The Enhanced Production of Hyaluronic Acid by Cultured Rat Fibroblast Cells Treated with Cyclic AMP and Its Dibutyryl Derivative HIDEKI KOYAMA, MIKIO TOMIDA AND TETSUO O N 0 Department of Biochemistry, Cancer Institute, Japanese Foundation for Cancer Research, Toshima-ku, Tokyo 170, Japan

ABSTRACT Cells of a newly established rat fibroblast line (SEN) in culture synthesize mucopolysaccharides, which have been identified as hyaluronic acid, chondroitin-4-sulfate and heparan sulfate. Treatment of the cells with adenosine 3':5'-cyclic monophosphate resulted in a marked stimulation of production of hyaluronic acid, but not of the other mucopolysaccharides. Treated cells also showed increased activity of hyaluronic acid synthetase, a reduction in growth rate, and morphological alteration. In addition, 5-bromodeoxyuridine was found to counteract greatly the cyclic AMP effect.

Adenosine 3':5'-cyclic monophosphate (CAMP)acts as a second messenger that is released within the cells of target tissues after stimulation by the first messengers (hormones). The tissues then exhibit characteristic responses to the enhanced levels of cAMP produced by the hormones (Robinson et al., '68). In cultured cells in vitro, treatment with this cyclic nucleotide or its derivative dibutyryl adenosine 3':5'-cyclic monosphosphate (dbc AMP) is known to cause many cellular changes. For example, cAMP treatment restores sensitivity to contact inhibition of transformed cells and alters their morphology to that of normal cells (Hsie and Puck, '71; Johnson et al., '71 ; Sheppard, '71). It enhances the expression of some tissue-specific functions such as collagen synthesis (Hsie et al., '71), sulfated mucopolysaccharide synthesis (Goggins et al., '72), melanin synthesis (Johnson and Pastan, '72), and nurite formation (Prasad and Hsie, '71). Moreover, it induces the activity of enzymes such as alkaline phosphatase (Koyama et al., '72; Nose and Katsuta, '74), glutamine synthetase (Chader, '71), phosphoenolpyruvate carboxykinase (Barnett and Wicks, '71), tyrosine aminotransferase (Butcher et al., '71), and serine dehydratase (Kapp et al., '73). The aim of this paper is to describe the enhancement by cAMP and dbc AMP of hyaluronic acid production by a rat fibroJ. CELL. PHYSIOL.,87: 189-198.

blast cell line newly established in this laboratory . MATERIALS AND METHODS

Cells and culture methods The rat fibroblast cell line used here was established by chance during cultivation of the Morris 5123TC hepatoma. On July 7, 1967, a solid tumor (58 transplant generations in this laboratory in Buffalo rats) was digested with 0.1 % Pronase (Kaken Kagaku Co., Tokyo) in phosphate-buffered saline (PBS). The cells were plated onto 60-mm petri dishes, containing 5 ml of a modified Eagle's MEM (Yamane et al., '68) supplemented with 10% calf serum and 0.1% Bacto-Peptone (Difco, Detroit), and cultured at 37°C in a humidified atmosphere of 5-10% C 0 2 in air. Confluent cultures were transferred at an appropriate dilution after digestion with 0.02% pronase plus 0.02% EDTA. During the first five transfers, the cultures were composed of epithelial-like fusiform cells, probably the hepatoma cells. However, thereafter fibroblastlike cells became gradually predominant, and epithelial cells disappeared with subsequent transfers. After ten transfers (132 days), the culture was cloned (inoculated at 100 cells per dish), and 12 days later, five clones isolated successfully. One of these clones, showing a typical fibroblastic morReceived Feb. 21,' 7 5 . Accepted July 3, '75.

