Proc. Nati. Acad. Sci. USA Vol. 74, No. 10, pp. 4173-4177, October 1977 Biochemistry
Cellular transformation and differentiation. Effect of Rous sarcoma virus transformation on sulfated proteoglycan synthesis by chicken chondrocytes* (chondroitin sulfate/tumorization/cartilage cells/developmental biology/oncogenic virus)
MASAHIRO MUTOt, MAKOTO YOSHIMURA, MINORU OKAYAMA*, AND AKIRA KAJI§ Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174
Communicated by Louis B. Flexner, June 21,1977
ABSTRACT Incorporation of sulfate into sulfated proteoglycans by isolated chicken chondrocytes was inhibited up to 74% by transformation with the Rous sarcoma virus, and a similar inhibitory effect was observed on acetate incoration into chondroitin sulfate. Slower sedimenting sulfated proteoglycans appear after the viral transformation. The ratio of chondroitin 4-sulfate to chondroitin 6-sulfate in these slower sedimenting sulfated proteoglycans was different from that of normal chondrocytes, but the chain lengths of sulfated glycosaminoglycans produced by normal chondrocytes and transformed chondrocytes were not significantly different. Chondrocytes were also infected with a temperature-sensitive mutant of RSV, ts LA24, which has a temperature-sensitive lesion in the transforming gene. Hyaluronic acid production by these cells was increased, and the slower sedimenting sulfated proteoglycan was produced only at the permissive temperature. Recently, we reported (1) that chondrocytes isolated from 10-day-old chicken embryos can be transformed by either wild-type Rous sarcoma virus (RSV) or RSV ts LA24, a class T temperature-sensitive mutant of RSV. Reversible morphological changes as well as changes in incorporation of [14C]acetate into hyaluronic acid by these cells was observed upon transformation by RSV ts LA24. In our previous studies (1-5), attempts had been made to correlate cell transformation with dedifferentiation, and the effect of transforming gene products on the maintenance of differentiation (2) was studied. In our continued effort to understand the effect of viral transformation on the differentiation of specific cells, we have turned our attention to the synthesis of sulfated proteoglycans and hyaluronic acid by chicken chondrocytes transformed by RSV. It had been shown that chondrocytes synthesize sulfated proteoglycans specific for cartilage and the recent study indicated that the sedimentation behavior of sulfated proteoglycans appears to vary depending on the stage of differentiation of the chondrocytes (6). These results indicate that the analysis of sulfated proteoglycans may provide a useful marker for the stage of differentiation of chondroblastic cells. In this communication we report that, when chicken chondrocytes are transformed by RSV, the amount of inorganic sulfate incorporated into sulfated proteoglycan is markedly reduced. In addition, sulfated proteoglycans synthesized by transformed cells are qualitatively different from those of normal chondrocytes, which suggests that a core protein of slower sedimenting sulfated proteoglycan may be changed to a different protein upon transformation. This is consistent with the notion that cell transformation causes dedifferentiation (1-5). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 4173
MATERIALS AND METHODS Labeling of Chondrocytes. Chondrocytes and RSV-transformed chondrocytes were prepared as described (1). After 5 days' incubation, 5 uCi of [14C]acetate (New England Nuclear Co., 220,uCi/umol) or 5 ,Ci of 35S04 (845 mCi/mmol) in 1 ml of medium was added to the chondrocyte culture dish. The incorporation of [14C]acetate or -3G4 into proteoglycans was carried out for 6 or 24 hr. The celllayer was then washed three times with minimum essential medium, and labeled proteoglycans were isolated from the cell layer as well as from the medium according to Kimata et al. (7). Analysis of Glycosaminoglycan by Chondroitinase ABC and Hyaluronidase Digestion. After the final precipitation with ethanol, the proteoglycans were suspended in 0.9 ml of 0.1 M Tris-HCI, pH 8.0/7% ethanol containing 1 mg of Pronase and incubated for 24 hr at 410. The mixture was then boiled for 5 min and centrifuged; 0.7 umol glucuronic acid equivalents of chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronic acid were added to the supernatant. After digestion, the glycosaminoglycans were washed by repeated precipitation with ethanol to eliminate labeled small molecules produced by Pronase digestion and dissolved in 230 ,l of 0.15 M NaCl/0.3 M Na acetate, pH 6.0, containing 30 units of Streptomyces hyaluronidase (Amano Co., Nagoya, Japan). After the completion of the hyaluronidase digestion (20 hr at 410), 60,ul of reaction mixture was spotted on Whatman 3MM paper which was developed by an ascending method with 1-butyric acid/0.5 M NH40H, 5:3 (vol/vol), for approximately 30 hr (8). The paper was cut into 1-cm-wide strips and each strip was then assayed for radioactivity in