[CANCER RESEARCH 39, 2802-2806, July 1979] 0008-5472/79/0039-0000$02.00 and of Normal and Tumoral of Humans and Rats1

Paulo A. S. Mourão,2 Yara M. Michelacci, and Olga M. S. Toledo

Laboratdrio de Investigacáo em Reumatologia, Faculdade de Medicina da Universidade de SãoPaulo, Caixa Postal 8100, 0 1246 SãoPaulo, SP, Brazil (P. A. S. M.. 0. M. S. Ti, and Departamento de Bioquirnica e Farmacologia da Escola Paulista de Medicina, SãoPaulo,SP, BrazilfY. M. M.j

ABSTRACT MATERIALS AND METHODS

Differences in the glycosaminoglycans and proteoglycans GAG. Fresh cartilageswere obtainedfrom young and adult synthesized by ‘‘young,‘‘‘‘adult,‘‘andtumoral chondrocytes mammals and checked for ‘‘normality'‘bymacroscopic ex are reported. Young and human chondrosarcoma amination. Human chondrosarcoma from the upper femoral contain 4- and 6-sulfates, whereas adult human end was the kind gift of Dr. 0. Gianotti Filho (Departamento de cartilage contains almost exclusively chondroitin 6-sulfate. Anatomia PatolOgica, Hospital A. C. Camargo, SãoPaulo, High keratan sulfate content is reported in adult cartilage, Brazil). Transplantable rat chondrosarcoma was provided by whereas it is almost absent in young and tumoral cartilages. Dr. R. R. Brentani (LaboratOrio de Oncologia Experimental, The electrophoretic pattern and keratan sulfate content in Faculdade de Medicina da Universidade de SãoPaulo). GAG these proteoglycans from adult cartilage are clearly distinct was extracted from cartilages as previously described (20). from those of the young and tumoral cartilages. The high Standard chondroitin 4- and 6-sulfate and molecular weight is the distinguishing property of the glycosa were purchased from Miles Laboratories (Elkhart, Ind.). Kera minoglycan synthesized by tumoral chondrocytes. tan sulfate was prepared from bovine cornea, according to the method of Meyer et al. (i 6). The barium salt of heparitin sulfate INTRODUCTION from bovine lung, obtained through the courtesy of Dr. L. L. Coleman, Upjohn Co., Kalamazoo, Mich., was converted to its Several groups of workers (12, 13, 25) have established the ammonium salt as described previously (6). Heparitin sulfates existence of variations in the GAG' content of articular carti B, C, and D from bovine lung tissues were prepared as previ lage, corresponding to distance from the articular surface. ously described (5). Increases in the relative proportion of the chondroitin 4-sulfate Proteoglycans. Fresh canilages were immersed in 10 ml of (19, 20) and in the average molecular weight of the chondroitin acetone and kept for 24 hr at 5°with 2 fresh changes of sulfates (i 0) were found to correspond with distance from the acetone. The dried tissue (50 mg) was macerated and sus articular surface of the epiphysis of young mammals. A species pended in 1.0 ml of 4.0 M guanidine hydrochloride. In some specific distribution of chondroitin 4- and 6-sulfates in the experiments, protease inhibitors [1 m@ phenylmethylsulfonyl epiphysial cartilages of adult mammals has been reported fluoride, 2 mM EDTA, and soy bean trypsin inhibitor (0.2 mg/ recently by Mourãoand Dietrich (i 9). Chondroitin 6-sulfate is ml)] were added to the guanidine hydrochloride solution. The present in high relative proportions in adult epiphysial carti mixture was maintained at 5°for 48 hr under agitation and lages in most of the mammals studied whereas, in some species then dialyzed for 4 hr against distilled water. of the order Rodentia, chondroitin 4-sulfate is the only chon Enzymes. Chondroitinases AC and ABC were purchased droitin sulfate present. Choi et al. (2) reported that chondroitin from Miles Laboratories. Chondroitinase C was prepared as 4-sulfate is the exclusive produced by a described by Michelacci and Dietrich (18). Crude extract from transplantable rat chondrosarcoma, while MourãoandDietrich Flavobacterium heparinum was prepared as previously de (19) observed that normal cartilages of young and adult rats scribed (3). Crude extract from Pseudomonas sp., which cat also contain almost exclusively chondroitin 4-sulfate. Although alyze an extensive cleavage of keratan sulfate, was prepared the chemical composition of human chondrosarcoma has been according to the method of Nakazawa et al. (22). Trypsin was reported by Meyer et al. (15) and by Anderson et al. (1), the purchased from Sigma Chemical Co. (St. Louis, Mo.). GAG have not been analyzed by newly developed biochemical Other Chemicals. Agarose was purchased from L'lndustrie methods. Biologique FrançaiseS.A.(Gennevilliers, Seine, France); 1.3- This paper gives a comparative analysis of GAG extracted diaminopropane was from Aldrich Chemical Co. (Milwaukee, from young, adult, and tumoral cartilages of humans and rats. Wis.); amino sugars and were from British Drug The objective of this study is to compare GAG synthesized by House Chemical Ltd. , ‘young, ‘‘‘‘old, ‘‘and tumoral chondrocytes of these 2 mam Identification and Quantitation of GAG and Proteoglycans. mals. The GAG and proteoglycans were identified by a combination of agarose gel electrophoresis (4) and enzymatic degradation

