Inhibition of precartilaginous chick somites by oncogenic virus

DEVELOPMENTAL BIOLOGY 97, ?o-80 (1983)

Inhibition of Precartilaginous Chick Somites by Oncogenic Virus

MAKOTO YOSHIMURA, JAMES W. LASH,~ NAGASWAMI VASAN,~ AND AKIRA KAJI

Departments of Anatomy and Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received July SO, 1980; accepted in revised form December 22, 1982

Infection of embryonic chicken notocbord-somite explants with ROW sarcoma virus inhibited the in vitro differentiation of somites into cartilage. Visual inspection of the explants revealed that viral infection reduced the size of cartilage nodule formation. Formation of the complex of sulfated proteoglycans with hyaluronic acid was inhibited by RSV infection, and sedimentation analysis of the sulfated proteoglycans showed that very little fast sedimenting proteoglycans were synthesized by RSV-infected explants. The infected explants primarily synthesize a slowly sedimenting sulfated proteoglycan which was chondroitinase resistant. These slow-sedimenting sulfated proteoglycans lack the ability to associate with hyaluronic acid and appear to be noncartilaginous. These effects of RSV are apparently due to the WC gene of this virus since the mutant tdlO8, which lacks part of the WC gene, has no detectable influence on the chondrogenic differentiation of somite explants. Similarly, infection with RAV-2 as well as with uv-irradiated virus had no detectable effect. The inhibition of synthesis of fast sedimenting proteoglycans was observed at 41°C with explants infected with tsNY68, suggesting that residual activity of transforming gene of this virus at the nonpermissive temperature is sufficient for this inhibition in the explants.

INTRODUCTION

The Rous sarcoma virus (RSV) contains at least four genes: gag (group-specific antigen), pal (reverse transcriptase), env (viral envelope glycoprotein), and src (responsible for transformation) (for review, see Han- afusa, 1977; Vogt and Hu, 1977). Thus, cells infected with a RSV mutant lacking the part of the src gene are not transformed, even though viruses are produced. Ex- tensive genetic as well as biochemical studies have been carried out with reference to the action of the src gene. The discovery of a temperature-sensitive mutant of the src gene has helped considerably in the understanding of the direct or indirect effect of the src gene. Cells infected with one such mutant (RSV tsNY68) produce virus regardless of the temperature, while cellular transformation can be observed only at the permissive temperature (36-37°C) (Kawai and Hanafusa, 19’71).

A number of reports have described the effects of oncogenic transformation in vitro of differentiated as well as differentiating cells (Kaighn et al., 1966; Fogel and Defendi, 1967; Lee et al., 1968; Easton and Reich, 1972; Graessmann et al., 1973; Fiszman and Fuchs, 1975; Holtzer et al., 1975; Hynes et al., 1976; Kaji et al., 1976; Arbogast et al., 1977; Muto et al., 1977; Okayama et al., 1977; Pacifici et al., 1977; Roth et al., 1977; Toole et al., 1977; Kobayashi and Kaji, 1978; Gross and Rifkin, 1979; Tanaka et al., 1980; Yoshimura et al., 1981). From these

’ To whom all correspondence should be addressed. * Present address: Department of Anatomy, College of Medicine and

Dentistry of New Jersey, Newark, N.J. 07013.

reports it can be generalized that oncogenic transformation frequently causes differentiated cells to lose some differentiated traits, and has an adverse effect on differentiating cells.

The Rous sarcoma virus genome has been shown to interfere with the normal function of many cell types. For example, it has been shown that activation of the transforming principles of RSV interferes with formation of multinucleated myotubes and induces DNA synthesis in myotubes (Kobayashi and Kaji, 1978). Fur- thermore, infection of differentiated chondrocytes with RSV resulted in the loss of a particular size proteoglycan molecule which could be identified in non-infected chondrocytes (Muto et al., 197’7). The possible relationship between cellular transformation and cellular differentiation has been studied in many laboratories (Abelev et al., 1963; Alexander, 1972; Bull et al., 1974; Coggin and Anderson, 1974; Criss, 1970; Fishman et al., 1968; Gold and Freedman, 1965; Kurth and Bauer, 1973; Ogawa and Ichihara, 1972; Potter, 1968; Schapira, 1973; Tanaka and Kaji, 1980). The results to be reported here are related to the effects of RSV infection on cells which have not yet expressed their development potential. It has been shown that oncogenic cells and differentiating embryonic cells have some features in common (Roth and Kaji, 1979).

The in vitro stimulation of somitic chondrogenesis by notochordal tissue presents a convenient system for the study of viral transformation and its effect upon differentiation. It has long been known that the embryonic notochord plays an important role in the stimulation

70 0912-1606/&l 33.00 Copyright Q 198.3 by Academic Press. Inc. All rights of reproduction in any form reserved.

YOSHIMURA ET AL. Precartiln@wus Chick Smites Inhibition 71

of vertebral (somitic) chondrogenesis (Lash, 1968; Lash and Vasan, 1977; Lash and Vasan, 1978).The embryonic somite contains precursor cells for muscle (myotome), dermis (dermatome), and cartilage (sclerotome). The sclerotome cells respond to the influence of the notochord by synthesizing and accumulating cartilage matrix products. It has recently been shown that the size distribution of cartilage proteoglycans can be used as a reliable index of chondrogenic differentiation (Lash and Vasan, 1978; Ovadia et al., 1980).