a Packard scintillation counter. Another portion of each sample (150,Al), after purification by alcohol precipitation, was digested with 5 units of chondroitinase ABC in 0.1 M Tris.HCI, pH 7.9, at 370 for 20 hr. The digest was spotted on Whatman 3MM paper and developed as described above. Sucrose Density Gradient Analysis of Proteoglycans. After the isolation of proteoglycans as described (7), 500,ul of the sample was layered onto 16 ml of 5-20% linear sucrose gradient containing 4 M guanidine.HCI/0.05 M Na acetate buffer, pH 5.8, and centrifuged in a SW 27.1 rotor of a Spinco L ultracentrifuge at 25,000 rpm at 200 for 28 hr; 0.5-ml fractions were collected from the bottom. In some experiments, inhibitors of Abbreviation: RSV, Rous sarcoma virus. * This is paper no. 6 in this series from this laboratory. The preceding five papers are refs. 1-5. t Present address: National Institute of Radiological Sciences, Anagawa, Chiba, Japan. * Present address: Blood Institute Center in Nagoya National Hospital, 3-no-Maru, Naka-ku, Nagoya, Japan. § To whom reprint requests should be addressed.
Proc. Nat. Acad. Sci. USA 74 (1977)
Biochemistry: Muto et al. Table 1. [35SJSulfate incorporation into sulfated proteoglycans
4174
of normal and transformed chondrocytes 35S, cpm/$tg of DNA RSV-transformed Normal Labeling time, hr chondrocytes chondrocytes 45,037 6 11,626 44,603 163,320 24 Extraction and purification of sulfated proteoglycans were carried out to the step prior to the proteolytic digestion.
proteolytic enzymes were added and identical results were obtained (9).
A
rial. Analysis of Sulfated Glycosaminoglycans. To examine the
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RESULTS Incorporation of [35S]Sulfate into Sulfated Proteoglycans by Normal Chondrocytes and RSV-Transformed Chondrocytes. When cells were infected by RSV for approximately 5-6 days prior to addition of 35SO4, total incorporation of 35SO4 into the sulfated proteoglycans fraction decreased to approximately 26% of control (Table 1). This decrease could be due to rapid degradation of synthesized sulfated proteoglycan by transformed chondrocytes. However, this is unlikely because the incorporation of sulfate is linear for 6 hr of incubation and steadily increases up to 24 hr or possibly more. In addition, the inhibitory effect of RSV transformation on the sulfate incorporation into sulfated proteoglycan was similar at 24 and 6 hr. Incorporation of [14CJAcetate into Hyaluronidase-Resistant Glycosaminoglycans. An experiment similar to that shown in Table 1, using [14C]acetate as a precursor of sulfated glycosaminoglycans, was performed. In this experiment, [14C]acetate-labeled glycosaminoglycans were treated with hyaluronidase. After a 24-hr incorporation period, hyaluronidaseresistant glycosaminoglycans from normal chondrocytes contained 68,796 cpm/,gg of DNA and those from RSV-transformed chondrocytes contained 32,121 cpm/,ug of DNA. Because chondroitin sulfate is the major glycosaminoglycan resistant to hyaluronidase, these values represent mostly chondroitin sulfate. Although inhibition (53%) was not as extensive as with sulfate incorporation (74%), the data are consistent with the concept that the transformation of the chondrocytes by RSV causes decreased formation of chondroitin sulfate. Sedimentation Behavior of Sulfated Proteoglycans Synthesized by Normal and Transformed Chondrocytes. In the experiment illustrated in Fig. 1, the cells were labeled with 35SO4 for 6 hr. Sulfated proteoglycans were isolated and subjected to sucrose gradient centrifugation. As shown in Fig. 1B, one additional peak of sulfated proteoglycan (peak II) and relative increase of a slower sedimenting sulfated proteoglycans (peak III) were observed in the material from the cell layer of the RSV-transformed chondrocytes. Peak III may correspond to the "ubiquitous" proteoglycan discussed by Goetinck et al. (10). The medium from transformed chondrocytes contained proportionally greater amounts of this slower sedimenting sulfated proteoglycan. It should be noted that the major peak of sulfated proteoglycan excreted into the medium by normal and transformed cells sedimented similarly to peak II of the cell-bound material of transformed chondrocytes. However, as discussed in the following section, analysis of chondroitin sulfate of excreted material clearly showed that this sulfated proteoglycan is not related to peak II of the cell-bound mate-
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Fraction
FIG. 1. Sucrose density gradient centrifugation analysis of
35SO4-labeled sulfated proteoglycans. Sedimentation was from right
to left. After centrifugation, 0.1 ml of each 0.5-ml fraction was assayed for radioactivity. (A) Control cell layer; (B) RSV (wild type)-infected cell layer; (C) control medium; (D) RSV (wild type)-infected cell's medium. Cells were labeled for 6 hr, and data are expressed as cpmhtg of DNA present in the total amount of sample applied to the gradient.