, This research was aided by Grant 77/0748 from Fundaçao de Amparo a with specific mucopolysaccharidases and trypsin. The prod Pesqulsa do Estado de SãoPaulo(FAPESP). ucts formed by the action of the enzymes on the GAG and 2 Supported by FINEP (Flnanciadora de Estudos e Projetos) and CNPq (Con selho Nacional de Desenvolvimento CientIfico e Tecnológico). proteoglycans were identified by paper chromatography (24) 3 The abbreviations used are: GAG, (s); aDi-4S, 2-aceta and agarose gel electrophoresis (4). The agarose gel slides mido-2-deoxy-3-O-(8-o-gluco-4-enepyranosyluronic acid)-4-0-sulfo-o-galac (5.0 x 7.5 cm) were prepared with 1.2% agarose in 0.05 M toss; ADi-6S, 2-acetamido-2-deoxy-3-O-(fl-o-gluco-4-enepyranosyluronic acid) 6-O-sulfo-o-galactose. 1,3-diaminopropane acetate, pH 9.0 (nondissociative condi Received October 11, 1978; accepted April 12, 1979. tions). In some experiments, 6 M urea was added to the buffer

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(dissociative conditions). The relative amounts of the products a mixture of chondroitinase ABC plus F. heparinum extract formed by chondroitinase degradation were quantitated by (which degrades all the GAG, except keratan sulfate). A band densitometry as previously described (7) or by hexosamine of chondroitinase ABC plus F. heparinum extract-resistant GAG determinations after elution of the chromatograms (5). Densi is present in adult cartilage. This band has the same electro tometry was performed by means of a Beckman Model R-i 12 phoretic mobility as the standard keratan sulfate and is sus scanning densitometer. The quantitation of the GAG and pro ceptible to Pseudomonas sp. extract action. The high gluco teoglycans was performed by densitometry of the agarose gel samine and galactose contents and low uronic acid content in electrophoresis after toluidine blue staining. The error of the this cartilage (Table 1) furnish additional evidence that the method was in the range of 4.8% for the GAG and 7.5% for resistant band is keratan sulfate. This band is practically absent their degradation products. In some experiments, the GAG in young cartilage and chondrosarcoma. were measured as uronic acid by the carbazole reaction (8). Chondroltin Sulfates from Young, Adult, and Tumoral Car Molecular Weight DetermInation. The molecular weight of tilages. Fig. 1 shows the products formed by the action of the GAG was measured by the method of Hilbom and Anastas chondroitinases AC and C upon the GAG of young, adult, and siadis (9) except that polyacrylamide gel slabs instead of tubes tumoral cartilages from rat and humans. Two unsaturated di were used. The following standards were used: chondroitin 4- saccharides, namely, @Di-4Sand @Di-6S,areformed by the sulfate [13,000 ±8,000 (S.D.)]; dermatan sulfate (19,000 ± chondroitinase AC. Only trace amounts of unsaturated nonsul 9,500); chondroitin 6-sulfate (65,000 ±25,000); heparitin fated and disulfated disaccharides are formed. sulfate B (25,000 ±I 0,000); heparitin sulfate C (9,300 ± It can be seen that glycosaminoglycans extracted from rat 500); and hepantin sulfate D (3,800 ±1,500). cartilages (young, adult, and tumoral) produce only @Di-4S. Other Methods. The percentages of glucosamine and galac GAG extracted from adult human cartilage produce only @Di tosamine were measured after acid hydrolysis (8 N HCI for 6 hr 6S, whereas human young and tumoral cartilages produce at 1000) by densitometry of chromatograms on Whatman No. both