to low doses of [3H]dThd or X-rays showed a 40-65% decrease in the frequency of chromatid and isochromatid deletions induced by a challenge dose of 2.5 # g / m l BLM. These observations, taken together, are interesting since all the 3 mutagens induce a similar kind of lesions - - strand breaks in DNA and the lymphocytes adapted to one mutagen became resistant to the challenge dose of the other two. This resistance and cross-resistance may be due to the

induction of the same or similar mechanism(s) involved in the repair of DNA strand breaks. Similar results have also been reported in cultured mammalian cells adapted to low doses of alkylating mutagens: cross-resistance was observed to be induced by challenge doses of other alkylating mutagens, but not by non-alkylating agents (Samson and Schwartz, 1980; Kaina, 1983). The mechanism(s) by which the adaptation is brought about has been the subject for several investigations by other authors. In the case of alkylating mutagens, pretreatment of the cells with low, non-toxic doses seems to induce repair enzymes such as DNA glycosylase and methyltransferase, making the cells more efficient to deal with the alkylated lesions thereby, rendering resistance to the mutagenic and lethal effects of a subsequent challenge dose (Evensen and Seeberg, 1982; Waldstein et al., 1982). The activity of the repair enzyme, poly(ADP-ribose) polymerase (Skidmore et al., 1979, Durkacz et al., 1980) has been indirectly implicated in the lymphocytes adapted to [3H]dThd and X-rays (Wiencke et al., 1986; Shadley and Wolff, 1987). The latter observations are important in the context of the present study since the action of BLM, [3H]dThd and X-rays on cellular DNA is similar. Whether or not such a mechanism is involved in the adaptation of lymphocytes to BLM is yet to be determined. There are data which suggest that BLM is inactivated by an inactivating enzyme, BLM-hydrolase (aminopeptidase B group), which hydrolyzes the carboxyl amide group of fl-aminoalanine moiety in the BLM molecule (Umezawa, 1973). In clinical studies, BLM is more effective in the treatment of certain types of cancer and not others and this has been attributed to a differential distribution of BLM-hydrolase enzyme among the tissues (Ichikawa, 1969; Clinical Screening Cooperative Group of the EORTC, 1970). Yoshioka et al. (1978) examined 3 transplantable tumor cell lines and found that the BLM-hydrolase activity was lowest in BLM-sensitive AH 66 and higher in AH 66F and L1210, which were resistant. They suggested that the sensitivity difference in these 3 tumor cells is due to BLM-hydrolase. Whether the adaptive dose of BLM given to the lymphocytes stimulates the activity of BLM-hydrolase, which could at least partially counteract the effect of the

challenge dose is not known. The role(s) of DNA repair enzyme(s) and BLM-hydrolase in the resistance and cross-resistance observed in the human blood lymphocytes merit further investigation. BLM has been widely used as a chemotherapeutic drug for a variety of human cancers. If the treatment is pursued for long periods and depending on the doses employed, the adaptive response of the cells and tissues involved may not be excluded. This might modify the efficiency of the treatment.

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