possibility that the sulfated glycosaminoglycans of RSVtransformed chondrocytes may be qualitatively different from those of normal chondrocytes, the fractions of each peak of Fig. 1 were pooled and the relative amounts of chondroitin 6-sulfate, chondroitin 4-sulfate, and other sulfated glycosaminoglycans were determined. This was done by digestion of the radioactive material with chondroitinase ABC followed by paper chromatography (Fig. 2). As shown in Table 2, the compositions of chondroitin sulfate from peaks I, II, and III of the material bound to the cell layer were different from each other. For example, chondroitinase ABC-resistant materials were as much as 30-40% in peak III but only 4-7% in peaks I and II. This material in peak I is probably keratosulfate, as has been reported to exist in cartilage (11), whereas that in peak III may be heparan sulfate (6). Relatively large amounts of chondroitin 4sulfate were observed in peak III; peak I contained a smaller amount of chondroitin 4-sulfate compared to chondroitin 6sulfate. Peak II was not similar to peak I with respect to relative amounts of chondroitin 6-sulfate and chondroitin 4-sulfate. .Q
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FIG. 2. Paper chromatographic analysis of chondroitinase ABC digests of labeled glycosaminoglycans free of hyaluronic acid. Cells were labeled with [W4C]acetate for 24 hr, and the sulfated glycosaminoglycans were digested with chondroitinase ABC and paper chromatographed for 40 hr. (Upper) Radioactivity of each strip of paper chromatogram. The values are per Ag of DNA present in the digests applied at the origin. 0, Normal; 0, transformed. (Lower) Spots as located by UV light.
Biochemistry:
Proc. Nat. Acad. Sci. USA 74 (1977)
Muto et al.
4175
Table 2. Analysis of cell-bound sulfated glycosaminoglycans fractionated on sucrose density gradient CH-ase ABC% of total Ratio: CH 4-SO4 CH 6-SO4 resistant %t cpm 6-SO4/4-SO4 cpm cpm cpm
Peak* III I
1,304 3,308
41.0 4.0
626 46,186
III II I
846 182 152
30.7 7.4 4.8
571 1,024
1,458
Normal chondrocytes 30.4 966 19.7 35.7 29,205 56.4 chondrocytes RSV-infected 38.9 1,071 20.7 42.7 1,044 41.8 37.0 48.0 1,122
0.65 1.58
2.2 84.8
0.53 0.98 1.30
15.7 37.0 28.3
CH-chondroitin.
* Peaks are numbered as in Fig. 1. t Percentage is of the total sulfated glycosaminoglycan of each peak.