In this communication, we have studied the effect of RSV on the notochord-stimulated in vitro differentiation of embryonic chick somites into cartilage. Criteria of differentiation included the visual observation of cartilage formation in living explants and the size distribution of cartilage proteoglycans. It was found that infection of the notochord-somite explants with RSV resulted in the loss of the large proteoglycan aggregates, with small proteoglycan monomers being the predominant proteoglycan detected. Consistent with this finding, proteoglycans from infected explants did not associate with hyaluronic acid in vitro whereas proteoglycans from control explants did associate. In addition, sucrose density gradient centrifugation indicated that the sulfated proteoglycan monomers from the infected explants were smaller than those in the controls. A surprising finding was that the effect of RSV infection could be observed regardless of the culture temperature, even when infected with a mutant of RSV having a temperature sensitive lesion of the src gene. Yet, infection with the transformation-defective mutant, td108 (Hanafusa et al., 1977; Kawai et al., 1977) or RAV-2 (Hanafusa, 1965) did not inhibit differentiation, suggesting that the src gene is responsible for the observed inhibitory effect on the cartilage formation.

MATERIAL AND METHODS

Culture Techniques

Somites and notochords were dissected from stages 17 and 18 (staging series of Hamburger and Hamilton, 1951) of leukosis-free SPAFAS White Leghorn chick embryos, according to the methods described by Ellison and Lash (1971) and Gordon and Lash (1974). The tissue explants, consisting of lo-12 clustered somites surrounding a 0.15mm length of notochord, were placed on Nuclepore filters (complete methodology in Lash and Vasan, 1978). The liquid nutrient medium consisted of Simms’ balanced salt solution, fetal calf serum, and the nutrient supplement F12X in the proportions 221 (Mi- nor, 1973). The fetal calf serum and the nutrient supplement F12~ were obtained from Gibco Grand Island, New York. All explants were maintained at an appro- priate temperature (37 or 41°C) for 6 days in a humid- ified atmosphere of 95% air, 5% CO,.

Infect&n of Explants and the Preparatim of Virus

The explants were infected within an hour after being placed on the Nucleopore filters. The filters containing the explants were placed in 1.0 ml of virus solution (containing lo6 FFU) for 2 to 6 hr, and then transferred to non-virus-containing liquid nutrient medium. The four strains of RSV used were Prague wild-type RSV, tsNY68 (Kawai and Hanafusa, 1971), tdlO8 of Schmidt Ruppin strain (Hanafusa et al., 1977; Kawai et al., 1977), and RAV-2 (Rous-associated virus 2) (Hanafusa, 1965). These viruses were generously given to us by Dr. Han- afusa. The viruses used in this study were prepared by harvesting the medium of chick embryo fibroblast culture infected with virus and media of noninfected fibroblast culture were used for mock infection. The virus solution (5 ml) of tsNY68 (lo6 FFU/ml) was kept in a lo-cm petri dish under ultraviolet lamp for 30 min from 10 cm distance, at room temperature. This treatment reduced the reverse transcriptase activity to l/200. The explants were exposed to the virus solution on the second day for another 2 to 6 hr.

Labeling of Proteoglycans

During the last 24 hr of culture, each dish of explant was exposed to 50 &i of [35S]sodium sulfate (New En- gland Nuclear, Boston, Mass.).

Extraction of Proteoglycans

All explants were extracted immediately after col- lection, never frozen and thawed. The filters containing the explants were transferred to 30 ml Corex (No. 8445) glass test tubes, to which was added 1.0 ml of 4.0 M guanidine hydrochloride (GuHCl) solution containing proteolytic inhibitors, as described by Lash and Vasan (1978). The tubes were sealed with Parawax film and placed on a rotary shaker in a cold room (4°C) for 48 hr. The extract was then dialyzed against cold-distilled water for 48 hr. After centrifugation at 10,OOOg for 30 min, the supernatant containing the extracted proteoglycans was lyophilized. If the proteoglycans exist in the tissues as aggregates, the high molarity salt (4.0 M GuHCl) will extract them as dissociated monomers, as well as nonassociated monomers. Dialysis under low ionic strength (distilled water) will cause the proteoglycans to reaggregate if the core protein contains the hyaluronic acid region, and if hyaluronic acid is present. Centrifugation or chromatography in 4.0 MGuHCl (dissociative conditions) will detect dissociated monomers whereas similar assays with low molarity salt (0.5 M NaCl) will detect monomers capable of aggregating in the presence of hyaluronic acid. See below for methods of centrifugation, chromatography, and detection of monomers capable of aggregating.

72 DEVELOPMENTAL BIOLOGY VOLUME 97,1983

Sucrose Density Gradient Centrifugation

The lyophilized proteoglycan extract was dissolved in 1.0 ml of 4.0 M GuHCl solution, and 0.5 ml of this was layered over a 16-ml linear sucrose gradient (5-20s) containing 4.0 MGuHCl and 0.05 MNa-acetate (pH 5.8). Centrifugation was performed in a Beckman Model L2- 65B ultracentrifuge with a swinging-bucket rotor (SW 27.1) at 20°C for 28 hr at 120,OOOg. An aliquot of 0.1 ml of each fraction was absorbed onto a 2.3-mm disk of Whatman 3MM filter paper and dried. The dried disks were placed into scintillation vials for radioactive de- terminations. We define large proteoglycan monomer as those molecules sedimenting beyond the position corresponding to Tube 15 under these circumstances.

Molecular Sieve Chromatography

Molecular sieve chromatography was performed according to the method of Lever and Goetinck (1976), as modified by Lash and Vasan (1978), using controlled- pore glass beads (CPG) (Electra-Nucleonics, Fairfield, N. J.).

The bead size was CPG-10-2500 (mean pore size 2753 A). We define large aggregates as those complex of proteoglycan with hyaluronic acid eluting earlier than fraction 48 under our fractionation conditions.