disaccharides by the action of the chondroitinase AC. The ‘lfilterpaper. These chromatograms were developed in bu quantitative data for this experiment are shown in Table 2. tanol:pyridine:water (4:3:1 , v/v/v) as descending solvent and Young human cartilage contains 55% chondroitin 4-sulfate, revealed by silver nitrate staining. The galactose contents in tumoral human cartilage contains 36% chondroitin 4-sulfate, the GAG were measured as described previously (22). Amino whereas adult human cartilage contains only chondroitin 6- sugars were measured after hydrolysis (4 N HCI for 6 hr at sulfate. The 3 cartilages from rat contain almost exclusively 1000) by a modified Elson-Morgan reaction (23). chondroitin 4-sulfate. The chondroitin sulfates extracted from rat cartilages were RESULTS resistant to the chondroitinase C action while the GAG from human cartilages formed @Di-6S,tetrasaccharide, and oligo Concentration and Chemical Composition of GAG in Dif saccharide. The relative amounts of these products and their ferent Cartilages. The amounts of GAG extracted from young, @Di-4S:@Di-6Sratioare shown in Table 3. adult, and tumoral cartilages, as well as the molecular weight, Proteoglycans of Different Human Cartilages. Chart 2 uronic acid content, and the relative concentration of gluco shows the electrophoretic mobilities of the proteoglycans ex samine, galactosamine, and galactose in these GAG, are tracted from young, adult, and tumoral human cartilages, be shown in Table 1. The GAG from adult cartilages contain more fore and after incubation with trypsin. Adult cartilage contains glucosamine and galactose and less uronic acid than those 2 fractions of proteoglycans, P1 and P2, according to their from young cartilage and chondrosarcoma. The total amounts electrophoretic mobility, whereas young cartilage and chon of the GAG extracted from young cartilages and from chondro drosarcoma contain only Fraction P2. The relative proportion sarcoma are lower than those from adult cartilages. The mo of the fractions is shown in Table 4. No significant lecular weight of the GAG from chondrosarcoma is greater difference was observed in this proportion whether the extrac than those from normal cartilages. tion was done with or without proteases inhibitors or whether Agarose Gel Electrophoresis of the GAG before and after the electrophoresis was carried out in the presence of 6 M urea Degradation with Enzymes. Chart 1 shows the electrophoretic (dissociative condition) or not (nondissociative conditions). mobilities of the GAG extracted from normal young and adult After trypsin action, the proteoglycans from adult cartilage human cartilages and from human chondrosarcoma before and formed 2 bands. The main one migrates as the standard after incubation with Pseudomonas sp. extract (which degrades chondroitin 4- or 6-sulfate and is degraded by the chondroiti keratan sulfate but not the other GAG), chondroitinase AC, and nase AC. The other one, which is resistant to the chondroitinase Table1 Concentration andchemical compositioncartilagesHexosaminesof GAG in different (%CartilageTotal and galactose acid GAG)MW.Galac-total GAG (% content (% dry wt of tie dry wt of tis Galac- Glucosa mineYoung7.62.9<5 sue)aUronic sue)in toss tosamine 10,ooobAdult12.81.718 72 2821,100 ± 10,000Chondrosarcoma7.23.9<5 47 3525,000 ± 76 24>100,000

a The quantitation of the GAG was performed by densitometry of the agarose slides after electrophoresis and toluidine blue staining. b Mean ± S.D.

