molecules characteristically produced by specialized ... - Europe PMC

EFFECT OF 5-BROMODEOXYURIDINE ON EXPRESSION OF CULTURED CHONDROCYTES GROWN IN VITRO* BY H. ScImuLTPE HOLTHAUSENI S. CHACKOt E. A. DAVIDSON,t AND H. HOLTZER§ MILTON S. HERSHEY MEDICAL CENTER, PENNSYLVANIA STATE UNIVERSITY, HERSHEY; AND UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA

Communicated by Karl Meyer, April 28, 1969

Abstract.-Exposure of cultured cartilage cells to 5-bromodeoxyuridine results in a rapid loss of the ability to synthesize the chondroitin 4-sulfate-protein complex characteristic of the extracellular matrix of differentiated cartilage. UDP-glucose dehydrogenase, UDP-N-acetyl hexosamine 4-epimerase and the enzyme system responsible for catalyzing 3'-phosphoadenosine-5'-phosphosulfate synthesis all progressively decline in activity, while several other cellular processes seem unaffected. However, there are marked morphological changes which may be associated with plasma membrane components or the synthesis of the protein-polysaccharide complex.

The effect of 5-bromodeoxyuridine (BUdR) on differentiating cells is unusual. Moderate concentrations of the analog reversibly depress the synthesis of many molecules characteristically produced by specialized cells but do not markedly depress the rate of cell multiplication.'-' After one S period in BUdR, myogenic cells fail to fuse to form multinucleated myotubes and fail to translate for myosin. Myogenic cells have been maintained in BUdR for over two weeks, during which time the population doubles approximately five times. When such repressed myogenic cells are allowed to replicate in normal medium, many of their progeny fuse and form typical multinucleated myotubes.2 4-I Similarly, replicating presumptive neuroblasts, replicating amnion cells, and replicating chondrocytes grown in BUdR are specifically, but reversibly, inhibited from sprouting neurites and from secreting hyaluronic acid and chondroitin sulfate, respectively.8 9 Mitotically quiescent, differentiated embryonic chondrocytes removed from their in vivo polysaccharide matrix and grown in culture are induced to re-enter the mitotic cycle and deposit polysaccharide matrix.'0 The chondroitin sulfate component of this matrix is cytologically and biochemically identical with that normally present in cartilage; in addition, these cells may synthesize hyaluronic acid and keratan sulfate.", 12 Normal chondrocytes in vitro are polygonal, sessile cells tightly adherent to one another but loosely bound to the plastic substrate of the culture dish. When chondrocytes are grown in BUdR, they transform into flattened, amoeboid cells which repel one another, are tightly bound to the plastic substrate, do not deposit metachromatic matrix, and fail to synthesize significant amounts of chondroitin sulfate.' 3, 8 The major nonfibrillar component of the extracellular matrix of cartilage is a protein-polysaccharide complex which contains chondroitin-4-sulfate chains covalently linked to a polypeptide backbone. The molecule can be visualized 864