Also, incorporation of a5SO4 into peak I was uniformly inhibited (>95%) upon transformation regardless of species of glycosaminoglycans. On the other hand, the incorporation into peak III was not significantly inhibited by RSV-transformation for any of the three sulfated glycosaminoglycans. Because peak III is qualitatively different from peak I, the relative increase of proportion of peak III upon transformation is not a result of degradation by a heightened proteolytic enzyme activity in the transformed chondrocytes (9). In sulfated glycosaminoglycans of proteoglycans excreted into the medium by normal chondrocytes, the ratio of chondroitin 6-sulfate to chondroitin 4sulfate was approximately the same. This suggests that the major sulfated proteoglycans found in the medium of normal cells were the direct degradation products of the cell-bound material but not related to peak II of the material bound to the transformed chondrocytes. C
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To examine the possibility that glycosidases of the transformed cells may have degraded the glycosaminoglycan chain, glycosaminoglycan portions of sulfated proteoglycans were analyzed on a Sephadex G-200 column. As shown in Fig. 3, the elution patterns of s5SO4-labeled- glycosaminoglycans were similar, regardless of transformation. These experiments appear to indicate, therefore, that the chain length of glycosaminoglycan portions has not been drastically altered by transformation with RSV. Temperature-Dependent Hyaluronic Acid Synthesis by Chondrocytes Infected with RSV ts LA24. In a previous communication (1), we established that the morphological change caused by transformation of chondrocytes is reversible by the use of the temperature-sensitive mutant, RSV ts LA24. To substantiate further that the chondrocytes' chemical characteristics are also reversibly changed, incorporation of [14C]acetate into hyaluronic acid was studied at two temperatures with chondrocytes infected with RSV ts LA24. As shown in Table 3, chondrocytes transformed by this mutant virus had a higher [14C]acetate incorporation into hyaluronic acid when incubated at 370 (the permissive temperature for the transforming gene) than when incubated at 41° (the nonpermissive temperature). The chondrocytes infected with RSV ts LA24 changed their characteristics in the expected direction within 24 hr after the temperature shift. Normal chondrocytes incorporated more [14C]acetate into hyaluronic acid at 410 than
0
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Table 3. Effect of temperature on incorporation of [14C]acetate into hyaluronic acid by normal and RSV ts LA 24-infected chondrocytes Radioactive hyaluronic acid formed Ratio: medium/cell Temp. shift cpm/ug of DNA
D
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370 - 370
Normal chondrocytes 1,246 3,349
370 - 370
6,293 Infected chondrocytes 16,732 13,800 6,544 8,351
15
410 ,.370 410 - 41° 370 - 41° 10
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F raction
FIG. 3. Sephadex G-200 gel filtration of chondroitin sulfate of normal and wild-type RSV-transformed chondrocytes. 35SO4-Labeled sulfated glycosaminoglycan samples were subjected to gel filtration on Sephadex G-200 column (1.5 X 70 cm). The arrows indicate the void volume of the column. (A) Control cell layer, 130 x 103 cpm; (B) RSV-infected cell layer, 171 X 103 cpm; (C) control medium, 31.2 X 103 cpm; (D) infected cell medium, 9.0 X 103 cpm.
410-37° 410 - 410 37°-41°
6,130
0.38 0.57 1.07 1.53 1.02 1.24 0.94 0.91
RSV-infected cells were kept at the first temperature for 5 days and then at either the same or a different temperature for 28 hr, followed by incubation with [14C]acetate for 24 hr.
4176
Biochemistry:
Proc. Nat. Acad. Sci. USA 74 (1977)
Muto et al.
Table 4. Effect of temperature on the chondroitin sulfate formed by RSV ts LA24-infected chondrocytes [14C]Acetate incorporated, cpm/,g of DNA Ratio: CH 6-SO44-SO4 1.54 1,572 4,780 370 370 (9.4%) (28.5%) 2.58 4,053 7,992 410 - 370 (11.0%) (21.6%) 2.95 6,129 8,746 25,766 410 - 410 (13.5%) (19.2%) (56.6%) (6.5%) 2.41 4,350 6,661 16,058 2,085 370 - 410 (14.1%) (21.5%) (51.9%) (6.7%) Experimental conditions were as in Table 3. Numbers in parentheses are percentage of radioactivity in total labeled hyaluronidase-resistant CH-ase ABCresistant GAG 1,697 (10.1%) 3,048 (8.2%) 2,948
Temp. shift -
CH 6-SO4 7,394 (44.1%) 20,586 (55.7%)
CH 4-SO4
glycosaminoglycans. Control experiment with normal chondrocytes showed no appreciable change in the ratio, 6-S04/4-S04, with shift of temperature. CH = chondroitin; GAG = glycosaminoglycan. at 370. Therefore, the effect of the activation of the transforming gene product can overcome the effect of the temperature alone. Effect of Temperature on the Synthesis of Chondroitin Sulfate by Cells Infected with RSV ts LA24. Chondrocytes infected with RSV ts LA24 were studied to determine if the qualitative change of sulfated proteoglycans observed with the transformed chondrocytes is reversible depending on the activity of the transforming gene product (Table 4). At 370, these cells incorporated less [14C]acetate into chondroitin 6-sulfate than into chondroitin 4-sulfate as compared to the normal chondrocytes. With a temperature shift from 410 to 370, the incorporation of [14C]acetate into chondroitin sulfate decreased but did not reach the level of those cells kept at 370 from the time of infection. The ratio of chondroitin 6-sulfate to chondroitin 4-sulfate also decreased during the temperature shift down and the opposite was observed when temperature was raised. Fig. 4 indicates that change in the sedimentation behavior of sulfated proteoglycans as described in Fig. 1 can also be demonstrated with cells infected with RSV LA ts 24 at the permissive (370) and at the nonpermissive (410) temperatures.
