Conn巴ctiveTissue Vo1. 11， No. 3， 91-105 (1979)

8ummary

Mucopolysaccharides and Proteoglycans of Chondrosarcomas

Naomi Kawabゲ andSadako Yamagata**

キ Departmentof Orthopaedic Surgery， Kawasaki Medical School ** Laboratory of Cell Biology， Aichi Cancer Center Research Institute

Biochemical analyses were made and in vitrοincorporation of 358引Ilfatewas studied in

samples from two chondrosarcomas of the pelvic bone. Sample日 ofadjacent joint cartilage

and human embryonic cartilage served as controls.

Incorporation of 358圃sulfateinto the total glycosaminoglycan of the tumors exceeded that

of controls by 3 to 17 times in case 1.

Chondroitin sulfates A and C existed in the two tumors. Keratan sulfate was absent.

Analyses of the proteoglycan from the tumors revealed that they consisted of two macro-

molecular species of different molecrilar weights.

1. Introduction

Significant amounts of mucopolysaccharide have been found to be present in

various animal and human tumors. Human tumors of mesodermal origin have been

reported to produce only one type of mucopolysaccharide， whi1e corresponding normal

tissues yield a variety of mucopolysaccharides1， 2).

Proteoglycans are present in the extracellular matrix of normal hyaline carti1age，

primari1y as aggregatesめ Solventswith 4M guanidinium chloride effectively dis-

sociate the aggregates and allow the proteoglycan molecules to be extracted from

the tissue引. The chemical and physical characteristics of such preparations isolated

from a variety of carti1age have been described1，5-7).

The present investigation was carried out to determine the uptake of 35S-sulfate

into chondrosarcoma mucopolysaccharides， the properties of the protein-polysaccharide，

and the isomers of the mucopolysaccharides in two cases of chondrosarcoma. To

date， few biochemical analyses of chondrosarcomas have been reported.

11. Materials and Methods

The material was obtained from two cases of chondrosarcoma of the pelvic bone.

For comparison， human adult articular carti1age from a hip and human embryonic

carti1age were used.

Received June 28， 1979; accepted for publication October 19， 1979.

- 92ー Connective Tissue

Case 1. Female， age 34. One year ago she had pains in the left hip; roentgeno司

grams showed cystic lesions in the acetabulum region. Biopsy was performed. His-

tological diagnosis: Chondrosarcoma. Replacement of the pelvis was performed by

endoprosthesis. There were no postoperative complications and the patient adapted

satisfactorily to her prosthesis.

Case 2. Male， age 33. Two years ago oliguria started and gradually worsened.

There was a nontender palpable mass in the lower abdomen. Laparotomy revealed

a tumor arising from the pubic bone: Histological diagnosis was chondrosarcoma.

The patient died of renal insufficiency. The tumor mass was sawed in two longitu-

dinally， and the cut was photographed.

One half of the bone taken from each patient was used for histological diagnosis

and the other for radioisotope studies. Tissue blocks measuring approximately 1 cm3

were cut from the tumors. In case 1， five tumor blocks， designated 1-5， were taken

for autoradiographic and morphologic examination (Fig. 1). Two blocks， one from

a peripheral and th巴 otherfrom a central area， were taken from the tumors for

biochemical analysis (Figs. 1，2).

Fig. 1. Case 1. R ight: In corporation of 35S.sulfate into various zones of the tumor and into normal joint cartilage. Left: Two blocks of peripheral (P) and central (C) area of tumors were taken for biochemical anal ysis.

N. I<awabe and S. Yamagata: Mucopolysaccharides and Proteoglycans of Chondr伺 arcomas - 93 -

Fig. 2. Case 2. Two blocks were taken for biochemical analysis.

Autoradiography and morphology

1‘umor blocks were incubated for two hr in 5 ml of Eagle's MEM solution

containing 100 μ1 of radioactive sulfate. The specimens were fixed in 10% buffered

formalin， embedded in paraffin wax， and 6-micron sections were stained with safranin

o fast green and iron haematoxylin (for acid mucopolysaccharides). For autoradio-

graphy， sections were coated with Sakura NR-M2 emulsion* and exposed at 40

C for

three or four weeks. The sections were then developed and poststained with haema-

toxylin-eosin.