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+ Chase ABC Control + kas. + Chase AC F.H.extract 4 ‘4 4 4 4,IJf 6-

4- 1 @@@@@ 2- .;___7___;___;.#I@@\4@.;._.UI @ , , , I 1 II

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@ @—I10 20III 30 ‘ ‘@0@20'30 ([ECTISPISRETICUIUATISI(UI) Chart 1. Densitometry of GAG in agarose gel electrophoresis before and after incubation with enzymes. GAG (100 @zg)wasincubated with 0.01 unit of chondroltinase AC (Chase AC). 100 @gofPseudomonas sp. extract (Kase), or 0.01 unit of chondroitinase ABC plus 100 @gofF. heparinum extract (Chase ABC ± F.H. extract@,in 0.05 @,iTrls-HCIbuffer, pH 8.0, at 37°for 8 hr, in a final volume of 20 @d.Appropriatecontrols were also incubated in the absence of enzymes. After incubation, 5-@oIaliquotswere submitted to agarose gel electrophoresis (0.05 M I ,3-diaminopropane acetate, pH 9.0). After staining, the GAG were quantitated by densitometry. Row 1, young cartilage; Row 2, adult cartilage; Row 3, chondrosarcoma; arrows, electrophoretic migration of standard keratan sulfate (I) and chondroltln 4- and 6-sulfate (10.

Table2 materials were precipitated with 2 volumes of ethanol. The DisaccharideproductsformedbyACupon theactionof chondroitinase electrophoretic mobilities of the products thus obtained are GAG from normal cartllages andchondrosarcomaChondroitinase shown in Fig. 2. The F. heparinum-resistant material migrates AC products as standard keratan sulfate, and the Pseudomonas sp. resistant (%)aCartilage material showed 2 bands: a strong one that migrates as chon @Di-6SYoung Mammal ADI-4S droitin sulfate; and a faint one, which migrates as keratan <5Adult Rat 100 sulfate. The faint band is susceptible to further incubation with <5Tumoral Rat 100 Pseudomonas sp. extract. The relative concentrations of ga <5Young Rat 100 lactosamine, glucosamine, and galactose in each sample are 45Adult Human 55 shown in Table 5. High glucosamine and galactose content in 100Tumoral Human <5 the slow-moving band furnishes additional evidence that this Human 36 64 band is keratan sulfate. This band is practically absent in GAG a The relativeamountsof the productswere quantitatedby densitometryof and proteoglycans extracted from young and tumoral carti the chromatograms after silver nitrate staining. lages. Table3 Percentagesofamountsand @.Di-4S:@Di-6Sratioof theproducth DISCUSSION formed by the action of chondroitinase C on the chondroitin sulfates fromhumancartilagesandchondrosarcomaCartilageYoung from The results reported in this paper strongly indicate that there are differences in the GAG and proteoglycans synthesized by adult, young, and tumoral chondrocytes. Keratan sulfate is TumoralChondroitlnase Adult present in much greater amounts in adult cartilages, confirming @Di-4S: aDi-4S: @Di-4S: earlier observations (1 1, 14), whereas it is almost absent in @Dl-6SOligosaccharideC productsa % aol-es % @Di-6S % young cartilages and chondrosarcoma. The proteoglycan elec 36 2.8 55 0.25 trophoretic pattern of young cartilage and of human chondro Tetrasacchartde 33 0.8 5 0.9 25 0.9 sarcoma and the keratan sulfate content in these proteoglycans ADI-6S 31 0 95 0 20 0 are clearly distinct from those from adult cartilage. Chondroitin a The relative amounts of the products were quantitated by hexosamine 4-sulfate is absent in adult human cartilage as previously determInations after elution of the chromatograms. reported (20), whereas young cartilage and human chondro ABC plus F. heparinum extract action, migrates as standard sarcoma synthesized chondroitin sulfate containing consider keratan sulfate and is degraded by Pseudomonas sp. extract. able amounts of 4-sulfated units. These results Trypsin digestion of proteoglycans extracted from young car indicate that the proteoglycans, keratan sulfate and chondroitin tilage and chondrosarcoma produces only chondroitin sulfate. 4- and 6-sulfate in the human chondrosarcoma reported in this Chmlcal Composition of GAG Samples from Adult Human paper resemble those of ‘‘young'‘chondrocytes but not Cartilage. GAG (I mg) and proteoglycans (I .5 mg) from normal , ‘adult― chondrocytes. The high molecular weight of the GAG adult cartilages were incubated with 1 ml of F. heparinum or from human chondrosarcoma is the distinguishing property of Pseudomonas sp. extracts. After incubation, the nondegraded the GAG synthesized by tumoral chondrocytes. The possibility

