Stimulatory effect of bleomycin on the synthesis of acidic glycosaminoglycans in cultured fibroblasts derived from rat carrageenin granuloma

359

Biochimica et Biophysica Acta, 444 (1976) 359--368 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 28020 STIMULATORY E F F E C T OF BLEOMYCIN ON THE SYNTHESIS OF ACIDIC GLYCOSAMINOGLYCANS IN CULTURED FIBROBLASTS DERIVED FROM RAT CARRAGEENIN GRANULOMA

KATSUHIRO OTSUKA ,,1, SEI-ITSU MUROTA 1 and YO MORI 2 I Department of Pharmacology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo-173, and 2Departnient of Biochemistry, Tokyo College of Pharmacy, Hachiouji-shi, Tokyo-192 (Japan)

(Received February 24th, 1976)

Summary Cultured fibroblasts derived from rat carrageenin granuloma were treated with bleomycin and the synthesis of hexosamine-containing substances was compared with that in control cells. Four day treatment with 0.1 pg bleomycin/ml resulted in a significant increase of the production of these macromolecules by the cells, though DNA synthesis was remarkably inhibited at this dose of bleomycin. The stimulatory effect could be seen as early as the second day of bleomycin treatment, and was enhanced with increasing treatment time. Further fractionation of the hexosamine-containing substances revealed that synthesis of acidic glycosaminoglycans was more sensitive to bleomycin than that of glycoproteins, i.e., acidic glycosaminoglycans increased by 80% and glycoproteins by 53% after four day treatment with 0.1 pg bleomycin/ml. The increased components of acidic glycosaminoglycans included n o t only hyaluronic acid but also sulphated glycosaminoglycans. Collagen synthesis was increased by 23% by the same dose of bleomycin. N-Acetyl-~-glucosaminidase, one of the degradation enzymes for acidic glycosaminoglycans released into the cultured medium, was decreased significantly by bleomycin.

Introduction Bleomycin, an antitumor antibiotic isolated from a strain of Streptornyces verticillus [1], has been found to be a most effective agent controlling a number of h u m a n cancers [2], particularly in the treatment of skin tumors. The absence of hematopoietic toxicity and immunosuppresive activity have been * Ph.D. Fellow of the Tokyo College of Pharmacy

360 specially mentioned as advantages of this therapeutic agent [3]. With increasing use, however, it has become apparent that diffuse pulmonary fibrosis has been recognized as a severe and puzzling complication of bleomycin therapy [4,5], and measures controlling the side effects of bleomycin are desired. It is well known that acidic glycosaminoglycans and glycoproteins play an important role on collagen fibrillogenesis and insolubility [6--9]. Several workers have reported that acidic glycosaminoglycans may influence fibrillogenesis and that interactions between acidic glycosaminoglycans and collagen fibers may be of importance in the structural organization of the tissue [6,7,10--13]. Some studies have been reported on the mechanism of fibrosis of collagen caused by bleomycin. Tetsuka et al. have reported that the antibiotic enhanced the activity of protocollagen proline hydroxylase during collagen biosynthesis [14], and Ichihashi et al. have demonstrated the reduction of cutaneous collagenase activity after administration of the antibiotic [15]. However, no report is available on the effects of bleomycin on acidic glycosaminoglycans except our previous preliminary work [16]. In this report, the effect of bleomycin on the hexosamine-containing substances was studied by means of cultured fibroblasts which were known to produce a large a m o u n t of intercellular substances [17,18]. Materials and Methods Culture conditions Cloned fibroblasts, line C4, which were isolated from a rat carrageenin granuloma by Murota et al. and shown to produce large amounts of acidic glyeosaminoglycans and collagen [17,18], were used. The cells were cultured in a Petri dish at 37°C in a 5% CO2 atmosphere with Ham's F-12 medium (Nissui Seiyaku Co., Tokyo) supplemented with 10% fetal bovine serum (Microbiological Associates, Bethesda) and antibiotics (Penicillin, 100 U/ml and Streptomycin, 100 pg/ml). As a rule, medium transfer and subculture were performed every 2--3 days and 7--10 days, respectively, and a mixture of 0.05% EDTA and 0.05% trypsin was used for detachment of the cells. All experiments were started just after the cells reached stationary phase, when the concentration of serum was lowered to 5% so that a constant cell number was maintained, unless otherwise stated. Bleomycin or hydroxyurea treatment A sterilized aqueous solution of bleomycin sulphate (a gift from Nippon Kayaku Co., Tokyo) or h y d r o x y u r e a (Tokyo Kasei Co., Tokyo) was stored at --20°C as stock solution (1 mg/ml). This was diluted with Ham's F-12 medium plus 5% fetal bovine serum just before use. The cells were exposed to various concentrations of bleomycin (0.01--1 pg/ml) or h y d r o x y u r e a (0.01--10/~g/ml) during their stationary phase for four days unless otherwise stated. The bleomycin or hydroxyurea-containing medium was changed every two days. Incorporation studies DNA synthesis: After bleomycin or h y d r o x y u r e a treatment, the cells were labelled with 2 pCi/ml of [6-3H] thymidine (specific activity, 5 Ci/mmol). After