189

190

H I D E K I KOYAMA, MIKIO TOMIDA A N D T E T S U O O N 0

phology, was designated SEN (fiber in Japanese) and used in this study. This SEN line was injected into 11 new born Buffalo rats at 1 4 x 106 cellslanimal and into 11 suckling ones at 106 cells per animal. No tumors arose during the observation period of more than four months. The cells show a modal chromosome number of 72 comprising 40 biarmed chromosomes and 32 acrocentrics. This karyotype is quite different from that of the 5123TC tumor reported by Nowell et al. ('67), and demonstrates that the SEN line is of rat origin. Treatment of cells with chemicals and assay of mucopolysaccharide content in medium Cells in logarithmic growth phase were dissociated with the Pronase-EDTA solution, counted in a hemocytometer, and inoculated into 60-mm petri dishes (4 X 1 0 5 cellsldish) containing the above mentioned medium supplemented with CAMP, dbc AMP or 5bromodeoxyuridine (all from Sigma, St. Louis), as previously reported (Koyama et al., '72). After various incubation times, the medium was removed and frozen for the assay of mucopolysaccharide. The cells were dissociated and counted as above. The total content of mucopolysaccharide secreted into the medium was assayed by the method of Davidson ('63). Since we found that the amount of mucopolysaccharide bound to cells was much less than that in the medium, the culture medium alone was used for the assay. Usually 4 ml of the thawed culture medium was assayed, and the quantity of glucuronic acid, a common component of mucopolysaccharide, was determined by the procedure of Bitter and Muir ('62). The results are expressed as the amount of glucuronic acid in terms of cell number or per culture dish. Extraction and electrophoresis of mucopolys acc haride The mucopolysaccharide was extracted from SEN cells grown with or without dbc AMP (10-3 M) for 48 hours as follows: the medium (10 ml) plus cells harvested from two petri dishes with a rubber-policeman was mixed with Na acetate to a concentration of 1% ; three volumes of alcohol were added and the mixture was kept at -20°C overnight. The mixture was centrifuged at

3,000 rpm for 20 minutes. The resulting pellet was suspended into 10 ml of 0.2 M Tris-HC1buffer (pH 7.8), heated in a boiling water bath for ten minutes and then digested at 50°C with 1 mglml of Pronase. This procedure was followed by an additional overnight treatment of the non-dialyzable material in 0.3 M NaOH at 37°C. Trichloroacetic acid was added to the resulting solution, and this mixture was centrifuged at 3,000 rpm for 20 minutes. The supernatant was taken and dialyzed against tap water for two days and against distilled water for one day. The mucopolysaccharide was precipitated by the addition of three volumes of alcohol in the presence of Na acetate (1%). This precipitate was redissolved in 25 pl of distilled water and 8 p1 of this solution was applied to cellulose acetate strips (Sartorius, West Germany) and run in 1 M acetic acid-pyridine buffer (pH 3.8) at d.c. 0.5 mA per centimeter for 20 minutes (Seno and Meyer, '63). For some experiments, electrophoresis was conducted in two other solutions: 0.1 M Ba acetate (Wessler, '68) and 0.2 M Ca acetate (Seno et al., '70). After electrophoresis the strips were stained for mucopolysaccharide with 0.5% alcian blue 8GX in 3% acetic acid for ten minutes, washed with water and rendered transparent by dipping into decaline. The bands which appeared were scanned using a Joyce-Loebl Autodensidater MK3.

Assay of hyaluronic acid synthetase activity The method of this enzyme assay has been described in a previous paper (Tomida et al., '74). Briefly, cells were harvested by scraping with a rubber-policeman, washed three times with cold saline and stored at -20°C until the assay was run. At the time of assay, the pellets were thawed and used without any purification, because such crude extracts showed the highest activity. The enzyme reaction was based on the incorporation of UDP-14C-glucuronicacid (3 18 cilmole, The Radiochemical Centre, Amersham) into the hyaluronic acid fraction in the presence of UDP-N-acetyl-glucosamine (Sigma) and enzyme extracts. The enzyme activity (specific activity) was expressed by cpm of *4C-glucuronic acid in hyaluronic acid per hour per milligram protein. Protein was determined by a slight modification of the method of Lowry et al. ('51).

cAMP EFFECT ON HYALURONIC ACID SYNTHESIS RESULTS

Demonstration of mucopolysaccharide synthesis Since the establishment of the SEN line, we noticed that the culture medium became viscous as the cells grew. Hyaluronic acid can be easily detected by mucin clot formation, a test which is specific for it, as described before (Koyama and Ono, '70). When the used medium from confluent SEN cultures was mixed with acetic acid at 1% , a thread-like precipitate was formed. However, the same medium, which had been treated with either 10 unitslml of bovine testicular hyaluronidase (Boehringer Mannheim Japan, Tokyo) for ten minutes at 3 7 ° C

I

cs

HA

I

n

I

I

-

-I

-

z

ln

m

ln

i\ I

I

1

2

3

4

Migration distance( cm) Fig. 1 Electrophoresis of mucopolysaccharides produced by SEN cells grown in the absence or presence ofdbc AMP. The methods for cell culture, extraction, and electrophoresis of mucopolysaccharide were described in MATERIALS A N D METHODS. (a) Authentic hyaluronic acid (HA; 0.8 pg) plus chondroitin-4-sulfate (CS; 0.4 p g ) ; (b) mucopolysaccharide extracted from untreated control cultures; (c) mucopolysaccharide from thecultures grown in the presence of dbc AMP at 1 0 - 8 M for 48 hours.