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Cellular transformation and differentiation. Effect of Rous sarcoma virus transformation on sulfated proteoglycan synthesis by chicken chondrocytes* (chondroitin sulfate/tumorization/cartilage cells/developmental biology/oncogenic virus)
MASAHIRO MUTOt, MAKOTO YOSHIMURA, MINORU OKAYAMA*, AND AKIRA KAJI§ Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174
Communicated by Louis B. Flexner, June 21,1977
ABSTRACT Incorporation of sulfate into sulfated proteoglycans by isolated chicken chondrocytes was inhibited up to 74% by transformation with the Rous sarcoma virus, and a similar inhibitory effect was observed on acetate incoration into chondroitin sulfate. Slower sedimenting sulfated proteoglycans appear after the viral transformation. The ratio of chondroitin 4-sulfate to chondroitin 6-sulfate in these slower sedimenting sulfated proteoglycans was different from that of normal chondrocytes, but the chain lengths of sulfated glycosaminoglycans produced by normal chondrocytes and transformed chondrocytes were not significantly different. Chondrocytes were also infected with a temperature-sensitive mutant of RSV, ts LA24, which has a temperature-sensitive lesion in the transforming gene. Hyaluronic acid production by these cells was increased, and the slower sedimenting sulfated proteoglycan was produced only at the permissive temperature. Recently, we reported (1) that chondrocytes isolated from 10-day-old chicken embryos can be transformed by either wild-type Rous sarcoma virus (RSV) or RSV ts LA24, a class T temperature-sensitive mutant of RSV. Reversible morphological changes as well as changes in incorporation of [14C]acetate into hyaluronic acid by these cells was observed upon transformation by RSV ts LA24. In our previous studies (1-5), attempts had been made to correlate cell transformation with dedifferentiation, and the effect of transforming gene products on the maintenance of differentiation (2) was studied. In our continued effort to understand the effect of viral transformation on the differentiation of specific cells, we have turned our attention to the synthesis of sulfated proteoglycans and hyaluronic acid by chicken chondrocytes transformed by RSV. It had been shown that chondrocytes synthesize sulfated proteoglycans specific for cartilage and the recent study indicated that the sedimentation behavior of sulfated proteoglycans appears to vary depending on the stage of differentiation of the chondrocytes (6). These results indicate that the analysis of sulfated proteoglycans may provide a useful marker for the stage of differentiation of chondroblastic cells. In this communication we report that, when chicken chondrocytes are transformed by RSV, the amount of inorganic sulfate incorporated into sulfated proteoglycan is markedly reduced. In addition, sulfated proteoglycans synthesized by transformed cells are qualitatively different from those of normal chondrocytes, which suggests that a core protein of slower sedimenting sulfated proteoglycan may be changed to a different protein upon transformation. This is consistent with the notion that cell transformation causes dedifferentiation (1-5). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 4173
MATERIALS AND METHODS Labeling of Chondrocytes. Chondrocytes and RSV-transformed chondrocytes were prepared as described (1). After 5 days' incubation, 5 uCi of [14C]acetate (New England Nuclear Co., 220,uCi/umol) or 5 ,Ci of 35S04 (845 mCi/mmol) in 1 ml of medium was added to the chondrocyte culture dish. The incorporation of [14C]acetate or -3G4 into proteoglycans was carried out for 6 or 24 hr. The celllayer was then washed three times with minimum essential medium, and labeled proteoglycans were isolated from the cell layer as well as from the medium according to Kimata et al. (7). Analysis of Glycosaminoglycan by Chondroitinase ABC and Hyaluronidase Digestion. After the final precipitation with ethanol, the proteoglycans were suspended in 0.9 ml of 0.1 M Tris-HCI, pH 8.0/7% ethanol containing 1 mg of Pronase and incubated for 24 hr at 410. The mixture was then boiled for 5 min and centrifuged; 0.7 umol glucuronic acid equivalents of chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronic acid were added to the supernatant. After digestion, the glycosaminoglycans were washed by repeated precipitation with ethanol to eliminate labeled small molecules produced by Pronase digestion and dissolved in 230 ,l of 0.15 M NaCl/0.3 M Na acetate, pH 6.0, containing 30 units of Streptomyces hyaluronidase (Amano Co., Nagoya, Japan). After the completion of the hyaluronidase digestion (20 hr at 410), 60,ul of reaction mixture was spotted on Whatman 3MM paper which was developed by an ascending method with 1-butyric acid/0.5 M NH40H, 5:3 (vol/vol), for approximately 30 hr (8). The paper was cut into 1-cm-wide strips and each strip was then assayed for radioactivity in a Packard scintillation counter. Another portion of each sample (150,Al), after purification by alcohol precipitation, was digested with 5 units of chondroitinase ABC in 0.1 M Tris.HCI, pH 7.9, at 370 for 20 hr. The digest was spotted on Whatman 3MM paper and developed as described above. Sucrose Density Gradient Analysis of Proteoglycans. After the isolation of proteoglycans as described (7), 500,ul of the sample was layered onto 16 ml of 5-20% linear sucrose gradient containing 4 M guanidine.HCI/0.05 M Na acetate buffer, pH 5.8, and centrifuged in a SW 27.1 rotor of a Spinco L ultracentrifuge at 25,000 rpm at 200 for 28 hr; 0.5-ml fractions were collected from the bottom. In some experiments, inhibitors of Abbreviation: RSV, Rous sarcoma virus. * This is paper no. 6 in this series from this laboratory. The preceding five papers are refs. 1-5. t Present address: National Institute of Radiological Sciences, Anagawa, Chiba, Japan. * Present address: Blood Institute Center in Nagoya National Hospital, 3-no-Maru, Naka-ku, Nagoya, Japan. § To whom reprint requests should be addressed.