361 incubation the medium was removed and the acid soluble DNA precursors were completely washed out as described in previous papers [19,20], followed by solubilizing the cells with 1 ml of Soluen TM-100 (Packard Inst. Co., Zurich) and transferred it into a vial containing 10 ml of a scintillation fluid, and the radioactivity was measured by a liquid scintillation counter. Acidic glycosaminoglycan and glycoprotein synthesis: After bleomycin or h y d r o x y u r e a treatment, the cells were labelled with 0.5 or 2 pCi/ml of D[6-3H]glucosamine hydrochloride (specific activity, 29 Ci/mmol) or 2 pCi/ml of [3SS]sulphuric acid (specific activity, 1.1 • 102 mCi/ml H2SO4) for 1--48 h. Acidic glycosaminoglycans and glycoproteins were extracted as mentioned below. The radioactivity of the extracted acidic glycosaminoglycans or glycoproreins was measured in a liquid scintillation counter. The scintillator cocktail was composed of toluene (1000 ml), Triton X-100 (500 ml) and 2,5-diphenyloxazole (6 g). Collagen synthesis: After bleomycin treatment, the cells were labelled with 2 pCi/ml of L-[2,3-3H2]-proline (specific activity, 29 Ci/mmol) for 24 h. The collagen fraction was extracted by the m e t h o d of Fitch [21] followed by hydrolysing the sample with 6 M HC1 at l l 0 ° C for 18 h and the radioactivity incorporated into hydroxyproline extracted by the m e t h o d of Kivirikko et al. [22] was counted by a liquid scintillation counter. All the radioactive precursors were purchased from The Radiochemical Centre, Amersham.

Extraction of acidic glycosaminoglycans and glycoproteins After cultivation, medium and harvested cells were treated separately with 0.5 M NaOH at 4°C for 18 h, followed by adjusting the pH to 7.5 by adding HC1 and one tenth volume of 0.5 M Tris • HC1 buffer containing 25 mM CaC12, pH 7.5. Pronase E (70 000 p.u.k./g, Kaken Chemicals Co., Tokyo) was added to the mixture in two doses at a 24 h interval to provide a total of 2% of protein, and proteolysis was carried out for 48 h at 50°C. Trichloroacetic acid was added to the digest to give a final concentration of 10% and the precipitated protein was removed by centrifugation. The supernatant was dialyzed against tap water for 48 h and then distilled for 24 h. The dialysate was designated as hexosamine-containing substances. NaC1 was added to the dialysate to give a final concentration of 0.04 M. The crude cetylpyridinium • acidic glycosaminoglycan complex was precipitated by adding 1% cetylpyridinium chloride to the dialysate. The supernatant was extracted with amyl alcohol at 0°C and the aqueous layer dialyzed against running tap water for 48 h and then distilled water for 24 h. The dialysate was evaporated using a rotary evaporator. This fraction was designated the glycoprotein fraction. The c e t y l p y r i d i n i u m , acidic glycosaminoglycan complex was dissolved in 1 ml of 4 M NaC1 and acidic glycosaminoglycans were reprecipitated with 14 ml of 80% ethanol. The precipitated acidic glycosaminoglycans were washed twice with 80% ethanol. The a m o u n t of hexosamine was determined by a slightly modified m e t h o d of the Elson-Morgan reaction [23] after hydrolysis of the sample in 2 M HC1 at 100°C for 16 h.