35S incorporation

The slices used for autoradiography were washed in alcohol to remove the free

35S0.-'， then dried， weighed on a microbalance and solubilized with NCS solubilizer

at 450C. Portions of the solubilized samples were taken for indirect determination

of 3oS-sulfate incorporation into the the total glycosaminoglycan. Radioactivity was

measured with a liquid scintillation counter using PCS solubilizer.

Biochemical analysis

Tissue blocks cut from the central and peripheral areas of the tumors were sliced.

The slices were extracted with a solution of 4 M guanidine hydrochloride， 0.05 M

sodium acetate， 0.01 M sodiumεDT A， 0.1 M 6-aminohexanoic acid， and 0.005 M benza-

midine hydrochloride， pH 5.8， at 40C for 48 hr. Five volumes of the solution were

used per g of tissue.

本 Madein ]apan by Konishiroku Photo Ind. Co. Ltd.

- 94- Connective Tissue

1) Determination of molecular size of proteoglycan substrate

The 4 M guanidine hydrochloride tissue extract was brought to a density of 1.50 g

per ml by the addition of只solidCsCl (0.59 g of CsCl per ml of solution) and centri-

fuged in a Hitachi RS-65T rotor at 40，000 rpm for 44 hr at 20'C to produce a

density gradient. Proteoglycan subunits (PGS)， as described by Hascall et al'"ペwere

isolated from the bottom two-fifths of the tubes (p> 1.53). The PGS were dialyzed

against 0.5 M guanidine hydrochloride， 0.05 M Tris-HC1， pH 8.5， at 4 'C. The PGS

fraction was loaded on the top of a sucrose density gradient ranging from 5 to 20%

containing 4 M guanidine hydrochloride， pH 5.8， in a cellulose nitrate tube. The

density gradient was centrifuged at 24，000 rpm for 16 hr at 20'C in a SW 27.1 rotor

in a Beckman ultracentrifuge. The gradient was divided into fourteen fractions from

bottom to top. After each fraction had been dialyzed against 0.5 M Tris-HCl bu任er，

the uronic acid concentration was determined by the carbazole method.

2) Determination of isometric chondroitin sulfates

The 4 M guanidine hydrochloride extracts were dialyzed against 0.05 M Tris同HCl

buffer at 4 'C. The resulting solution was digested with pronase P (Kaken Kagaku

Company， Tokyo) in 0.05 M Tris-HCl buffer， pH 7.3 for 24 hr at 37'C， then treated

with 0.3 N NaOH at 37'C for 4 hr. The solution was brought to approximately pH 7

with hydrochloric acid and 60% trichloroacetic acid solution was added to a final

concentration of 5%. The solution was held at O'C for 12 hr， then centrifuged. The

supernatant was dialyzed against running tap water.

The crude acid mucopolysaccharides were precipitated with 3 vol of ethanol in

the presence of 1% potassium acetate overnight. The precipitate was washed with

ethanol and ether and dried in a vacuum over P2058).

For simultaneous determination of the three isomeric chondroitin sulfates， two

reaction mixtures and a blank mixture were prepared9，1O). The reaction mixtures

contained， in 50μ1， 10μ1 of enriched Tris-HCl bu妊er(pH 8.0， 0.05 M)， the test sample

(0.5 pmoles as uronic acid)， and either 0.2 units of chondroitinase-ABC* (tube 1) or

0.3 units of chondroitinase-ACネ (tube2). The blank lacked the enzymes. These

mixtures were incubated at 37'C for 30 min and then applied to Whatman No. 1

filter paper (60 cm long) in a stream of hot air. Descending development of the

chromatograms with 1-butyric acid-0.5N ammonia (5: 3 v jv) was normally performed

overnight at room temperature. After drying， the disaccharides were visualized with

a Minerali号ht. The areas containing disaccharides were cut out. Each excised sec-

tion was cut into small segments and placed in a centrifuge tube， to which was

added 2 ml of 0.01 N HCl. The tube was sealed and held at 50'C for 10 min， then

centrifuged. The absorbance of the supernatant solution was measured at 232 mμ

against the similarly treated blank.