Determination of Radioactive Proteoglycans

The scintillation fluid contained 6.0 g of PPO/liter, 0.3 g POPOP/liter (Packard Instruments, Downers Grove, Ill.). Radioactivity was determined using a Model 4200 Intertechnique scintillation counter which was programed to present the disintegrations per minute in bar graph form (IN/US Service Corp., Fairfield, N. J.). In some cases radioactivity was determined in a Packard scintillation counter.

DNA Analysis

The aliquot of dissociated sample in 4.0 M GuHCl was used for DNA assays (Hinegardner, 1971).

Histological Procedures

For histological procedures, the explants were removed from the Nuclepore filters and transferred onto the surface of nutrient agar (Lash, 1963). After 3 hr the explants had rounded up and were then removed and fixed in Karnovsky formaldehyde-glutaraldehyde solution as described by Belsky et al. (1980). Paraffin- embedded tissues were sectioned and stained with Al- cian blue-Delafield’s hematoxylin (Ellison and Lash, 1971).

Assay for Hyaluronic Acid Binding

For complete methodology and terminology, see Hei- negard (1972), Faltz et al. (1979), and Vasan and Lash (1979). The radioactive proteoglycans were extracted under dissociative conditions (4.0 M GuHCl). After dialysis, these extracts were then centrifuged in a cesium chloride gradient under associative conditions (0.5 M GuHCl) in a Beckman 40 (fixed angle) rotor at 38,000 rpm. The bottom one-fourth of the tube was recovered, and represented the Al, or associated, fraction. After dialysis and lyophilization, the Al fraction was centrifuged as above in a cesium chloride gradient under dissociative (4.0 M GuHCl) conditions. The bottom two- fifths of the tube was retained as the purified proteoglycan monomer fraction (Al-Dl). After dialysis and lyophilization, the Al-D1 fraction was dissolved in 2.5 ml of 4.0 M GuHCl. To this was added 10 ~1 of hyaluronic acid (stock solution 1 mg/ml in deionized water) and 0.25 ml of a nonradioactive Al-D1 sample (250 mg in 4.0 M GuHCl) known to be able to bind with HA. After mixing on a Vortex, the solution was left at room temperature for 1 hr, then dialyzed overnight against 2 liters of 0.5 M NaCl. The sample was then applied to the CPG column and assayed as described above.

RESULTS

Morphological Observations of Infected and Noninfected Explants

Observations were made on over 500 control and 600 virus-treated explants. Invariably the control explants formed two to five prominent cartilage nodules. Under the dissecting microscope the diameter of these nodules ranged from 200 to 300 pm. The virus-treated explants seldom possessed more than one nodule whose diameter was 150 pm or less. Histological sections through rep- resentative explants are shown in Fig. 1. Thus, the virus infection inhibited, but did not completely prevent cartilage formation.

The E#ect of RSV Infection of the Sedimentation Behavior of Sulfated Proteoglycans

Notochord-somite explants were infected with wild- type RSV (lo6 FFU/ml) as described under Materials and Methods, and permitted to develop in culture for 6 days. After exposure to radioactive sulfate for the last 24 hr of the culture period, the sulfated proteoglycans were extracted with 4.0 M GuHCl. Under these dissociative extraction conditions, the large sulfated proteoglycan aggregates (complex of hyaluronic acid and proteoglycans) are dissociated into hyaluronic acid and proteoglycan monomers (Sajdera and Hascall, 1969). The sulfated proteoglycans from the control explants (Fig.

YOSHIMURA m AL. Precartilaginm Chick Smites Inhibition 73

FIG. 1. Histological sections of notochord-somite explants. (A) Con- trol, noninfected explant showing large cartilage nodule (arrow). (B) Explant infected with Prague strain RSV wild-type virus. Note that

fewer and smaller nodules are present (arrow). 250X.

15

10

5

0

6.

0 10 20 30

FRACTION NU llBER

P D 1 ,

0 10 20 30

FRACTlON NUMBER

FIG. 2. Sucrose density gradient centrifugation of %04-labeled pro-

teoglycans extracted from control and infected notochord-somite explants (as in Figs. 1A and B). Centrifugation was done under dissociative conditions. Samples were applied to a 5 to 20% sucrose density

gradient (16 ml) and spun at 120,OOOg for 28 hr (see Materials and Methods). Sedimentation was from right to left. Fractions (0.5 ml)

were collected from the bottom of the tube. (A) A fast-sedimenting proteoglycan is the predominant molecule in the control explants. (B) A slower sedimenting, small proteoglycan is the predominant molecule in the infected explants (material applied to the gradient in A,

46,500 cpm; in B, 1000 cpm).

2A) sedimented faster than those extracted from RSV wild-type infected explants (Fig. 2B). Virus infection thus inhibited the normal differentiation of cartilage in the sense that the fast-sedimenting sulfated proteoglycans, which are characteristic of chondrogenic differentiation, are not formed.