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as containing a polypeptide core of approximately 10,000 mol wt, to which are attached five or six chondroitin-4-sulfate chains of approximately 15,000 mol wt each.'3, 14 The carbohydrate components of the chondroitin-4-sulfate chains are D-glucuronic acid and N-acetyl-D-galactosamine4-O-sulfate. There are three enzyme systems essential for the production of these specific components: UDP-glucose dehydrogenase, UDP-N-acetyl-D-glucosamine 4-epimerase, and the sulfate activating system responsible for the synthesis of 3'-phosphoadenosine-5'-phosphosulfate. This report is concerned with the effect of 5-bromodeoxyuridine on the level of these enzymes as well as on the protein-polysaccharide end product when chondrocytes are grown in its presence. Materials and Methods.-A pure population of suspended 10-day vertebral chondrocytes was obtained as described." 8 Cultures were plated at 1 X 103 cells/60-mm plastic Petri dish (Falcon Plastics, tissue culture grade) in 4 ml of medium. The culture medium consisted of Ham's" F-10 with twice the amino acid concentration, supplemented with 10% calf serum and 1% bovine serum albumin (Grand Island Biological or Microbiological Associates). Cultures were fed either daily or on alternate days: 2 ml of medium was decanted and 2 ml of fresh medium added. Cultures were incubated for specified periods of time under control conditions, and then subsets were cultured in the presence of BUdR (10 ,g/ml) for varying periods or in the presence of -*S-labeled inorganic sulfate (sodium salt, carrier-free, Abbott Lab.), or both. In all cases, control cultures containing sibling cells of approximately equivalent numbers were cultured for a period equal to the total time of culture, Whether in the BUdR or sulfate. Cultured cells were harvested by scraping and centrifuging and were disrupted in an all-glass homogenizer with 0.15 M NaCl as a suspending medium. Cell debris was removed by centrifugation at 12,000 X g for 15 min, and aliquots of the supernate were employed for enzyme assays. The protein content of the supernatant was determined by the method of Lowry.'6 All enzyme assays were conducted at several levels of enzyme and at least two time periods to permit direct measure of specific activities. 10-3 M BUdR had no effect on the activity of control enzymes. UDP-glucose dehydrogenase was assayed in extracts of cultured cells by using 14Clabeled UDP-glucose as a substrate and chromatographically separating the UDP-glucuronic acid produced. This was assayed for radioactivity, initially on a strip scanner and subsequently, for quantitation, in a scintillation counter. The UDP-N-acetyl hexosamine 4-epimerase was assayed with radioactively labeled UDP-N-acetyl galactosamine as a substrate. After incubation, the nucleotides were hydrolyzed and the N-acetyl amino sugars resolved by chromatography on borate-impregnated paper which was subsequently assayed for radioactivity. The sulfate-activating system was assayed as previously described.'7 The remainder of the supernate from the cell extract was combined with the growth medium and the sulfated polysaccharides isolated by precipitation with cetyl pyridimium chloride after addition of unlabeled chondroitin 4-sulfate as carrier. The polysaccharide fraction was extensively purified and the location of the ester sulfate group determined by the rate of release of `5S inorganic sulfate during mild acid hydrolysis. Control material for this latter experiment was prepared biosynthetically by injecting 10-day-old chick embryos with inorganic sulfate 3"S and isolating chondroitin 4-sulfate from the cartilage of the embryos after 4 additional days of development. Results and Discussion.-A quantitative measure of either the UDPG dehydrogenase or the hexosamine epimerase as a function of time of exposure to BUdR revealed that there is a progressive decrease in enzyme activity beginning with a 24-hour sample and decreasing to very low levels after five days. Cultures grown for extended time periods have slight but detectable activity which,

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however, is 10 per cent or less than 10 per cent of control values. Similar results are observed for the sulfokinase. Data are summarized in Figure 1. The release of I'S inorganic sulfate from labeled polysaccharide is indicative of the locus of these sulfate groups on the polymer. The comparison of hydrolysis curves for polysaccharide isolated from control and BUdR grown cells is shown in Figure 2. In general, there is no significant difference between the two groups, although the total amount of sulfate incorporated into chondroitin sulfate is far less for the BUdR group (Table 1 and Fig. 3). The BUdR-altered cells show essentially no increase over a ten-day period in terms of sulfate incorporation into polysaccharide. However, it should be noted that the nature of the isolation procedure employed would tend to lose low-molecular-weight fractions such as tetra- or hexasaccharide and, in addition, would be unable to detect the presence of protein core as yet unsubstituted with polysaccharide chains. The continued appearance of low levels of radioactivity in sulfated polysaccharide (approximately 10% of control after seven days) and of very low levels of specific enzymes, may be artifacts of the assay procedures or may be due to the presence in the cultures of cells that have not yet divided and hence have not formed BU-DNA. It is not known how many divisions chondrocytes have to undergo in BUdR to partially or totally depress the activity of the enzymes. 12