Two dimensional electrophoresis A b o u t 5--10 pg of acidic glycosaminoglycan samples were subjected to two

362 dimensional electrophoresis on cellulose acetate strips (Separax, Joko Sangyo Co., Tokyo) with 0.1 M pyridine/0.47 M formic acid buffer (pH 3) for the first dimension and 0.1 M barium acetate (pH 8) for the second dimension as reported by Hata and Nagai [24]. Location of individual acidic glycosaminoglycan was detected by staining the strip with alcian blue. Chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid were obtained from Seikagaku Kogyo Co. T o k y o , heparin from Novo, Copenhagen and heparan sulphate and keratan sulphate were generous gift from Dr. R. Hata of Tokyo Medical and Dental University, Tokyo. A more detailed method for the identification of acidic glycosaminoglycans produced by the culture cells was described in the previous papers [17,18].

Assay o f N-acetyl-~-glucosaminidase activity After cultivation, 0.5 ml of the cultured medium were incubated for 30 min at 37°C with 0.5 ml of 1 mM p-nitrophenyl-N-acetyl@D-glucosaminide (BDH Chemicals Ltd., Poole) in 0.05 M citrate buffer (pH 4.5) containing 0.1% Triton X-100. The a m o u n t of p-nitrophenol released during incubation was measured by spectrophotometry at 400 nm. Results

Effect o f bleomycin on the [ZH]glucosamine incorporation into hexosaminecontaining substances by cultured fibroblasts Cells in the stationary phase were exposed to various doses of N e o m y c i n for 8 days. The activity of the treated cells in incorporating [3H]glucosamine into hexosamine-containing substances during the following 24 h was compared with those in control cells. As shown in Fig. 1, a marked increase, about 80% over control, was seen in the bleomycin-treated cells at the concentration of 0.1 pg bleomycin/ml. Microscopic observation showed no significant difference between control and Neomycin-treated cells. Effect of bleomycin on the kinetics of hexosamine-containing substances production by cultured fibroblasts After exposure of the cells to 0.1 pg bleomycin/ml for 8 days, the activity of the cells in incorporating [3H]glucosamine into hexosamine-containing substances was followed for 48 h. Results shown in Fig. 2 indicate that bleomycintreated cells retained a kept higher activity in incorporating the labelled precursor t h r o u g h o u t the experimental period. However, there is no difference in the incorporation pattern between control and bleomycin-treated cells. It was also noted in both cases that the hexosamine-containing substances produced and accumulated in the cells were secreted into the culture media at 6--8 h. Effect o f bleomycin on the acidic glycosaminoglycans contents in cultured fibro blasts Fig. 3 shows the time course for the acidic glycosaminoglycans contents of the cell layer in both control and bleomycin-treated cultures. The stimulatory effect of bleomycin on acidic glycosaminoglycans synthesis was seen as early as the second day, and the stimulation was enhanced with increasing time.

363

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I 0.01

il 0.1

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13 68 24 48 TIME OF EXPOSURE TO 3H.GLUCOSAMINE

BLEOMYCIN CONCENTRATION

(h)

tpO/roll

Fig. 1. Dose r e s p o n s e of c u l t u r e d f i b r o b l a s t s t r e a t e d w i t h b l e o m y c i n . Cells w e r e e x p o s e d t o 0.5 p C i / m l of [ 3 H ] g l u c o s a m i n e f o r 24 h a f t e r 8 - d a y t r e a t m e n t w i t h b l e o m y c i n d u r i n g t h e i r s t a t i o n a r y p h a s e . R e s u l t s i n d i c a t e t h e r a d i o a c t i v i t y i n c o r p o r a t e d i n t o h e x o s a m i n e - c o n t a i n i n g s u b s t a n c e s in cell l a y e r (106 cells) or c u l t u r e m e d i a . E a c h p o i n t i n d i c a t e s m e a n -+ S.E. (n = 5): c~ cell l a y e r ; e , c u l t u r e m e d i a . Fig. 2. E f f e c t of b l e o m y c i n o n t h e k i n e t i c s of h e x o s a m i n e - c o n t a l n i n g s u b s t a n c e s p r o d u c e d b y c u l t u r e d fibroblasts. Cells w e r e e x p o s e d t o 0.5 p C i / m l of [ 3 H I g l u c o s a m i n e f o r 1 - - 4 8 h a f t e r 8 - d a y t r e a t m e n t w i t h 0,1 p g b l e o m y c i n / m l . R e s u l t s i n d i c a t e t h e r a d i o a c t i v i t y i n c o r p o r a t e d i n t o h e x o s a m i n e - c o n t a i n i n g subs t a n c e s in cell l a y e r o r c u l t u r e m e d i a . E a c h p o i n t i n d i c a t e s m e a n +- S.E. (n = 3). B r o k e n lines, c o n t r o l ; solid lines, b l e o m y c i n t r e a t m e n t ; o cell l a y e r ; • c u l t u r e m e d i a .