Proc. Nat. Acad. Sci. USA 74 (1977)
Biochemistry: Muto et al. Table 1. [35SJSulfate incorporation into sulfated proteoglycans
4174
of normal and transformed chondrocytes 35S, cpm/$tg of DNA RSV-transformed Normal Labeling time, hr chondrocytes chondrocytes 45,037 6 11,626 44,603 163,320 24 Extraction and purification of sulfated proteoglycans were carried out to the step prior to the proteolytic digestion.
proteolytic enzymes were added and identical results were obtained (9).
A
rial. Analysis of Sulfated Glycosaminoglycans. To examine the
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RESULTS Incorporation of [35S]Sulfate into Sulfated Proteoglycans by Normal Chondrocytes and RSV-Transformed Chondrocytes. When cells were infected by RSV for approximately 5-6 days prior to addition of 35SO4, total incorporation of 35SO4 into the sulfated proteoglycans fraction decreased to approximately 26% of control (Table 1). This decrease could be due to rapid degradation of synthesized sulfated proteoglycan by transformed chondrocytes. However, this is unlikely because the incorporation of sulfate is linear for 6 hr of incubation and steadily increases up to 24 hr or possibly more. In addition, the inhibitory effect of RSV transformation on the sulfate incorporation into sulfated proteoglycan was similar at 24 and 6 hr. Incorporation of [14CJAcetate into Hyaluronidase-Resistant Glycosaminoglycans. An experiment similar to that shown in Table 1, using [14C]acetate as a precursor of sulfated glycosaminoglycans, was performed. In this experiment, [14C]acetate-labeled glycosaminoglycans were treated with hyaluronidase. After a 24-hr incorporation period, hyaluronidaseresistant glycosaminoglycans from normal chondrocytes contained 68,796 cpm/,gg of DNA and those from RSV-transformed chondrocytes contained 32,121 cpm/,ug of DNA. Because chondroitin sulfate is the major glycosaminoglycan resistant to hyaluronidase, these values represent mostly chondroitin sulfate. Although inhibition (53%) was not as extensive as with sulfate incorporation (74%), the data are consistent with the concept that the transformation of the chondrocytes by RSV causes decreased formation of chondroitin sulfate. Sedimentation Behavior of Sulfated Proteoglycans Synthesized by Normal and Transformed Chondrocytes. In the experiment illustrated in Fig. 1, the cells were labeled with 35SO4 for 6 hr. Sulfated proteoglycans were isolated and subjected to sucrose gradient centrifugation. As shown in Fig. 1B, one additional peak of sulfated proteoglycan (peak II) and relative increase of a slower sedimenting sulfated proteoglycans (peak III) were observed in the material from the cell layer of the RSV-transformed chondrocytes. Peak III may correspond to the "ubiquitous" proteoglycan discussed by Goetinck et al. (10). The medium from transformed chondrocytes contained proportionally greater amounts of this slower sedimenting sulfated proteoglycan. It should be noted that the major peak of sulfated proteoglycan excreted into the medium by normal and transformed cells sedimented similarly to peak II of the cell-bound material of transformed chondrocytes. However, as discussed in the following section, analysis of chondroitin sulfate of excreted material clearly showed that this sulfated proteoglycan is not related to peak II of the cell-bound mate-
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FIG. 1. Sucrose density gradient centrifugation analysis of
35SO4-labeled sulfated proteoglycans. Sedimentation was from right
to left. After centrifugation, 0.1 ml of each 0.5-ml fraction was assayed for radioactivity. (A) Control cell layer; (B) RSV (wild type)-infected cell layer; (C) control medium; (D) RSV (wild type)-infected cell's medium. Cells were labeled for 6 hr, and data are expressed as cpmhtg of DNA present in the total amount of sample applied to the gradient.