74 DEVELOPMENTAL BIOLOGY VOLUME 97, 1983

The Efect of RSV Infection upon Proteoglycan Aggregate Synthesis

The extracellular matrix of cartilage consists primarily of collagen and proteoglycans. The proteoglycans exist as monomers, aggregates (monomers linked to hyaluronic acid), and nonaggregating monomers. Employing molecular sieve chromatography, it is possible to determine the distribution of aggregates and monomers in GuHCl extracts of cartilage (Lever and Goetinck, 1967; Lash and Vasan, 1978). When such a

100 f-

A

molecular distribution profile was examined from the cartilage nodules of notochord-somite explants, a het- erogeneous display of proteoglycans typical of embryonic cartilage characterizes such explants (Fig. 3A). This means that the relatively large complex of sulfated proteoglycan molecules and hyaluronic acid was produced by the somites undergoing chondrogenic differentiation. When the infected explants were analyzed, a markedly different pattern was seen (Fig. 3B). The molecular size was much more homogeneous, with the small sulfated proteoglycans predominating. Since most of the labeled

40 t 50 60 70

“0 FRACTION NUMBER “t

“0 FRACTION NUMBER “t

FIG. 3. Molecular sieve chromatography on controlled-pore glass beads of %01-labeled proteoglycans extracted from control (A) and infected (B) explants. The column was prepared as under Materials and Methods and run under associative conditions to detect proteoglycan aggregates and monomers. Control explants (A) contain three classes of proteoglycans, large aggregates (passing through the column in void volume (V,), intermediate-size proteoglycans in the included volume, and small monomers at the total volume (V,). These three classes of proteoglycans characterize differentiating cartilage (see Lash and Vasan, 19’78; Ovadia et al., 1980). Proteoglycans from the infected explants (B) consist mostly of small molecules (eluting just before V,) and a minor component eluting at the position of proteoglycan aggregates (near V,) (material applied to column in A, 16,800 cpm; in B, 4100 cpm). In Figs. 3 and 4 the values of each fraction are plotted as a percentage of the highest value fraction.

YOSHIMURA ET AL. Precartilagiwus Chick Somites Inhibition 75

I I I 1 40 t 50 60 70 I30 )

"0 FRACTION NUMBER "t

a FRACTION NUMBER "t

FIG. 4. Molecular sieve chromatography (as in Fig. 3) of “SOA-labeled proteoglycans from control (A) and infected (B) explants assaying for the ability of Al-D1 monomers to associate in vitro with hyaluronic acid (HA). The in vitro association of proteoglycans with HA was performed as under Materials and Methods. (A) Proteoglycans from noninfected explants plus HA, showing that most of the monomers are capable of binding to HA, forming large proteoglycan aggregates (shown at V,). (B) Proteoglycans from RSV-infected explants contain only a small proportion of molecules that bind with HA. Most of the molecules remain unassociated (near VJ (material applied to column A, 4300 cpm; in B, 2000 cpm).

proteoglycans from transformed explants behave as monomers in molecular sieve chromatography, the ability of these proteoglycans to bind with hyaluronic acid in vitro was examined. The results shown in Fig. 4 suggest that the proteoglycans synthesized by RSV-infected culture did not associate with hyaluronic acid while most (but not all) of those from noninfected culture did. The sulfated proteoglycan monomers of normal cells were converted to very large aggregates by the addition of exogenous hyaluronic acid (Fig. 4A). Such a change was observed only to a very small extent in Fig. 4B, which involves RSV-infected tissue. Control ex-

periments indicated that in the absence of added HA no such aggregates were observed.

From the results presented we can conclude that in control explants (Fig. 3A) there are at least three kinds of proteoglycans. One population can aggregate with HA and this material is represented in the first peak of Fig. 3A (as an aggregate). The second peak is nonaggregated, yet aggregatable (upon addition of exogenous hyaluronic acid) monomer. The third peak does not aggregate even with added hyaluronic acid. This nonaggregating material can be seen to remain after hyaluronic acid (small peak between fractions 70 and


80 in Fig. 4A). This nonaggregating, and as yet un- identified, molecule has been observed in other analyses of embryonic cartilage (Lash et al., 1982). The viral infection eliminates the synthesis of the aggregatable monomer leaving only the small nonaggregatable proteoglycan. The small molecules between fractions 70 and 80 in Fig. 3B represent nonaggregated proteoglycans in the total extract of infected explants. After purification of Al-D1 monomers, and rechromatography in the presence of hyaluronic acid, these molecules remain unassociated (Fig. 4B). To show that the aggregating effects were due to the presence of hyaluronic acid, Al- Dl monomers were prepared from both control and infected somites. Neither of these monomer populations showed any aggregation when chromatographed in the absence of hyaluronic acid (data not shown). Their position in the elution pattern was the same regardless of whether they were chromatographed under associative conditions (i.e., 0.5 M GuHCl) or dissociative conditions (i.e., 4.0 M GuHCl). Under both conditions the proteoglycan monomers eluted at the fraction toward the end of molecular sieve column (data not shown). These experiments indicate that the proteoglycans from RSV-infected explants lack a hyaluronic acid-binding region and are incapable of forming aggregates. The Al proteoglycan fraction of infected explants (Fig. 3B) contains a small amount of large proteoglycan aggregate. Comparable aggregates are also seen when Al-D1 monomers from these extracts are rechromatographed in the presence of hyaluronic acid (Fig. 4B).

FRACTION NUMBER

FIG. 5. Sucrose density centrifugation of sulfated proteoglycans from noninfected explants cultured at 41°C for 6 days. Centrifugation performed under dissociative conditions as in Fig. 2. The predominant molecule is the large proteoglycan monomer characteristic of differentiating cartilage (material applied to gradient, 49,500 cpm). In this and following figures, the radioactivity of each fraction is expressed as per pg DNA corresponding to the tissue from which the fraction came. DNA was measured by the method of Hinegardner (1971).

” o..I ul 0 10 a

FRACTION NUMBER \

I

t

500- B.

0 0 10 20 30

FRACTION NUMBER

FIG. 6. Sucrose density centrifugation of sulfated proteoglycans extracted from notochord-somite explants infected with tsNY68 virus and cultured at either 37°C (A) or 41°C (B). In both instances only small proteoglycans are detected (material applied to gradient in A, 18,000 cpm; in B, 19,500 cpm).