191

or 10 units/ml of Streptomyces hyaluronidase (Seikagaku Kogyo, Tokyo) for ten minutes at 55"C, gave completely negative results in this test. The former hyaluronidase is not specific for hyaluronic acid alone, but the latter is. This result demonstrates the presence of hyaluronic acid. To examine whether SEN cells produced other mucopolysaccharides, the total mucopolysaccharide fraction was extracted from the medium and cells and analyzed by electrophoresis on cellulose acetate strips. This result is shown in figure 1. Figure l a shows the pattern of a mixture of authentic hyaluronic acid (Boehringer Manheim Japan) and chondroitin-4-sulfate (Seikagaku Kogyo) obtained commercially. It is clear in figure l b that the mucopolysaccharide from the SEN cells grown in normal medium gives three bands. The most anodal band coincides with that of authentic chondroitin-4-sulfate. Conversely, the most slowly migrating and most dense band is assigned to hyaluronic acid, although it moves slightly faster than the authentic hyaluronic acid. This could be due to the existence of impurities contained in the commercial product. These results were also supported by results of the electrophoretic patterns obtained in 0.1 M Ba acetate and 0.2 M Ca acetate solutions. In addition, the intermediate band (fig. lb) is likely to be heparan sulfate. judging from the comparison of its migration patterns on electrophoresis in the three different solutions mentioned above.

Stimulation of hyaluronic acid production by cAMP and dbc A M P Preliminary observations had shown an increased viscosity of the medium upon treatment of the SEN line with dbc AMP, suggesting that the drug treatment could stimulate at least hyaluronic acid production. SEN cells were grown in the presence of this nucleotide (10-3 M) for 48 hours, and their mucopolysaccharides were extracted and analyzed on electrophoresis as described above. The resulting pattern is shown in figure lc. Since this experiment was performed using replicate cultures of those in figure Ib, one can compare the two results almost quantitatively. It is evident that in the dbc AMP treated sample the amount of hyaluronic acid increased

:I b

a

100-

100 -

-

1= V I , .-

m

\ (3\

7 v

-0

.-U 50 m U

.-

c 0 L

3 U 3

a

-152

VI .U

0

-10-

X

v) d

50

r

-

m '

W U

LU0

-5

0

ty('

0

1

2

3l

/

l

:

about two-fold, but the production of the two sulfated mucopolysaccharides seemed unaffected . The total mucopolysaccharide content was assayed in the medium of SEN cultures grown in the presence of varying concentrations of either cAMP or dbc AMP for three days. Figure 2 shows that there was an enhanced production of glucuronic acid in these cultures, and that this effect was dose-dependent. Treatment with cAMP at 3 X 10-4 M or more resulted in a 80 to 280% increase in the glucuronic acid content per dish (fig. 2a). At these concentrations, however, cell growth, as estimated by cell counts, was reduced only 10 to 1 7 % . The derivative dbc AMP was more effective than cAMP in stimulating mucopolysaccharide production (fig. 2b) and this effect was also dose-dependent: at 1-2 X 10-3 M,the amount of glucuronic acid per dish became four-fold that of the control. In parallel to this greater stimulatory effect, this derivative showed a greater inhibition of cell growth than did CAMP. The cell numbers in the cultures treated with 1-2 X 10-3 M were 4 3 5 7 % of those in the untreated cul-

i

'

0

1

2

5

tures. Therefore, the glucuronic acid content in terms of cell number was increased by seven- to nine-fold. Addition of 5'-AMP, ADP, or ATP at 10-3 M also increased the glucuronic acid production, to a lesser but significant degree. Similarly, prostaglandin El (1-10 pglml), which can activate adenylcyclase located on the cell membrane to elevate the intracellular cAMP levels, was somewhat active. However, theophylline, an inhibitor of phosphodiesterase, and dibutyryl guanosine 3': 5'-cyclic monophosphate, at 10-3 M had no significant effect on either mucopolysaccharide production or cell multiplication under the present experimental conditions. Na butyrate (Wako Pure Chem., Osaka) was tested, since it can be formed by degradation of dbc AMP added to cells. At concentrations greater than 10-3 M, this compound increased slightly the glucuronic acid content in the medium of the treated cultures. Since the assay method used here is not specific for hyaluronic acid alone, the values presented above as glucuronic acid represent the sum of the three mucopolysac-