possibility that the sulfated glycosaminoglycans of RSVtransformed chondrocytes may be qualitatively different from those of normal chondrocytes, the fractions of each peak of Fig. 1 were pooled and the relative amounts of chondroitin 6-sulfate, chondroitin 4-sulfate, and other sulfated glycosaminoglycans were determined. This was done by digestion of the radioactive material with chondroitinase ABC followed by paper chromatography (Fig. 2). As shown in Table 2, the compositions of chondroitin sulfate from peaks I, II, and III of the material bound to the cell layer were different from each other. For example, chondroitinase ABC-resistant materials were as much as 30-40% in peak III but only 4-7% in peaks I and II. This material in peak I is probably keratosulfate, as has been reported to exist in cartilage (11), whereas that in peak III may be heparan sulfate (6). Relatively large amounts of chondroitin 4sulfate were observed in peak III; peak I contained a smaller amount of chondroitin 4-sulfate compared to chondroitin 6sulfate. Peak II was not similar to peak I with respect to relative amounts of chondroitin 6-sulfate and chondroitin 4-sulfate. .Q
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FIG. 2. Paper chromatographic analysis of chondroitinase ABC digests of labeled glycosaminoglycans free of hyaluronic acid. Cells were labeled with [W4C]acetate for 24 hr, and the sulfated glycosaminoglycans were digested with chondroitinase ABC and paper chromatographed for 40 hr. (Upper) Radioactivity of each strip of paper chromatogram. The values are per Ag of DNA present in the digests applied at the origin. 0, Normal; 0, transformed. (Lower) Spots as located by UV light.
Biochemistry:
Proc. Nat. Acad. Sci. USA 74 (1977)
Muto et al.
4175
Table 2. Analysis of cell-bound sulfated glycosaminoglycans fractionated on sucrose density gradient CH-ase ABC% of total Ratio: CH 4-SO4 CH 6-SO4 resistant %t cpm 6-SO4/4-SO4 cpm cpm cpm
Peak* III I
1,304 3,308
41.0 4.0
626 46,186
III II I
846 182 152
30.7 7.4 4.8
571 1,024
1,458
Normal chondrocytes 30.4 966 19.7 35.7 29,205 56.4 chondrocytes RSV-infected 38.9 1,071 20.7 42.7 1,044 41.8 37.0 48.0 1,122
0.65 1.58
2.2 84.8
0.53 0.98 1.30
15.7 37.0 28.3
CH-chondroitin.
* Peaks are numbered as in Fig. 1. t Percentage is of the total sulfated glycosaminoglycan of each peak.
Also, incorporation of a5SO4 into peak I was uniformly inhibited (>95%) upon transformation regardless of species of glycosaminoglycans. On the other hand, the incorporation into peak III was not significantly inhibited by RSV-transformation for any of the three sulfated glycosaminoglycans. Because peak III is qualitatively different from peak I, the relative increase of proportion of peak III upon transformation is not a result of degradation by a heightened proteolytic enzyme activity in the transformed chondrocytes (9). In sulfated glycosaminoglycans of proteoglycans excreted into the medium by normal chondrocytes, the ratio of chondroitin 6-sulfate to chondroitin 4sulfate was approximately the same. This suggests that the major sulfated proteoglycans found in the medium of normal cells were the direct degradation products of the cell-bound material but not related to peak II of the material bound to the transformed chondrocytes. C
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-
5C
To examine the possibility that glycosidases of the transformed cells may have degraded the glycosaminoglycan chain, glycosaminoglycan portions of sulfated proteoglycans were analyzed on a Sephadex G-200 column. As shown in Fig. 3, the elution patterns of s5SO4-labeled- glycosaminoglycans were similar, regardless of transformation. These experiments appear to indicate, therefore, that the chain length of glycosaminoglycan portions has not been drastically altered by transformation with RSV. Temperature-Dependent Hyaluronic Acid Synthesis by Chondrocytes Infected with RSV ts LA24. In a previous communication (1), we established that the morphological change caused by transformation of