The Eflect of RSV tsNY68 on Chondrogenesis at Permissive and Nonpermissive Temperatures

Since the src gene activity of RSV tsNY68 is dependent upon temperature, it was of interest to examine the effect of temperature on chondrogenic inhibition by this virus. Uninfected explants kept at 41°C synthesize the fast-sedimenting sulfated proteoglycans, indistin- guishable from uninfected explants cultured at 37°C (cf. Figs. 5 and 2A). Thus, temperature alone has no dis- cernible effect upon chondrogenesis as measured by the formation of fast-sedimenting sulfate proteoglycans. Figures 6A and B show sedimentation behavior of sulfated proteoglycans from explants infected with RSV tsNY68. It can be seen that the inhibitory effect of RSV on the synthesis of fast-sedimenting sulfated proteo-

YOSHIM~JRA ET AL. Precartilaginous Chick Somites Inhibition 77

4

01 d / 0 10 20 30

FRACTION NUMBER

FIG. 7. Sucrose density gradient centrifugation of sulfated proteoglycans extracted from control explants (A) and explants infected with a transformation-defective mutant of the Schmidt-Ruppin strain of RSV (td108) (B). Both explants were maintained at 37°C for 6 days. In both instances the predominant molecule is the large proteoglycan monomer characteristic of differentiating cartilage (material applied to gradient in A, 38,000 cpm; in B, 30,000 cpm).

glycans is apparent at both permissive (37°C) and nonpermissive (41°C) temperatures. From this result we conclude that the total expression of the src gene is not necessary for the inhibitory effect of RSV on somite chondrogenesis. These conclusions were supported by the very much reduced presence of cartilage nodules at both 37 and 41°C with explants infected with tsNY68.

The Eflect of Transformation-Defective Virus, Row-Associated Virus 2, and W-Irradiated Virus on Swmite Chondrogenesis

As discussed in the preceding section, the temperature-sensitive virus (RSV tsNY68) could cause an inhibitory effect on somite chondrogenesis even at the nonpermissive temperature. It appeared, therefore, possible that the effect we observed may simply be due to virus infection and not to the src gene’s effect per se as reported with the limb bud chondrogenesis system by Gross and Rifkin (1979). We, therefore, examined the effect of a transformation-defective virus (td108) (Hanafusa et al., 1977; Kawai et al., 1977) on somite chondrogenesis, using the sedimentation behavior of sulfated proteoglycans as a chondrogenic assay. As shown in Figs. ?A and B, fast-sedimenting sulfated proteoglycans were synthesized in both explants even though a slight reduction (23%) was observed in explants infected with the td virus (Fig. 7B). From this

result, we conclude that the src gene of td108, which is shorter than that in the wild type or the ts mutant, is not enough to prevent chondrogenic differentiation. It should be pointed out that in these experiments production of virus took place as much as the case of infection with tsNY68, as measured by the level of reverse transcriptase in the culture media (data not shown).

To substantiate our notion that the inhibition of the appearance of differentiation markers is dependent on the action of the src gene and not dependent on infection itself, we examined the effect of RAV-2 (Rous-associated virus 2 having no src gene) and uv-irradiated RSV on the formation of large sulfated proteoglycan as examined by the molecular sieve chromatography. The result of this experiment is summarized in Table 1. It is clear from this table that only under the conditions where the src gene is present was the inhibition of formation of large sulfated proteoglycan aggregates observed. Visual observation of cartilage formation in these experiments substantiated this conclusion.

DISCUSSION

There have been many attempts to study the rela- tionships between oncogenesis and differentiation. This is understandable, since changes in transformed tissue are sometimes described as “dedifferentiation.” In this report we have studied the effect of viral infection of chondrogenic tissues during the process of differentiation, as we have done on the myotube differentiation (Kobayashi and Kaji, 1978) and epidermal cell differentiation (Yoshimura et al., 1981).

In this communication, we have been able to show that viral infection results in an inhibition of chondrogenesis. The infected somites synthesize very little of the cartilage-characteristic proteoglycan, and the predominant proteoglycan is a small monomer lacking the ability to bind with hyaluronic acid. We cannot discount the possibility that the cartilage nodules in the infected explants are derived from cells that escaped the initial infection. With cell cultures an analysis for complete

TABLE 1 LACK OF EFFECT OF RAV-2 ON UV-IRRADIATED RSV ON THE

SYNTHESIS OF LARGE-SULFATED PROTEOGLYCAN-AGGREGATES

Virus infection Large PG/small PG”

None 1.09 RAV-2 1.25 NYts6Sb 0.24 W/-irradiated virus 1.06

a Radioactive sulfated proteoglycans eluting between fractions 40 and 61 from the CPG column are regarded as large aggregates of proteoglycans. These values are from two to six repeat experiments.

*After infection the explants were incubated at 37°C.