3) Electrophoretic separation of acid mucopolysaccharides

The acid mucopolysaccharides of the connective tissue of the tumor were separa-

* Made in ]apan by Seikagaku Kogyo Co. Ltd.

，~目 Kawabe and S. Yamagata: Mucopolysaccharides and Prote岨 Iycansof Chondrosarccmas - 95ー

ted by eJectrophoresis on a cellulose acetate membrane with various solutions (0.2 M

calcium acetate solution' り， 1M acetic acid-pyridine solution'へor0.1 M ammonia-

veronal buffer13l).

Standard substances employed were commercial samples of chondroitin sulfates

A， B and C， heparin， hyaJuronic acid (Seikagaku Kogyo Co.)， bovine corneal kerato-

sulfate and shark cartilage keratopolysulfate， the last two a gift from Dr. Nakasawa

(Department of Biochemical Pharmacology， University of Meijo).

Cellulose acetate strips were dried in air， then stained with 0.5% toluidine blue

for 5 min， washed with 1% acetic acid for 1 min and tap water for 10 min， and dried.

111. Results

ド[orphologyand autoradiography

The histological examination of the central and peripheral areas of the tumors

verified that both cases were di釘erentiatedchondrosarcomas.

The central areas of case 1 showed a variation in cell size and lobulation without

a significant increase in the number of cells (Grade I-II). Staining with safranin 0

was marked (Fig. 3). The peripheral areas showed marked variation in cell size and

an increase in the number of cartilage cells， some of them with bizarre shapes (Grade

II). Staining of the matrix was moderate (Fig. 4).

The uptake of 35S-sulfate took place as a massive cellular incorporation. Dense

accumulations of silver grains were located over the matrix surrounding the cells.

The peripheral areas of the tumor showed a Iτ10St pronounced uptake of the isotope

Fig. 3. Chondrosarcoma， Grade [-ll. The central area of case 1 showed variation in size of cells and lobulation

without significant increase in number. (x 100)

- 96ー Connective Tissue

Fig. 4. Chondrosarcoma， Grade 11. The central area of case 1 showed marked variation in size and increased numbers

of cartilage cells， some of them with bizarre shapes. (x 100)

Fi冨.5. Coated autoradiogram showing the incorporation of SoS.sulfate into a normal

cartilage. (x 100)

N. Kawabe and S. Yamagata: Muco回 lysaccharidesand ProteoglYClns of Chondrosarcomas - 97ー

Fig. 6. Coated autoradiogram showing the incorporation of 35S.sulfate into a

chondrosarcoma (Grade 11). (x 200)

Fig. 7. Chondrosarcoma， Grade II-III. Markedly increased number of cartilage cells with pleomorphism of the nucl巴i.(xlOO)

- 98- Connective Tissue

Fig. 8. Chondrosarcoma， Grade [[1. Marked spindling and dedifferentiation of neoplasm. (x 200)

compared with that of normaI cartilage (Figs. 5，6).

The centraI area of case 2 showed a marked increase in cellularity and pleomor-

phism (Fig. 7)， and the peripheraI area showed marked spindling ancl dedifferentiation

of the neoplasm (Fig. 8).

35S incorporation

Table 1 shows the incorporation of radioactive sulfate into the total mucopoly-

saccharide of various areas of case 1， adjacent hip joint cartilage and human embr-

yonic cartilage (3 months).

The concentrations of isotope retained per mg of dry tissue are expressed as

counts per minute (cpm) and percentages of the corresponcling samples of control

Table 1. Incorporation of 35S.sulfate

Case 1 cpm/mg

4293

2 3560

3 726

4 2669

5 1114

Embryonic cartilage 6605

Articular cartilage 246

% of control

1745

1447

295

1085

453

2684

100

Incorporation of 35S.sulfat巴 (totalmucopolysaccharide fraction) into various zones of the tumor (1-5) and into normal cartilage and human embryonic car. tilage as controls. Radioacti、ityexpressed as counts per minute per mg dry tLssue.