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CONTROL + TRYPSIN @ P1 P2 ll@ 14 6 4 1 2

C.) 6

4 a 2 ‘I, 2

6

4 3 2

10 ‘ 20 30 10 20 ELECTROPHORETICMIGRATIONliii Chart 2. Densitometry of proteoglycans in agarose gel electrophoresis before and after incubation with trypsin. Proteoglycans (100 @g)wereincubated with trypsin in 0.05 M Tris-HCI buffer, pH 8.0, at 37°for 8 hr. in a final volume of 20 @tl.Appropriatecontrols were also incubated in the absence of trypsin. After incubation, 5-@tlaliquots were submitted to agarose gel electrophoresis (0.05 M 1,3-diaminopropane acetate, pH 9.0). After staining, the proteoglycans were quantitated by densitometry. Row 1, adult cartilage: Row 2, chondrosarcoma; Row 3, young cartilage: arrows, electrophoretic migration of standard keratan sulfate (I) and chondroitin 4- and 6-sulfate (II).

Table 4 The relative proportion of isomeric chondroitin sulfates has ProteoglycansfromnormalhumancartilagesandchondrosarcomaProteoglycanbeen studied in various cartilages from young and adult mam mals (19, 20). The presence of chondroitin 4-sulfate in growing (%)bElectrophore fractions cartilages, which disappears in adult cartilages (after the cal cification process is over), suggests that this GAG might be p2WithoutExtractionCartilagep1 one of the components of the matrix for the calcification proc 100inhibitors+proteaseYoung<5 66 ess. In agreement with this hypothesis is a chemical defect of Tumoral <5 100 the chondroitin 6-sulfate in a mucopolysaccharidosis, charac 6 M urea Young <5 100 terized by platyspondyly and irregularities of articular surfaces, + 6 M urea Adult 31 69 100With + 6 M ureaAdult Tumoral34 <5 where no apparent changes in the growth of long were observed (21). The increase in the relative amount of 4-sulfate 100hibitors+protease In Young<5 73 disaccharide units in the human chondrosarcoma, in which the Tumoral <5 100 calcification process commonly occurs, is in agreement with 6 M urea Young <5 100 this line of reasoning. + 6 M urea Adult 23 77 + 6 M ureaAdult Tumorai27 <5 100 Recently, studies on chondroitin sulfate structure have mdi a Agarose gel electrophoresis was performed in 0.05 M 1,3-diaminopropane cated that chondroitin 4- and 6-sulfates from whale, shark, and acetate, pH 9.0 (nondissociative conditions). In some experiments, 6 M urea was human cartilages are copolymers (17). The results presented added to the buffer (dissociative conditions). in this paper indicate also that the chondroitin sulfate from the b The quantitation of the proteoglycans was performed by densitometry of the agarose slides after electrophoresis and toluidine blue staining. human chondrosarcoma reported in this paper is a copolymer.