CAMP EFFECT O N HYALURONIC ACID SYNTHESIS

193

charides demonstrated by electrophoresis in figures lb,c. However, since the electrophoretic analyses have shown that only hyaluronic acid production is enhanced in the cells treated with cyclic nucleotides, it c a n therefore be concluded that the increases in glucuronic acid content (fig. 2) c a n be attributed to increases in hyaluronic acid content of the medium.

Growth curve and time course of mucopol ysacc haride production The growth curve of the cells and the time course of mucopolysaccharide production was studied in the absence or presence of dbc AMP. In addition, the effect of 5brornodeoxyuridine (BrdU) was studied, because this compound has been found to inhibit mucopolysaccharide synthesis in some cultured cells under conditions where cell growth is minimally affected (Abbott and Holtzer, '68; Bischoff and Holtzer, '68; Koyama and Ono, '71; Bischoff, '71). Figure 3 shows typical results of such an experiment. Following the initial lag period of 2 4 hours, during which no increase in cell number occurred, the cells in normal medium multiplied almost exponentially (fig. 3a). The mean doubling time in the period from 24 to 72 hours was 26.1 hours. When the cells were grown in medium containing dbc AMP at 10-3 M, a reduction in growth rate was found with increasing incubation time. The mean doubling time rose to 45.6 hours, and, at 72 hours, the number of cells was only 41 % of that in the control cultures. In cultures grown with BrdU alone, there was a 13% reduction in cell number, while in the cultures with BrdU plus dbc AMP, the cell yield was the same as that found in the cultures with the dibutyryl nucleotide alone. The production of mucopolysaccharide was time-dependent as shown in figure 3b. In control cultures, the amount of glucuronic acid secreted increased linearly with the time of incubation. The cells, however, propagated logarithmically after the first 24 hour lag. Therefore the rate of mucopolysaccharide synthesis, defined as the amount of glucuronic acid synthesized per cell per hour, gradually fell (table 1). On the other hand, the cultures grown in the dbc AMP continued to produce mucopolysaccharide at two and one-fifth to five and one-half-fold higher rate in each

Fig. 3 Growth curve and time course of mucopolysaccharide production by S E N cells grown i n the absence OT presence of dbc A M P . Cells were cultured in the medium with or without dbc AMP ( 1 0 - 3 M) and BrdU (1.6 X 1 0 - 5 M). Cell counts and mucopolysaccharide assay (glucuronic acid content) were carried out at 24, 48, and 72 hours. A , No addition (control); 0,dbc AMP; A, BrdU; 0 ,dbc AMP and BrdU.

corresponding interval (fig. 3b, table 1). In particular, a rise in the rate found in the treated cultures was in apparent contrast to its reduction in the control. Addition of BrdU to the cells growing in normal medium resulted in a 14% decrease in the yield of glucuronic acid per cell after 72 hour cultivation. However, when added

194

HIDEKI KOYAMA, MIKIO TOMIDA AND TETSUO ON0 TABLE 1

Comparison of the rate of mucopolysaccharide synthesis in S E N cells grown in the absence or presence of dbc AMP Rate of mucopolysaccharide synthesis 1 Time interval

Untreated cells

Treated cells

0.70 0.54 0.24

1.5 2.1 1.3

(hours) a24 24-48 4a72

The values were calculated from the results shown in figure 3 by dividing the amount of glucuronic acid by the mean cell number in the interval of 24 hours. 1 pg of glucuronic acid per cell hour.

to the cells with dbc AMP, BrdU blocked by as much as 40% the production of mucopolysaccharide. This change should be attributed mainly to the reduction of enhanced hyaluronic acid synthesis. Figure 4a shows a photograph of living SEN cells grown in normal medium for 48 hours. They are fibroblastic, and the cellular arrangement is extremely random like that of transformed cell lines. Cells grown in dbc AMP at 10-3 M for the same period (fig. 4b) are characterized by scant cytoplasm and by the extension of very long, thin processes, appearing more fibroblastic than the control cells above. These morphologic alterations are consistent with those reported by others (Hsie and Puck, '71;

Johnson et al., '71; Sheppard, '71). The cellular orientation of the treated SEN cells, however, still remained as random as that of the untreated cells.