N. Kawabe and S. Yamagata: Mucopolysaccharides and Proteoglycans of Chondrosarcomas - 99ー

articular cartilage. These data showed the generally high uptake of the isotope in

embryonic tissues and in chondrosarcomas. Hawever， there were also considerable

variations between samples of the same tumor. The radioactivity of the tumor sam-

ples was 3 to 17 times that of control cartilage.

Molecular size of proteoglycan substrate

When minced tissue was extracted with 10 volumes of solvents containing 4 M

guanidine hydrochloride， 95% of the total hexouronic acid was solubilized in 5 hr.

The sedimentation behavior of these materials (PGS) on a sucrose density gradient

is shown in Fig. 9 for cases 1 and 2 and embryo tissue. These profiles show a

bimodal distribution. The gradients of tube Nos. 3-5 show the heavy proteoglycan

(component H) and of Nos. 10-12 the light proteoglycan (component L). About 70%

of the proteoglycan was component H.

0.6

0.5

0.4

。円 0.3<0

《

0.2

o . 1

Case 1

Case 2

一一一一 Embryo Cart i I age

2 3 4 5 6 7 8 9 10 11 12 13 14

Tube No.

Fig. 9. Th巴 curvesshow h巴xouronicacid from sucrose density gradient of cases 1 and 2， and human巴mbryoniccartilage.

Analysis of isometric chondroitin sulfates

The results of isometric chondroitin sulfate analyses of these samples are shown

in Table 2. Silver nitrate staining of paper chromatographic separation of disac-

charides is shown in Fig. 1011). 1n case 1， the L1Di-4S and L1Di-6S were present in

comparable amounts to each other and to those in embryonic cartilage. 1n case 2，

the L1Di-6S was more prevalent than the L1Di-4S especially in the central area， re-

-100 - Connective Tissue

Table 2. Unsaturated disaccharides generated by digestion of proteoglycan with chondroitinase.

Crude AMPS LlDi.4S LlDi-6S LlDi-OS HU μmole/mg % % % %

Center 240 42_2 48.9 7.4 1.5 Case 1

Periphuy 257 42.0 36. 0 19_ 8 2.2

Center 225 34.5 51. 7 11. 0 1.8 Case 2

P号riphery 231 7.6 81. 0 7.5 3. 1

Embryonic cartilage 188 36. 5 46.0 17.5

Articular cartilage 152 11. 2 80.1 3.0 5. 7

ChS-A， B and C: calculated as isometric chondroitin sulfate digested by chondroitinase from various samples. Expressed as percentage of total.

む

/ぷシ 示 β。 ぷ， 〆 ι 〆 J 〆 red〆〆グ62f，c ぷ?c

mMk Casel C<lse 1 Cas.・2 C・se2 EmbflO Embr10 "'~~I:t f I ca'fi.1

Fig. 10. Paper chromatographic separation of disaccharides obtained by digestion of samples with chondroitinase-ABC and chondroitinase-AC.

sembling adult articular cartilage. Nonsulfated disaccharides of the tumors were

found to comprise about 10-20% of total disaccharides. Residues not digested by

chondroitinase were not measured.

Electrophoretic determination of mucopolysaccharide

On electrophoresis with calcium acetate solution as the electrolytic medium，

N. Kawabe and S. Yamagata: MucoDOlysaccharides and Proteoglycans of Chondrosarcomas -. 101ー

μ

i

s

H

一MN

・h

Fig. 11. Electrophoretic separation of sample mucopolysaccharides on cellulose ac巴tatepaper.

cases 1 and 2 showed only one bandspot， which corresponded to chondroitin sulfate

A or C (Fig. 11). With other electrolytic media， the same compact spots were ob-

tained. Keratosulfate was not found in either case 1 or 2.