Table5 REFERENCES Chemical compositioncartilageHexosamine of GAG from adult human 1. Anderson, C. E., Ludowieg, J., Eyring, E. J., and Horowitz, B. Ultrastructure (%)Sampl? and galactose content and chemical composition of chondrosarcoma. Report of one case. J. Jt. 5urg. Am. Vol., 45: 753-764, 1963. Galactose1 Galactosamine Glucosamine 2. Choi, H. U., Meyer, K., and Swarm, R. Mucopolysaccharide and protein 11 54 35 of a transplantable rat chondrosarcoma. Proc. Natl. Acad. 2 63 21 16 Sd. U. 5. A., 68: 877-879, 1971. 3 6 53 41 3. Dietrich, C. P. Enzymatic degradation of . A sulphamidase and a 9a 4 80 ii sulphoesterase from Flavobacterium heparinum. , 11 1: 91 -95, 1969. 4. Dietrich, C. P., McDuffie, N. M., and Sampaio, L. 0. IdentifIcation of acidic The samples are the same as indicated in Fig. 2. mucopolysaccharides by agarose gel electrophoresis. J. Chromatogr., 130: 299-304,1977. that the GAG and proteoglycan pattern depends on the type or 5. Dietrich, C. P., and Nader, H. B. Fractionation and properties offour heparitin origin of chondrosarcoma cells must be considered, and a sulfates from beef lung tissue: isolation and partial characterization of a homogeneous species of heparitin sulfate. Biochim. Biophys. Acta, 343: more extensive study on GAG and proteoglycan composition 34-44, 1974. of chondrosarcoma of various origins should be done. 6. Dietrich, C. P., Nader, H. B., Britto, L. R. G., and Silva, M. E. Chemical

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composition of heparitin sulfate. Fractionation and characterization of four 16. Meyer, K., Linker, A., Davidson, E. A., and Weissman, B. The mucopolysac acidic mucopolysaccharides in heparitin sulfate from beef lung tissue. charides of bovine cornea. J. Biol. Chem., 205: 61 1-620, 19@3. Biochim. Biophys. Acta, 237: 430-441 , 1971. 17. Michelacci, Y. M., and Dietrich, C. P. Structure of chondroitin sulfates: 7. Dietrich, C. P., Nader, H. B., and Mourão,P.A. S. Differentiation of Hunter's analyses of the products formed from chondroitin sulfates A and C by the and Hurler's syndromes by the analysis of the excreted mucopolysaccha action of the chondroitinases C and AC from Flavobacterium heparinum. rides. Biochem. Med., 8: 371 -379, 1973. Biochim. Biophys. Acta, 45 1: 436—443.1976. 8. Dische, Z. A new specific color reaction of hexuronic acids. J. Biol. Chem., 18. Michelaccl, Y. M., and Dietrich, C. P. Chondroitinase C from Flavobacterium 167: 189—192,1947. heparinum. J. Biol. Chem., 251: 1154—1158, 1976. 9. Hilborn, J. C., and Anastassiadis, P. A. Acrylamide gel electrophoresis of 19. Mourão,P.A. S., and Dietrich, C. P. Chondroitin sulfates of the epiphysial acidic mucopolysaccharides. Anal. Biochem., 31: 51-55, 1969. cartilages of different mammals. Comp. Biochim. Physiol., 62: 115—117, 10. Jones, I. L, and Lemperg, R. Chondroitin sulphate of calf knee-joint carti 1979. lage. Biochim. Biophys. Acta, 392: 310—318,1975. 20. Mourão,P.A. S.. Rozenfeld, S.. Laredo, J., and Dietrich, C. P. The distri 11. Kaplan, D., and Meyer, K. Ageing of human cartilage. Nature (Lond.), 183: bution of chondroitin sulfates in articular and growth cartilages of human 1267-1268, 1959. bones. Biochim. Biophys. Acta, 428: 19-26, 1976. 12. Lemperg, R., Larsson, S. E., and Hjertquist, 5. 0. The glycosaminoglycans 21 . Mourão,P. A. 5., Toledo, 5. P. A., Nader, H. B., and Diefrich, C. P. Excretion of bovinearticularcartilage.I.Concentrationanddistributionindifferent of chondroitln sulfate C with low sulfate content by patients with generalized layers in relation to age. Calcif. Tissue Res., 15: 237—251,1974. platyspondyly (brachyolmia). Biochem. Med., 7: 4 15—423,1973. 13. Maroudas, A., Muir, H., and Wingharn, J. The correlation of fixed negative 22. Nakazawa, K., Suzuki, N., and Suzuki, S. Sequential degradation of keratan charge with glycosaminoglycan content of human articular cartilage. sulfate by bacterial enzymes and purification of a in the enzymatic Blochim. Biophys. Acta, 177: 492-500, 1969. system. J. Blot. Chem.. 250: 905-91 1, 1975. 14. Mathews, M. B., and Glacov, S. Acid mucopolysaccharide patterns in aging 23. ROndIe,C. J. M., and Morgan, W. I. J. The determinationof glycosamine human cartilage. J. Clin. Invest., 45: 1103—1111, 1966. and galactosamine. Biochem. J., 61: 586-589, 1955. 15. Meyer, K., Davidson, E., Linker, A., and Hoffman, P. The acid mucopolysac 24. Saito, H., Yamagata,T., and Suzuki, S. Enzymaticmethodsfor the deter charides of connective tissue. Biochim. Biophys. Acta, 2 1: 506—518,19@6. minatlon of small quantities of isomeric chondroitin sulfate. J. Biol. Chem., 243: 1536—1542, 1968. 25. Stockwell, R. A., and Scott, J. E. DistributIOnof acid glycosaminoglycans in human articular cartilage. Nature (Lond.). 215: 1376-1 378, 1969.