78 DEVELOPMENTALBIOLOGY VOLUME97,1983

infection is feasible, but this cannot be easily done for Although the RSV-infected somites form small but tissues. These proteoglycans are both chondroitinase recognizable cartilage nodules, the matrix appears to and nitrous acid resistant, indicating that they do not contain aberrant molecular forms. Under the conditions contain the glycosaminoglycans characteristic of em- that we have employed, it is not possible to say whether bryonic cartilage (chondroitin sulfates and heparan sul- this cartilage would have the normal physiological fate) (Abrahamsohn et al., 1975). The control somites properties attributed to cartilage (cf. Muir, 1977). synthesized the glycosaminoglycans characteristic of It is surprising that the RSV mutant which has tem- embryonic cartilage, as described in Kosher and Lash perature-sensitive lesions at the src gene can influence (1975). This is an example of the inhibition of chon- the synthesis of fast-sedimenting sulfated proteogly- drogenic differentiation rather than the modulation of cans even at a nonpermissive temperature. In this re- already differentiated chondrocytes by oncogenic trans- gard, it should be mentioned that vertebral chondro- formation. Viral infection was found to result in the cytes transformed by tsNY68 proliferate three to five synthesis of small proteoglycan monomers which lacked times faster than control chondrocytes, even at 41°C the ability to associate with hyaluronic acid. The small (Tanaka et al., 1980). Similarly, with neuroretinal cells, proteoglycan synthesized by precartilaginous tissue has infection with some of class t RSVts mutants stimulate never been characterized except as to size. Scarcity of cell proliferation even at nonpermissive temperature material prevented us from characterizing the proteo- (Calothy and Pessac, 1976). It should be pointed out that glycan resulting from virus infection further than transformation of vertebral chondrocytes with tsLA24 showing that it lacks a HA-binding region and does not resulted in the reversible changes of differentiated contain the typical cartilage glycosaminoglycans. The characteristics which were dependent upon tempera- infected explants showed visually recognizable but small ture (Muto et al., 1977). We have preliminary results cartilage nodules. The small proportion of proteoglycan which indicate that infection of vertebral chondrocytes aggregates extracted from these explants is underrep- with tsNY68 leads to the decreased synthesis of fast- resented considering the number of cartilage nodules sedimenting sulfated proteoglycans even at a nonper- seen in the explants. Technically it is not feasible to missive temperature. Whether the apparent growth analyze the nodules separately from the surrounding stimulation by the src gene is involved in preventing “soft” tissue. We suggest that the nodules may contain the chondrogenic differentiation of somites has not yet small sulfated proteoglycans which lack a hyaluronic been determined. It appears that the ts lesion of tsNY68 acid-binding region. Similar results have been reported is a leaky mutation and the residual level of the src by Gross and Rifkin (1979), who studies viral inhibition protein which may function at 41°C may be enough to of limb bud chondrogenesis. They too report that the prevent differentiation, but not enough to express other infected tissues synthesize smaller proteoglycans than transformation markers (Tanaka et at., 1980). A tem- the uninfected limb buds. Since they did not use mo- perature-sensitive mutant tsGl201 (clone 9) has been lecular sieve chromatography it is not possible to tell reported to produce a definite temperature-sensitive from their studies whether the proteoglycans synthe- growth effect on differentiated chondrocytes (Tanaka et sized in the infected explants differ in the hyaluronic al., 1980). Whether this mutant will have a comparable acid-binding region. The observed change could be ei- effect on our explants (precartilaginous somitic tissue) ther due to a change of chondrocytes in the nodule or cannot be safely predicted for the reasons stated above. due to the overgrowth of noncollagenous cells in the Further studies will be carried out to explore these pos- nodule. sibilities.

Although we did not measure the production of hy- It should be emphasized that the inhibitory effect of aluronic acid in these experiments, it is unlikely that RSV on differentiation in this system is dependent on the absence of proteoglycan aggregates is due to a the presence of the src gene. Thus, td108 which has only shortage of hyaluronic acid. Thus, even if exogenous partial src gene, RAV-2 having no src gene, and uv- hyaluronic acid is added to the proteoglycans, aggre- irradiated virus had no appreciable effect, It, therefore, gation still does not occur. In addition, it has recently makes a sharp contrast to the effect of avian tumor been reported that virus-infected chondrocytes (Okay- virus on chondrogenesis of limb bud cells (Gross and ama et al., 1977) and myotubes (Yoshimura and Kaji, Rifkin, 1979) in which RAV-2 and td RSV had appre- 1979) have elevated levels of hyaluronic acid synthesis. ciable inhibitory effect on differentiation. Elevated levels of hyaluronic acid synthesis may be a It appears as though the same virus, in this case RAV- factor in the inhibition of differentiation, as indicated 2, can have different effects on different tissues. Even by the work of Toole et al. (1977) and Yoshimura and though somites and limb buds both form cartilage, there Kaji (1979). Even if there is a rise in hyaluronic acid are major differences in how this is effected. Somites production in the viral-infected somites, these somites required specific tissue interactions whereas limb bud do not synthesize proteoglycans capable of aggregating. chondroeenic tissue does not. Furthermore. tsNY68. ~~ ~~ _ ~~

YOSHIMURA ET AL. Precartilaginws Chick Somites Inhibition 79

whose effect on somite-notochord explants or on differentiated chondrocytes is not temperature sensitive, exerts a temperature-dependent effect on differentiated myotubes (Kobayashi and Kaji, 1979). From these con- siderations it appears that the intricate relationship between the host target proteins and the protein kinase of RSV is important in determining the expression of the transformed phenotype.

This work was supported by Grants HD-00380 (JWL) and CA-19497 (AK). We gratefully acknowledge the assistance of Gladys Treon in performing the dissection of embryos, and N. S. Chandrasekar in performing the biochemical analyses.

REFERENCES

ABELEV, G. I., PEROVA, S. D., KHRAMROVA, N. I., POSTNELCOVA, Z. A., and IRLIN, I. S. (1963). Production of embryonal a-globulin by trans- plantable mouse hepatomas. Transplantation 1, 174-180.

ABRAHAMSOHN, P. A., LASH, J. W., KOSHER, R. A., and MINOR, R. R. (19’75). The ubiquitous occurrence of chondroitin sulfates in chick embryos. J. Exp. ZooL 194, 511-518.

ALEXANDER, P. (1972). Foetal “antigens” in cancer. Nature (London) 235,137-140.

ARBOGAST, B. W., YOSHIMURA, M., KEFALIDES, A., HOLTZER, H., and KAJI, A. (1977). Failure of cultured chick embryo fibroblasts to in- corporate collagen into their extracellular matrix when transformed by Rous Sarcoma virus. J. Biol. Chem. 252, 8863-8868.