chondrocytes is reversible by the use of the temperature-sensitive mutant, RSV ts LA24. To substantiate further that the chondrocytes' chemical characteristics are also reversibly changed, incorporation of [14C]acetate into hyaluronic acid was studied at two temperatures with chondrocytes infected with RSV ts LA24. As shown in Table 3, chondrocytes transformed by this mutant virus had a higher [14C]acetate incorporation into hyaluronic acid when incubated at 370 (the permissive temperature for the transforming gene) than when incubated at 41° (the nonpermissive temperature). The chondrocytes infected with RSV ts LA24 changed their characteristics in the expected direction within 24 hr after the temperature shift. Normal chondrocytes incorporated more [14C]acetate into hyaluronic acid at 410 than
0
U~~~~~~~~~~~~~ ~
Table 3. Effect of temperature on incorporation of [14C]acetate into hyaluronic acid by normal and RSV ts LA 24-infected chondrocytes Radioactive hyaluronic acid formed Ratio: medium/cell Temp. shift cpm/ug of DNA
D
B
E
370 - 370
Normal chondrocytes 1,246 3,349
370 - 370
6,293 Infected chondrocytes 16,732 13,800 6,544 8,351
15
410 ,.370 410 - 41° 370 - 41° 10
20
30
20
30
F raction
FIG. 3. Sephadex G-200 gel filtration of chondroitin sulfate of normal and wild-type RSV-transformed chondrocytes. 35SO4-Labeled sulfated glycosaminoglycan samples were subjected to gel filtration on Sephadex G-200 column (1.5 X 70 cm). The arrows indicate the void volume of the column. (A) Control cell layer, 130 x 103 cpm; (B) RSV-infected cell layer, 171 X 103 cpm; (C) control medium, 31.2 X 103 cpm; (D) infected cell medium, 9.0 X 103 cpm.
410-37° 410 - 410 37°-41°
6,130
0.38 0.57 1.07 1.53 1.02 1.24 0.94 0.91
RSV-infected cells were kept at the first temperature for 5 days and then at either the same or a different temperature for 28 hr, followed by incubation with [14C]acetate for 24 hr.
4176
Biochemistry:
Proc. Nat. Acad. Sci. USA 74 (1977)
Muto et al.
Table 4. Effect of temperature on the chondroitin sulfate formed by RSV ts LA24-infected chondrocytes [14C]Acetate incorporated, cpm/,g of DNA Ratio: CH 6-SO44-SO4 1.54 1,572 4,780 370 370 (9.4%) (28.5%) 2.58 4,053 7,992 410 - 370 (11.0%) (21.6%) 2.95 6,129 8,746 25,766 410 - 410 (13.5%) (19.2%) (56.6%) (6.5%) 2.41 4,350 6,661 16,058 2,085 370 - 410 (14.1%) (21.5%) (51.9%) (6.7%) Experimental conditions were as in Table 3. Numbers in parentheses are percentage of radioactivity in total labeled hyaluronidase-resistant CH-ase ABCresistant GAG 1,697 (10.1%) 3,048 (8.2%) 2,948
Temp. shift -
CH 6-SO4 7,394 (44.1%) 20,586 (55.7%)
CH 4-SO4
glycosaminoglycans. Control experiment with normal chondrocytes showed no appreciable change in the ratio, 6-S04/4-S04, with shift of temperature. CH = chondroitin; GAG = glycosaminoglycan. at 370. Therefore, the effect of the activation of the transforming gene product can overcome the effect of the temperature alone. Effect of Temperature on the Synthesis of Chondroitin Sulfate by Cells Infected with RSV ts LA24. Chondrocytes infected with RSV ts LA24 were studied to determine if the qualitative change of sulfated proteoglycans observed with the transformed chondrocytes is reversible depending on the activity of the transforming gene product (Table 4). At 370, these cells incorporated less [14C]acetate into chondroitin 6-sulfate than into chondroitin 4-sulfate as compared to the normal chondrocytes. With a temperature shift from 410 to 370, the incorporation of [14C]acetate into chondroitin sulfate decreased but did not reach the level of those cells kept at 370 from the time of infection. The ratio of chondroitin 6-sulfate to chondroitin 4-sulfate also decreased during the temperature shift down and the opposite was observed when temperature was raised. Fig. 4 indicates that change in the sedimentation behavior of sulfated proteoglycans as described in Fig. 1 can also be demonstrated with cells infected with RSV LA ts 24 at the permissive (370) and at the nonpermissive (410) temperatures.
5
..A 5-
4
~~B
4-