Hyaluronic acid synthetase activity The activity of hyaluronic acid synthetase was compared in cells grown in the absence and presence of dbc AMP ( 1 0 - 3 M) for 48 hours. As shown in table 2, the nucleotide treatment elevated the activity of the enzyme by three and one-fifth-fold, while cell protein was reduced 1 2 % . This result provides additional evidence, at the enzyme level, for the enhancement of hyaluronic acid production by the cyclic nucleotides. TABLE 2

Hyaluronic acid synthetase activity in S E N cells grown in the absence o r presence of dbc AMP Cells grown

Hyaluronic acid synthetase (Spe. act.) 1

Cell protein (mgldish)

dbc AMP + d b c AMP

4770 f 30 15100

* 3100

0.564

* 0.039

0.495

* 0.042

Cells were grown in the absence or presence of dbc AMP (10-3 M) for 48 hours. The data wereobtained from three independent experiments. Expressed a s cpm of 14-glucuronic acid incorporated into hyaluronic acid per hour per milligram of protein. Mean SEM.

*

Fig. 4 (a) Living SEN cells grown in normal medium for 48 hours. Phase-contrast, 320 x . (b) Living SEN cells grown in medium containing dbc AMP ( 1 0 - 3 M) for 48 hours. Phase-contrast, 320 x .

cAMP EFFECT ON HYALURONIC ACID SYNTHESIS DISCUSSION

The data presented here shows that a newly established rat fibroblast cell line h a s the ability to produce hyaluronic acid, chondroitin-4-sulfate, and heparan sulfate, a n d the producton of hyaluronic acid alone is significantly enhanced upon treatment with cAMP or dbc AMP. This was demonstrated by electrophoretic analysis of the mucopolysaccharide fraction extratcted from cultures, determination of its content in the medium, and assay of hyaluronic acid synthetase activity. The first point to be discussed is the origin of the SEN line used here, because it was by chance established during cultivation of the Morris 5123TC hepatoma. However, we conclude that this line did not arise from the tumor tissue, but from normal connective tissue contaminating it. There are four arguments in support of this conclusion: (1) The morphology of the SEN line is typically fibroblastic, being quite different from that which we observed in the primary culture of the tumor. (2) In general, hyaluronic acid synthesis is considered to be a differentiated function characteristic of fibroblast cells in culture. ( 3 ) An extensive test for tumorigenicity of the S E N cells proved completely negative. (4) The cells had neither tyrosine aminotransferase nor other liver-specific enzymes that have been reported to exist in the 5123TC tumor (Ono, '66), although these enzyme activities might be lost during prolonged cultivation. cAMP treatment has been demonstrated not only to restore normal morphology and contact inhibition to malignant, transformed cell lines (Hsie and Puck, '71; Johnson et al., '71; Sheppard, '71), but also to promote the expression of many differentiated functions in vitro. The present work adds one more example to the latter effects of CAMP.It seems therefore that cAMP acts on the cell to enhance or stabilize the expression of differentiated functions, the potential for which has already been determined and the expression of which is maintained in vitro. These observations have been made on cells cultured in vitro, but probably even in vivo cells of tissues maintain their fully differentiated states by synthesizing the nucleotide or utilizing that from the circulatory system. Goggins et al. ('72) reported that treat-