BELSKY, E., VASAN, N. S., and LASH, J. W. (1980). Extracellular matrix components and somite chondrogenesis. Den. BioL 79,159-180.

BULL, D. L., TAYLOR, A. T., AUSTIN, D. M., and JONES, 0. W. (1974). Stimulation of fetal thymidine kinase in culture human fibroblasts transformed by SV 40 virus. Virology 57, 279-284.

CALOTHY, G., and PESSAC, B. (1976). Growth stimulation of chick embryo neural retinal on cells infected with Rous Sarcoma virus: Re- lationship to viral replication and morphological transformation. Virology 71, 336-345.

COGGIN, J. H., JR., and ANDERSON, N. G. (1974). Cancer, differentiation and embryonic antigens. Some central problems. Advan Cancer Res. 19, 105-165.

CRISS, W. E. (1970). A review of isozymes in cancer. Cancer Res. 31, 1523-1542.

EASTON, T. G., and REICH, E. (1972). Muscle differentiation in cell culture. J. Biol Chem. 247, 64206431.

ELLISON, M. L., and LASH, J. W. (1971). Environmental enhancement of in vitro chondrogenesis. Dev. BioL 26, 486-496.

FALTZ, L. L., CAPUTO, C. B., KIMURA, J. H., SCHRODE, J., and HASCALL, V. C. (1977). Structure of the complex between hyaluronic acid, the hyaluronic acid-binding region, and the link protein of proteoglycan aggregates from the swarm rat chondrosarcoma. J. BioL Chem 254, 1381-1387.

FISHMAN, W. H., INGLIS, N. I., STOLBACK, L. L., and KRANT, M. J. (1968). A serum alkaline phosphatase isoenzyme of human neoplastic cell origin. Cancer Res. 28, 150-154.

FISZMAN, M. Y., and FUCHS, P. (1975). Temperature sensitive expression of differentiation of transformed myoblasts. Nature (London) 254,429-431.

FOGEL, M., and DEFENDI, V. (1967). Infection of muscle cultures from various species with oncogenic DNA viruses (SV 40 and Polyoma). Proc. Nat. Acd Sci. USA 58, 967-973.

GOLD, P., and FREEDMAN, S. 0. (1965). Specific carcinoembryonic antigens of the human digestive system. J. Exp. Med. 122, 467-481.

GORDON, J. S., and LASH, J. W. (1974). In vitro chondrogenesis and differential cell viability. Dev. BioL 36, 88-104.

GRAESSMANN, A., GRAESSMANN, M., and FOGEL, M. (1973). The relationship of Polyoma virus-induced tumor (T) antigen to activation of DNA synthesis in rat myotubes. Dev. BioL 35,180-186.

GROSS, J. L., and RIFKIN, D. B. (1979). The effect of avian retroviruses on limb bud chondrogenesis in vitro. Cell 18, 707-718.

HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. MorphoL 88,48-92.

HANAFUSA, H. (1965). Analysis of the defectiveness of Rous Sarcoma virus. III. Determining influence of a new helper virus on the host range and susceptibility to interference of RSV. Virology 25, 248- 255.

HANAFUSA, H. (1977). Cell transformation by RSV tumor viruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 10, pp. 401. Plenum Press, New York/London.

HANAFUSA, H., HALPERN, C. C., BUCHHAGEN, D. L., and KAWAI, S. (1977). Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants. J. &p. Med. 146, 1735-1747.

HEINEGARD, D. (1972). Extraction, fractionation and characterization of proteoglycans from bovine tracheal cartilage. Biochim. Biophys. Acta 285, 181-192.

HINEGARDNER, R. T. (1971). An improved fluorometric assay for DNA. Anal. B&hem. 39,197-201.

HOLTZER, H., BIEHL, J., YEOH, G., MEGANATHAN, R., and KAJI, A. (1975). Effect of oncogenic virus on muscle differentiation. Proc. Nat. Acad. Sci. USA 72, 4051-4055.

HYNES, R. O., MARTIN, G. S., SHEARER, M., CRITCHLEY, D. R., and EPSTEIN, C. J. (1976). Viral transformation of rat myoblasts: Effects on fusion and surface properties. Dev. BioL 48, 35-46.

KAIGHN, M. E., EBERT, J. D., and STOTT, P. M. (1966). The susceptibility of differentiating muscle clones to Rous Sarcoma virus. Proc. Nat, Acad. Sci. USA 56, 133-140.

KAJI, A., YOSHIMURA, M., MUTO, M., CHI, J. S., ROTH, S. L., DELOTTO, R., and OKAYAMA, M. (1976). Differentiation and cancerization: Ex- periments using a temperature sensitive Rous Sarcoma virus mutant. Cancer Chemother. Rep. (Suppl.) 4, 35-46.

KAWAI, S., and HANAFUSA, H. (1971). The effect of reciprocal changes in temperatures on the transformed state of cells infected with a Rous Sarcoma virus mutant. Virology 46,470-479.

KAWAI, S., DEUSBERG, P. H., and HANAFUSA, H. (1977). Transfor- mation-defective mutants of Rous Sarcoma virus with srcgene dele- tions of varying length. J ViroL 24, 910-914.

KOBAYASHI, N., and KAJI, A. (1978). Induction of DNA synthesis in terminally differentiated myotubes by the activation of the src gene of Rous Sarcoma virus. Proc. Nat. Acad Sci USA 75, 5501-5505.

KOSHER, R. A., and LASH, J. W. (1975). Notochordal stimulation of in vitro somite chondrogenesis before and after removal of perino- tochordal materials. Dev. BioL 42, 363-3’78.