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0 2 4 6 8 10 T I M E OF EXPOSURE T O BLO(days)

TIME

OF EXPOSURE T O B L E O M Y C I N (days)

Fig. 3. Effect of bleomycin on the acidic glycosaminoglycan contents in cultured fibroblasts. Cells were exposed to 0.1 ,ag bleomycin/ml for 0--10 days during their stationary phase. T h e results indicate the a m o u n t of hexosamine per 10 6 cells, m e a n ± S.E. (n = 3). B r o k e n hncs, control; solid lines, bleomycin treatment. Extraction and analysis of acidic glycosarninoglycans axe described in the Methods. Fig. 4. Effect of bleomycin on the N-acetyl-~-glucosaminidase activity. Cells were exposed to 0.1 ~tg bleom y c i n / m l for 0--8 days during their stationary phase. E n z y m e activity released into culture media during the last t w o days in each g~oup w a s measured. Results indicate the a m o u n t of p-nitrophenol released from p-nitrophenyl-N-acctyl-~-D-glucosaminide during incubation period in culture m e d i u m per 10 6 cells. Each point indicates m e a n +- S.E. (n = 4). B r o k e n lines, control; solid lines, bleomycin treatment.

364

Effect o f bleomycin on the synthesis o f acidic glycosaminoglycans, glycoproteins and collagen by cultured fibroblasts Cells were exposed to [3H]glucosamine, H23~SO4 or [~H]proline for 24 h af. ter 4 day treatment of 0.1 pg bleomycin/ml. Acidic glycosaminoglycans, glycoproteins and collagen were extracted from cell layers and cultured media and the incorporated radioactivities were measured. As shown in Table I, the synthesis of these macromolecules was markedly enhanced by bleomycin, i.e., by 80% for total acidic glycosaminoglycans, by 53% for total glycoproteins and 23% for total collagen. However, no significant increase was seen in levels of noncollagenous proteins. It was also revealed that [3H]glucosamine was incorporated mostly into acidic glycosaminoglycans rather than glycoproteins, and that the stimulatory effect of bleomycin was greater on acidic glycosaminoglycans than on glycoproteins.

Analysis o f acidic glycosaminoglycans increased by treatment with bleomycin The extracted acidic glycosaminoglycan fraction was subjected to twodimensional electrophoresis. The m e t h o d used enables one to separate and identify seven kinds of acidic glycosaminoglycan, namely chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate, heparan sulphate, keratan sulphate, heparin and hyaluronic acid, owing to its high resolving power and sensitivity. As shown in Table II, relative ratios of individual acidic glycosaminoglycans did not statistically change as a result of bleomycin treatment.

Effect o f bleomycin on N-acetyl-t3-glucosaminidase activity o f cultured fibroblasts N-acetyl-~-glucosaminidase activity released into culture medium during cultivation of the cells in the presence of 0.1 pg bleomycin/ml was compared with that in control culture. As shown in Fig. 4, the enzyme activity decreased with increasing period of treatment time.

Effect o f bleomycin on [ZH]thymidine incorporation into DNA in cultured fibroblasts Cells in the logarithmic growth phase were exposed to various doses of bleomycin for 23 h and activity of the treated cells in incorporating [3H]thymidine into DNA during the following hour was compared to that of control cells. The activity of DNA synthesis of cells in the stationary phase was also examined after 4 day-treatment with bleomycin: in this case the cells were exposed to [3H]thymidine for 24 h instead of I h. The latter experimental condition was identical with that in other experiments concerning acidic glycosaminoglycans in this report. As shown in Table III, in both cases DNA synthesis was significantly decreased by bleomycin treatment at a concentration of as low as 0.01 pg/ml.