IV. Discussion

SるSuptake into total glycosaminoglycan was found to te hgher in tr.e chon-

drosarcomas than in normal joint cartilage. The chondrosarcomas exhibited the most

intense uptake at the periphery， with weak uptake in the central zones. Similar

autoradiographic patterns have been reported in human studi.es in ViV015・16-2り andin

vi tro22 • 23)

The higher uptake by tumor tissue seems to be due to its greater cellularity and

presumably an accelerated turnover of glycosaminoglycans. One indicator of a higher

synthetic rate was the greater accumulation of the sil ver grains found over the tumor

matrix than in normal cartilage. Probably， 35S-sulfate incorporation in tumor tissue

in vitro adequately reflects the corresponding process in vivo.

-102- Connective Tissue

Proteoglycans are present in the extracellular matrix of normal hyaline cartilages

as aggregates3). The chemical and physical characteristics of proteoglycan prepara-

tions isolated from a variety of cartilages have been described5，2J). The isolation of

proteoglycans from chondrosarcomas by procedures described by Hascal1 et al. yielded

preparations containing partial1y degraded proteoglycansJ，め.

Kimata et al.25) revealed that the epiphysis of 12-day-old chick embryo consisted

of two macromolecular species of different molecular weight by linear sucros巴density

gradient analysis. The more rapidly sedimenting component wi1l be referred to here

as component H and the more slowly sedimenting component as component L. Com-司

ponent H comprised about 90% of the total uronic acid. They stated that the chon-

droitin sulfate chains of component L always had a larger average size and a higher

4-to 6-sulfate ratio. When the cartilage slices were pulse-labeled with 35S-1abeled

sulfate， component L had a much higher specific radioacti vity than component H.

Their chase experiments indicated that the radioactivity in component L associated

with the Golgi membranes decreased rapidly.

The bimodal curve presented here is similar to those obtained by other authors26，27).

Palmoski et al. concluded that peak 1 represents cartilage-specific proteochondroitin

sulfate while peak II represents non-specific proteochondroitin sulfate， from the results

obtained for cultured chondrocytes treated with 5-bromo-2'-deoxyuridine (BUdR) and

sternal cartilage from nanomelic chick embryos. This conclusion is consistent with

the hypothesis that BUdR inhibits synthesis of cel1-specific products in chondrocytes28).

If our data correspond to the sedimenting components of Kimata and Palmoski et al.，

the two macromolecular species from the chondrosarcomas might represent differen-

tiated phenotypic expression. ln this study， the slower component of proteoglycan

was more prevalent in case 2， in which high malignancy was found， than in case l.

The higher proportion of the component L in case 2 seems to be due to a decrease

in the amount of component H， which is regarded as the cartilage-specific component.

This may be the result of dedifferentiation of cartilage cel1s.

Reports concerning the composition of the mucopolysaccharide of chondrosarcomas

are very scarce and differ in respect to the

N. Kawabe and S. Yamagata: Mucopolysaccharides and Proteoglycans of Chondrosarcomas -103

In case 2， the higher content of chondroitin sulfate C than in case 1 is interes-

ting in view of the fact that the patient died and histology showed a high malignancy

of grade III. Hasegawa reported that chondroitin sulfate C is the main constituent

of chondrosarcomas and that the activity of chondroitin transferase increases with

the malignancy of the chondrosarcoma山.

TheムDiOScontent was found to be approximately 7 to 20% in cases 1 and 2.

The content of acid mucopolysaccharide as a polymer was detected by electrophoresis.

Cases 1 and 2 showed only one band-spot of chondroitin sulfate A and C. Pr巴su-

mably， theムDiOScontent seems to be low sulfation of Ch-SA， or ChS-C.

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← 104 Connective Tissue

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N. Kawabe and S. Yamagata: MucopoJysaccharides and ProteogJycans of Chondrosarcomas - 105-

別刷請求先:(干701-01) 岡山県倉敷市松島577

川崎医科大学整形外科

川部直巳

Reprint requests to，'

Dr. Naomi Kawabe

Department of Orthopaedic Surgery， Kawasaki Medical School， Matsushima 577， Kurashiki 701-01， japan