KURTH, R., and BAUER, H. (1973). Avian oncornavirus-induced tumor antigens of embryonic and unknown origin. Virology 56,496-504.

LASH, J. W. (1963). Studies on the ability of embryonic mesonephros explants to form cartilage. Dev. BioL 6, 219-232.

LASH, J. W. (1968). Chondrogenesis: Genotypic and phenotypic expression. J. Cell PhysioL 72 (Suppl. l), 35-46.

LASH, J. W., and VASAN, N. S. (1977). Tissue interactions and extracellular matrix components, In “Cell and Tissue Interactions” (J. Lash and M. Burger, eds.), pp. 101-113. Raven Press, New York.

LASH, J. W., and VASAN, N. S. (1978). Somite chondrogenesis in vitro: Stimulation by exogenous extracellular matrix components. Dev. BioL 66, 151-171.

LASH, J. W., PARKER, C. H., and OVADIA, M. (1982). Patterns of proteoglycan synthesis during chondrogenesis. In “The Extracellular Matrix” (S. P. Hawkes and J. L. Wang, eds.), pp. 71-88, Academic Press, New York.

LEE, H. H., KAIGN, M. E., and EBERT, J. D. (1968). Induction of thymidine aH incorporation in multinucleated myotubes by Rous Sar- coma virus. Int. J. Cancer 3, 126-136.


LEVER, P. L., and GOETINCK, P. F. (1976). Molecular sieve chromatography of proteoglycans. A comparative analysis. Anal. Biachem 75.67-76.

MINOR, R. R. (1973). Somite chondrogenesis. A structural analysis. J. Cell Biol 56, 27-50.

MUIR, H. (1977). Structure and function of proteoglycans of cartilage and cell-matrix components. In “Cell and Tissue Interactions” (J. W. Lash and M. M. Burger, eds.), pp. 87-100. Raven Press, New York.

MUTO, M., YOSHIMURA, M., OKAYAMA, M., and KAJI, A. (1977). Cellular transformation and differentiation. Effect of Rous Sarcoma virus transformation on sulfated proteoglycan synthesis by chicken chondrocytes. Proc Nat. Acad. Sk USA 74,4173-4177.

OGAWA, K., and ICHIHARA, A. (1972). Isozyme patterns of branched- chain amino acid transaminase in various rat heptomas. Cancer Res. 32, 1257-1263.

OKAYAMA, M., YOSHIMURA, M., MUTO, M., CHI, J., ROTH, S., and KAJI, A. (1977). Transformation of chicken chondrocytes by RSV. Cancer Res. 317, 712-717.

OVADIA, M., PARKER, C. H., and LASH, J. W. (1980). Changing patterns of proteoglycan synthesis during chondrogenic differentiation. J. En&y01 Exp. Morphol 56. 59-70.

PACIFICI, M., BOEITIGER, D., ROBY, K., and HOLTZER, H. (1977). Trans- formation of chondroblasts by RSV and synthesis of the sulfated proteoglycan matrix. Cell 11,891-900.

POTTER, V. R. (1978). Summary of discussion on neoplasms. Cancer Res. 28, 1901-1907.

ROTH, S. L., and KAJI, A. (1979). Change of isozyme pattern during activation of a transforming gene product. Its relation to other biochemical markers of cellular transformation and differentiation. B&him. Biophgs. Acta 583,241-252.

ROTH, S. L., DELO?TO, R., and KAJI, A. (1977). Changes in leucine

osminotransferase isozymes by viral transformation and its cor- relation with the isozyme changes occurring during differentiation. Cancer Res. 37,1147-1153.

SAJDERA, S. W., and HASCALL, V. C. (1969). Protein polysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures. J. Biol Chem 244,77-87.

SCHAPIRA, F. (1973). Isozymes and cancer. Advan. Cancer Res. 18,77- 153.

TANAKA, A., and KAJI, A. (1980). Aggregation properties of chondrocytes infected with a temperature sensitive mutant of Rous Sar- coma virus. J. Cell Sci 43, 407-417.

TANAKA, A., PARKER, C., and KAJI, A. (1980). Growth stimulation of chondrocytes by RSV: Stimulation of growth rate of chondrocytes by RSV is not coordinated with other expression of src gene phenotype. J. vird 35, 531-541.

TOOLE, B. P., OKAYAMA, M., ORKIN, R. W., YOSHIMURA, M., MLITO, M., and KAJI, A. (1977). Developmental roles of hyaluronate and chondroitin sulfate proteoglycans. In “Cell and Tissue Interactions” (J. W. Lash and M. M. Burger, eds.), pp. 139-151. Raven Press, New York.

VASAN, N. S., and LASH, J. W. (1979). Monomeric and aggregate proteoglycans in the chondrogenic differentiation of embryonic chick limb buds. J. Ednyol. Exp. MmrphoL 49.47-59.

VOGT, P. K., and Hu, S. S. F. (1977). The genetic structure of RNA tumor viruses. Annu. Rev. Gent 11, 203-238.

YOSHIMURA, M., IWASAKI, Y., and KAJI, A. (1981). In vitro differentiation of chicken embryo skin cells transformed by RSV. J. Cell Physiol 109,373-385.

YOSHIMURA, M., and KAJI, A. (1979). The possible role of elevated hyaluronic acid in inhibition of differentiation of myogenic cells transformed by Rous Sarcoma virus. XIth International Congress of Biochemistry Abstracts, 499.

Inhibition of precartilaginous chick somites by oncogenic virus

Documents

Transcript of Inhibition of precartilaginous chick somites by oncogenic virus