195

ment of SV40 transformed 3T3 cells with dbc AMP and theophylline stimulates the synthesis of chondroitin-4- and -6-sulfates and dermatan sulfate. In the SEN cells, chondroitin-4-sulfate is also synthesized, but is not affected by the dbc AMP treatment. B-6 cells, which were derived by hybridization of mouse mammary carcinoma and Chinese hamster lung cells (Koyama et al., '70) are capable of producing hyaluronic acid (Koyama and Ono, '70), but their treatment with dbc AMP did not influence the amount produced. Such differences in response to the nucleotide have been seen as well for other cellular properties, but the reason remains to be determined. The effects of cAMP on cellular metabolism appears to be so pleiotropic that it would be very difficult to determine at what step or level and how cAMP acts on SEN cells to stimulate their hyaluronic acid production. Even at the step directly involved in hyaluronic acid biosynthesis, there are as many as ten enzymes that convert sequentially the first precursor glucose into the two carbohydrate nucleotides (UDP-Nacetyl-glucosamine and UDP-glucuronic acid) and then finally into hyaluronic acid (Jacobson, '70). We have not yet studied the activities of these enzymes except the last one, hyaluronic acid synthetase. The fact that the activity of this synthetase is elevated significantly in the treated cells may be at least one of the mechanisms for the stimulation of hyaluronic acid production. The direct involvement, as one of the key steps, of this enzyme in the regulation of hyaluronic acid synthesis has been supported by our findings that its production occurs only at the active stage of growth cycle where the rate is parallel with the enzyme levels in this cell line and the B-6 line (Tomida et al., '74). Recently, we have seen that addition of actinomycin D or cycloheximide blocks completely the increase in hyaluronic acid synthetase activity following exposure of SEN cells to CAMP,suggesting that new synthesis of this enzyme may be involved in this elevation (Tomida, Koyama and Ono, in preparation). BrdU inhibits slightly mucopolysaccharide production by SEN cells growing in normal medium, but greatly suppresses it by dbc AMP-treated cells. This thymidine analog is known to suppress many differentiated functions in animal cells (Wilt

196

HIDEKI KOYAMA, MIKIO TOMIDA AND TETSUO ONO

and Anderson, ’72). The present system having a character that the CAMP effect is counteracted by BrdU may provideone more useful system to study the mechanism of differentiation. ACKNOWLEDGMENTS

We wish to thank Mr. H. Kodama for his technical assistance. This work was supported by grants for Cancer Research from the Ministry of Education, Japan. LITERATURE CITED Abbott, J., and H. Holtzer 1968 The loss of phenotypic traits by differentiated cells. V. The effect of 5-bromodeoxyuridineon cloned chondrocytes. Proc. Nat. Acad. Sci., (U.S.A.), 5 9 : 1144-1151. Barnett, C. A,, and W. D. Wicks 1971 Regulation of phosphoenolpyruvate carboxykinase and tyrosine transaminase in hepatoma cell cultures. I. Effects of glucocorticoids, Ne,OZ‘-dibutyrylcyclic adenosine-3’,5’-monophosphateand insulin in Reuber H35 cells. J. Biol. Chem., 246: 72017206. Bischoff, R. 1971 Acid mucopolysaccharide synthesis by chick aminion cell cultures. Inhibition by 5-bromodeoxyuridine. Exp. Cell Res., 66 : 224236. Bischoff, R., and H. Holtzer 1968 Inhibition of hyaluronic acid synthesis by BUdR in cultures of chick amnion cells. Anat. Rec., 160: 317. Bitter, T., and H. M. Muir 1962 A modified uronic acid carbazole reaction. Anal. Biochem., 4: 3 3 0 434. Butcher, F. R., J. E. Becker and V. R. Potter 1971 Induction of tyrosine aminotransferase by dibutyryl cyclic-AMP employing hepatoma cells in tissue culture. Exp. Cell Res., 66: 321328. Chader, G. J. 1971 Hormonal effects on the neural retina: induction of glutamine synthetase by cyclic-3’,5’-AMP. Biochem. Biophys. Res. Commun.,43: 1102-1105. Davidson, E. H. 1963 Heritability and control of differentiated function in cultured cells. J. Gen. Physiol., 46: 983-998. Goggins, J. F., G. S. Johnson and I. Pastan 1972 The effect of dibutyryl cyclic adenosine monophosphate on synthesis of sulfated acid mucopolysaccharides by transformed fibroblasts. J. Biol. Chem., 247: 57595764. Hsie, A. W., C. Jones and T. T. Puck 1971 Further changes in differentiation state accompanying the conversion of Chinese hamster cells to fibroblastic form by dibutyryl adenosine cyclic 3’:5’-monophosphate and hormones. Proc. Nat. Acad. Sci., (U.S.A.),68: 1648-1652. Hsie, A. W., and T. T. Puck 1971 Morphological transformation of Chinese hamster cells by dibutyryl cyclic 3’:5’-monophosphate and testosterone. Proc. Nat. Acad. Sci., (U.S.A.), 68: 358361. Jacobson, B. 1970 The biosynthesis of hyaluronic acid. In: Chemistry and Molecular Biology of the Intracellular Matrix. Vol. 11. E. A. Balasz, ed. Academic Press, Inc., New York, pp. 763-781. Johnson, G. S., R. W. Friedman and I. Pastan