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4 5 6 lhA 4 S @[email protected]*sIss Fig. 2. Agarose gel elecfrophoresis of purified GAGsamplesfrom adult human D@O$.AC 0101. C cartilage. To about I .0 mg of GAG and 1.5 mg of 4.0 PAguanidinehydrochloride extractfromadultnormalcartilagewereadded1.0mlofcrudeextractfromF. heparinum (to prepare a pure sample of keratan sulfate) and 1.0 ml of crude extract from Pseudomonas sp. (to prepare a pure sample of chondroitin sulfate). Theincubationconditionsaredescribedin“MaterialsandMethods.―After Incubation, the precipitate formed was removed by centrifugation, and 2 volumes of ethanol were added to the clear supematant. After 12 hr at —10@,the -@4$ precipitate formed was collected by centritugation, washed once with 3.0 ml of 80% ethanol, and dried. About 100 @gofeach sample were submitted to agarose gel electrophoresls in 0.05 M 1,3-diaminopropane acetate buffer, pH 9.0, and stained with toluidine blue as previously described. Column 1, GAG plus F. hepannum extract; Column 2, GAG plus Pseudomonas sp. extract: Column 3, proteoglycan (P0) pIus F. heparinum extract: Column 4, proteoglycan plus Pseudomonas sp. extract; ST. mixture of standard keratan sulfate (KS) and chondroitin 4- and 6-sulfate (CS).

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Fig. 1. Paper chromatogram of the chondroitinase AC and C digest of the GAG from normal cartilages and chondrosarcoma. Three x 1o@' unit of the chondroitinase AC (Chase AC) and 2.8 x 1O@ unit of the chondroitinase C (Chase C) were incubated with 100 @igofGAG in 0.05 PAethylenediamineacetate buffer, pH 8.0, in a final volume of 20 4 After 12 hr incubation at 37°for the chondroitinase AC and at 200 for the chondroitinase C, the mixtures were spotted on Whatman No. 1 paper and subjected to descending chromatography in isobutyric acid: 1 M NH3(5:3, v/v) for 48 hr. After the run, the reducing products were located by silver nitrate staining. GAG from rat cartilages: 1, young: 2, adult: 3. tumoral. GAG from human cartilages: 4, young: 5, adult: 6, tumoral. ChA, chondroitin 4-sulfate: ChC, chondroitin 6-sulfate.

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Paulo A. S. Mourão, Yara M. Michelacci and Olga M. S. Toledo

Cancer Res 1979;39:2802-2806.

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