Effect o f hydroxyurea on the [3H]glucosamine incorporation into hexosaminecontaining substances by cultured fibroblasts To know whether the above-stated stimulatory effects of bleomycin on the production of hexosamine-containing substances by cultured fibroblasts are specific to bleomycin or are a general response of these cells to the substances

GLYCOPROTEINS

AND COLLAGEN

BY C U L T U R E D

FIBRO-

76373 ± 2489 137617 ± 6547 b (180)

(181)

20425 ± 633 36920 ± 3726 b

9700± 54 14635 ± 1559 d (151)

(156)

9297 ± 723 14486 ± 1245 d

92905± 6760 135468 ± 4054 c (146)

(156)

25007 ± 914 38984 ± 2433 c

Acidic glycosaminogtycans

Acidic glycosaminoglycans

Glycoproteins

H35SO4 (cpm/culture)

[3H] Glucosamine (cpm/culture)

a Values f o u n d in h y d r o x y p r o l i n e f r a c t i o n . Statistical significance: b p < 0 . 0 0 1 ; c p < 0 . 0 1 ; d p < 0 , 0 2 ; e p < 0 . 0 5 .

Medium Control Bleomycin

Control Bleomycin

CeU layer

Group

1975± 122 2809 ± 139 c (142)

(131)

2518 ± 251 3294 ~ 268

Glycoprotcins

107276 ± 3934 120243 ± 2766 (112)

(107)

I67594 ± 9072 179226 ± 14123

Total protein

(133)

4 2 0 ± 33 5 5 7 +- 4 4 e

867 ± 41 1025 ± 42 e (118)

Collagen a

[3It] Proline (cpm/culture)

Cells w e r e e x p o s e d t o [ 3 H ] g l u e o s a m i n e , H 2 3 5 S O 4 o r [ 3 H ] p r o l i n c f o r 24 h a f t e r 4 - d a y t r e a t m e n t w i t h 0.1 p g b l e o m y c i n / m l . R e s u l t s axe e x p r e s s e d as m e a n ± S.E, (n = 3), Figures in p a r e n t h e s i s i n d i c a t e p e r c e n t a g e s t o t h e c o n t r o l s .

E F F E C T O F B L E O M Y C I N ON T H E S Y N T H E S I S O F A C I D I C G L Y C O S A M I N O G L Y C A N S , BLASTS

TABLE I

¢O O3 C.n

TABLE

II

EFFECT OF BLEOMYCIN AND CULTURE MEDIA

ON THE

ACIDIC

GLYCOSAMINOGLYCAN

PATTERN

OF CELL LAYEt(

Cells were exposed to [3H]glueosamine or H235SO4 for 24 h after 4-day treatment with 0.1 pg blcomycin/ml. Details are described in Materials and Methods. Results are expressed as relative ratios of each acidic glycosaminoglycan component. Cell l a y e r

Medium

[3H]-

H35SO4

[3H]-

Glucosamine

Origin Hyaluronic acid C h o n d r o i t i n 4sulphate Dermatan sulphate C h o n d r o i t i n 6sulphate Heparan sulphate Heparin Keratan sulphate

TABLE

H35SO4

Glucosamine

Control

Bleomycin

Control

0 82

1 88

0 0

6 2

3 1

0 8 1 1

0 6 1 0

Bleomycin

Control

Bleomycin

Control

Bleomycin

0 0

0 82

0 75

0 0

0 0

43 5

53 4

14 2

20 2

84 5

80 9

0 41 11 0

0 37 6 0

0 2 0 0

0 2 1 0

0 5 6 0

0 6 5 0

III

EFFECT OF BLEOMYCIN FIBROBLASTS

ON

[3H]THYMIDINE

T h e cells w e r e e x p o s e d t o [ 3 H ] t h y m i d i n e stationary phase (1.1 - 105 cells/cm 2) or for rithmic growth phase (2.9 - 104 cells/cm2). e x p r e s s e d a s m e a n + S . E . (n = 5, s t a t i o n a r y dicate percentages to the controls. Bleomycin

INCORPORATION

INTO

DNA

IN

CULTURED

for 24 h after 4-day treatment with bleomyein during their 1 h after 23-h treatment with bleomycin during their logaDetails are described in Materials and Methods. Results are p h a s e ; n = 8, g r o w i n g p h a s e ) , T h e f i g u r e s in p a r e n t h e s i s in-

(pg]ml)

Stationary phase ( e p m p e r 1 • 1 0 6 cells)

Growth phase ( c p m p e r 1 • 1 0 6 cells)

0 0.01 0.1 1.0

4969 3639 1615 818

174760 141120 68239 24040

± 415 -+ 2 3 4 c ( 7 3 ) ~_ 1 0 2 a ( 3 3 ) ± 144 a(16)