1971 Restoration of several morphological characteristics of normal fibroblasts in sarcoma cells treated with adenosine-3’:5’-cyclic monophosphate and its derivatives. Proc. Nat. Acad. Sci., (U.S.A.), 68: 425429. Johnson, G. S., and I. Pastan 1972 N6,02’-Dibutyryl adenosine 3’,5’-monophosphate induces pigment production in melanoma cells. Nature New Biol., 237: 267-268. Kapp, L. N., J. A. Remington and R. R. Klevecz 1973 Induction of serine dehydratase activity by cyclic AMP is restricted to S phase in synchronised CHO cells. Biochem. Biophys. Res. Commun., 52: 1206-1212. Koyama, H., R. Kato and T. Ono 1972 Induction of alkaline phosphatase by cyclic AMP or its dibutyryl derivative in a hybride line between mouse and Chinese hamster in culture. Biochem. Biophys. Res. Commun., 46: 305-31 1. Koyama, H., and T. Ono 1970 Initiation of a differ en tiated function (Hy aluronic acid synthesis) by hybrid formation in culture. Biochim. Biophys. Acta, 21 7: 477487. 1971 Effect of 5-bromodeoxyuridine o n hyaluronic acid synthesis of a clonal hybrid line of mouse and Chinese hamster in culture. J. Cell. Physiol., 78: 265-272. Koyama, H., I. Yatabe and T. Ono 1970 Isolation and characterization of hybrids between mouse and Chinese hamster cell lines. Exp. Cell Res., 62: 455463. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-2 75. Nose, K., and H. Katsuta 1974 Induction of alkaline phosphatase activity by dibutyryl adenosine 3’:5’-cyclic monophosphate in aneuploid rat liver cells. Exp. Cell Res., 87: 8-14. Nowell, P. C., H. P. Morris and V. R. Potter 1967 Chromosomes of “minimal deviation” hepatomas and some other transplantable rat tumors. Cancer Res., 27: 1565-1579. Ono, T. 1966 Enzyme patterns and malignancy of experimental hepatomas. In: Biological and Biochemical Evaluation of Malignancy in Fxperimental Hepatomas (GANN Monograph). Vol. I. T. Yoshida, ed. The Japanese Foundation for Cancer Research and Japanese Cancer Association, Tokyo, pp. 189-205. Prasad, K. N., and A. W. Hsie 1971 Morphologic differentiation of mouse neuroblastoma cells induced in vitro by dibutyryl adenosine 3’:5‘-cyclic monophosphate. Nature New Biol., 233: 141142. Robinson, G. A,, R. W. Butcher and E. W. Sutherland 1968 Cyclic AMP. Ann. Review Biochem., 37: 149-174. Seno, N., K. Anno, K. Kondo, S. Nagase and S. Saito 1970 Improved method for electrophoretic separation and rapid quantitation of isomeric chondroitin sulfates on cellulose acetate strips. Anal. Biochem., 37: 197-202. Seno, N., and K. Meyer 1963 Comparative biochemistry of skin: The mucopolysaccharides of shark skin. Biochim. Biophys. Acta, 78: 258-264. Sheppard, J. R. 1971 Restoration of contactinhibited growth to transformed cells by dibutyryl adenosine 3’:5’-cyclic monophosphate.

CAMP E F F E C T ON HYALURONIC ACID SYNTHESIS PrOc. Nat. Acad. Sci., (U.S.A.), 68: 1316-1320. Tomida, M., H. Koyama and T. On0 1974 Hyaluronic acid synthetase in cultured mammalian cells producing hyaluronic acid: Oscillatory change during the growth phase and suppression b y 5-bromodeoxyuridine. Biochim. Biophys. Acta, 338: 352-363. Wessler, E. 1968 Analytical and preparative separation of acidic glycosaminoglycans by elec-

197

trophoresis in barium acetate. Anal. Biochem., 26:43-44, Wilt, F. H., and M. Anderson 1972 The action of 5-bromodeoxyuridine on differentiation. Develop. Biol., 28: 443-447. Yamane, I., Y. Matsuya and K. Jimbo 1968 An autoclavable powdered culture medium for mammalian cells. Roc. SOC. Exp. Biol. Med., 127: 335336.