-+ 7 5 9 9 -+ 5 4 4 0 b ( 8 1 ) ± 3759 a (39) ± 680 a (14)

a S t a t i s t i c a l s i g n i f i c a n c e : a P <: 0 . 0 0 1 ; b P < 0 . 0 1 ; c p < 0 . 0 5 . TABLE

IV

EFFECT OF HYDROXYUREA ON AMINE-CONTAINING SUBSTANCES

THE [3H]GLUCOSAMINE INCORPORATION BY CULTURED FIBROBLASTS

INTO

HEXOS-

Dose response of cultured fibroblasts treated with hydroxyurea. Ceils were exposed to 0.5 ~tCi/ml of [3H]glucosamine for 6 h after 4-day treatment with hydroxyurea during their stationary phase or 2 #Ci/ml of [6-3H]thymidine for 1 h after 23-h treatment with hydroxyurea during their logarithmic growth phase. Results indicate the radioactivity incorporated into hexosaminc-containing substances or DNA. E a c h v a l u e i n d i c a t e s m e a n ± S . E , (n = 3). T h e f i g u r e s in p a r e n t h e s i s i n d i c a t e p e r c e n t a g e s t o t h e c o n t r o l s . Hydroxyurea (pg/ml)

0 0.01 0.1 1.0 10.0

[3H] Glucosamine

[3tI] Thymidinc ( c p m p e r 1 • 1 0 6 cells)

Cell l a y e r ( c p m p e r 1 • 1 0 6 cells)

Medium ( c p m p e r 1 - 1 0 6 cells)

1453-+ 7 1 4 5 0 _+ 4 1 1 4 3 0 _+ 6 6 1545_+ 22 1508 ± 35

1383 1324 1351 1274 1207

* S t a t i s t i c a l s i g n i f i c a n c e : P "< 0 . 0 0 1

+- 8 5 + 31 ± 73 ± 40 +- 4 7

141 840-+ 1240 --119920 ± 1260 * (85) 73920 ± 1820 * (52)

367 interfering with DNA synthesis, the effect of another inhibitor of DNA, hydroxyurea, was examined instead of bleomycin. Table IV shows that hydroxyurea had no stimulatory effect on the production of hexosamine-containing substances by the cells. Microscopic observation showed no significant difference between control and hydroxyurea-treated cells. Discussion

Bleomycin has been reported to inhibit DNA synthesis without any apparent effects on RNA and protein synthesis in E. coli, Ehrlich ascites, and HeLa cells [25]. At low concentrations, bleomycin was also found to inhibit cell division in cultured HeLa cells [26]. In this study, bleomycin showed strong inhibitory effect on DNA synthesis in cultured fibroblasts (Table III}. On the other hand, bleomycin, at the same dose showed a marked stimulatory effect on acidic glycosaminoglycan and glycoprotein production as well as collagen synthesis (Table I). However, the stimulatory effect does not seem to be the result of inhibition of DNA synthesis, since another p o t e n t inhibitor of DNA synthesis, hydroxyurea, showed no potency of increasing acidic glycosaminoglycans (Table IV). Besides, there has been no evidence that m y t o m y c i n , one of the other potent inhibitors of DNA synthesis, caused fibrosis. From these facts it can be concluded that bleomycin specifically stimulates acidic glycosaminoglycan synthesis and that the increased acidic glycosaminoglycan and inhibited DNA production were not a coupling reaction. These in vitro findings seem to reflect the p h e n o m e n o n of pulmonary fibrosis frequently observed in the in vivo treatment of bleomycin in mouse [27], dog [28] and man [4,5,29]. Conversely, they indicate that this culture system is the most appropriate experimental model for studying the mechanism of fibrosis caused by bleomycin. For this purpose, alteration of intercellular substances by bleomycin was first examined. When the cells were cultured under bleomycin exposure with one of the certain radioactive precursors of the macromolecules, specific and remarkable increases of acidic glycosaminoglycans, glycoproteins and collagen were observed. The increment of acidic glycosaminoglycans was higher than that of glycoproteins or collagen (Table I). The addition of dermatan sulphate proteoglycan or chondroitin sulphate proteoglycan to the tropocollagen solution has been shown to modify the kinetics of collagen fibril formation from collagen molecules [30]. It seems likely therefore that the action of sulphated proteoglycans on tropocollagen may be important in collagen fibrillogenesis. The increased acidic glycosaminoglycan components were then examined in the expectation that some, such as dermatan sulphate, to have interaction with collagen fibrillogenesis [6,7,11, 12], had been specifically affected by bleomycin. However, as shown in Table II, no clear change was observed in the experimental design. From the viewpoint of degradation of acidic glycosaminoglycans, N-acetyl~-glucosaminidase activity was determined. The level of enzyme released into culture media during cultivation decreased in proportion to the duration of bleomycin treatment. From the results mentioned above, it is concluded that bleomycin has a spe-

368 cific potency to enhance acidic glycosaminoglycan components in cultured fibroblasts.

Acknowledgement The authors express their gratitude to Nippon Kayaku Co., Ltd., for the supply of bleomycin.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Umezawa, H. (1969) J. Jap. Med. Assoc. 62, 144--153 Clinical Screening Group, EORTC. (1970) Br. Med. J. 2, 643--645 Umezawa, H., Ishizuka, M., Maeda, K. and Takeuchi, T. (1967) Cancer 20, 891--895 Rudders, R.A. and Hensley, G.T. (1973) Chest 63, 626- -628 Bedrossian, C.W.M., Luna, M.A., Mackay, B. and Lightiger, B. (1973) Cancer 32, 44--51 Toole, B.P. and Lowther, D.A. (1966) Arch. Biochem. Biophys. 128, 567--578 Toole, B.P. and Lowther, D.A. (1968) Biochem. J. 109, 857--866 Lowther, D.A. and Natarajan, M. (1972) Bioehem. J. 127, 607--608 Obrink, B. and Wasteson, A. (1971) Bioehem. J. 1 2 1 , 2 2 7 - - 2 3 3 Greenwald, R.T., Schwartz, C.E. and Cantor, J.O. (1975) Biochem. J. 145, 601--605 Mathcws, M.B. (1965) Biochem. J. 96, 710--716 Mathews, M.B. and Decker, L. (1968) Biochem. J. 1 0 9 , 5 1 7 - - 5 2 6 Wood, G.C. (1960) Bioehem. J, 75, 605--612 Tetsuka, T., Kawai, S., Konno, K. and Endo, H. (1970) Seikagaku 4 2 , 4 6 8 Ichihashi, M., Shinkai, H., Takei, M. and Sano, S. (1973) J. A nt i bi ot i c s T o k y o 2 6 , 2 3 8 - - 2 4 2 Otsuka, K., Murota, S. and Mori, Y. (1975) Chem. Pharm. Bull. 23, 3038--3039 Murota, S., Mitsui, Y., Koshihara, Y. and Tsurufuji, S. (1974) Connective Tissue 6, 55--65 Murota, S., Koshihara, Y. and Tsurufuji, S. (1975) Bioehem. Pharmac. 25, 1107--1113 Baltimore, P. and Franklin, R.M. (1962) Proc. Natl. Acad. Sei. U.S. 48, 1 3 8 3 - - 1 3 9 0 Ohtsubo, K., Yamada, M. and Saito, M. (1968) Jap. J. Med. Sci. Biol. 2 1 , 1 8 5 - - 1 9 4 Fitch, S.M., H a r k n e ~ , M.L.R. and Harkness, R.D. (1955) Nature 176, 163 Kivirikko, K.I., Laitinen, O. and Prockop, D.J. (1967) Anal. Biochem. 19, 249--255 Gatt, R. and Berman, E.R. (1966) Anal. Biochem. 15, 167--171 Hata, R. and Nagai, Y. (1972) Anal. Biochem. 45, 462--468 Suzuki, H., Nagai, K., Yamaki, H., Tanaka, N. and Umezawa, H0 (1968) J. Antibiotics Tokyo 21, 379--386 Terashima, T. and Umezawa, H. (1970) J. Antibiotics T o k y o 20, 300--304 Adamson, I.Y.R. and Bowden, D.H. (1974) Am. J. Pathol. 7 7 , 1 8 5 - - 1 9 0 Fleischman, R.W., Baker, J.R., Thompson, G.R., Schaeppi, U.H., Illicvski, V.R., Cooney, D.A. and Davis, R.D. (1971) Thorax. 2 6 , 6 7 5 - - 6 8 2 Krous, H.F. and Hamlin, W.B. (1973) Arch. Pathol. 95, 407--410 Obrink, B. (1973) Eur. J° Biochem. 34, 129--137