-----*-u

10

~

CONTROL

> 8

6-

CONTROL

0 4

**} DEHYDROGENASE ~

*x)

2 is @

2

\\

O

2

~

x_

4

~ L

6

~

EPIMERASE

x4

~ ~

8 10 TIME (Days)

BUd R 12

,

14

FIG. n-Specific activities (expressed as micromoles of product formed/mg protein/min X 103) of enzymes prepared from control and BUdR-exposed cells. Time refers to length of exposure to 10 ;&g/ml of BUdR following preculture under standard conditions. Control cells were handled in an identical manner except for treatment with BUdR. The amount of soluble protein for control and BUdR-exposed cells was generally the same. Data are shown for UDPGdehydrogenase and UDP-N-acetyl hexosamine 4-epimerase. Quantitative data for 3'-phosphoadenosine-5'-phosphosulfate synthesis were more difficult to obtain reproducibly, but the activity of this enzyme system was always less than 5% of control values after 6-day exposure to BUdR. A semilog plot of the data suggests a first-order reaction with a half-time of 38 hr, comparable to the rate of cell division

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.0I 0:

0

FIGe. 2.-Release of 35S0O4 from isolated polysaccharide during mild acid hydrolysis. The rate of hydrolysis islindicative of the location of the sulfate ester group.18

\CHONDROITIN

4- SULFATE STANDARD

Z

o P* x 1.80 BUdR

o

POLYSACCHARIDE

*) CONTROL

1.7or 20

100 40 60 80 TIME OF HYDROLYSIS

120

(MIN.)

There are gross effects on cellular morphology after two days in BUdR. The control, functioning chondrocytes are polygonal and nonmotile, exhibit metachromatic matrix, and adhere to one another. In contrast, although the levels of BUdR employed were such as not to significantly affect the rate of cell division, the BUdR-altered cells are exceedingly flattened and amoeboid, do not deposit metachromatic matrix, and do not adhere to one another. The surface area of BUdR-altered cells is anywhere from four to eight times that of control cells. These pronounced morphological changes are apparent after one or two rounds of DNA synthesis in BUdR. The tight adhesion of the BUdR-altered cells to the plastic substrate frequently results in mitosis, yielding daughter cells of unequal sizes. These striking morphological changs in BUdR-altered cells may reflect some primary alteration in the plasma membrane structure and suggest that the formation of the characteristic protein-polysaccharide matrix product is interrelated with the morphological integrity of the chondrocyte. Alternatively, failure to synthesize the protein-polysaccharide may lead to the morphological changes. It is a provocative observation that BUdR-altered chondrocytes, BUdR-altered myogenic cells, and BUdR-altered neurogenic cells are essentially indistinguishable under the light microscope. The similar fibroblast-like appearance of many dedifferentiated cells in vitro may be associated with a comparable absence of functional expression. The cell membrane has a recognition function, and the extracellular matrix may be involved in cellTABLE;1. Total counts in isolated chondroitin 4-sulfate fraction. Time

Control IBUdR

-(days)-

1

2

4

6

9

11

27,000

40,000

41,000

40,000

45,500

40,500

18,000

5,000

3,500

11,500

6,500

5,000

Time refers to length of exposure to BUdr. (See legend of Fig. 3 for details of experimental

conditions.)


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PROC. N. A. S.

FIG. 3.-Radioactivity of chondroitin 4-sulfate after exposure of cultured chondrocytes to 35S0460 with and without 10 Ag/ml of BUdR. Cells were grown under 50 conditions identical to those described for Fig. 1, except that n5S --------. . ----. CONTROL inorganic sulfate (10 IACi/ml) was 40 ' included in the culture medium for / the final 72 hr of the incubation 0 30 period. Data are expressed as total / _ counts recovered in the polysac/charide fraction after purification. 0. The 6-day BUdR sample may have 20 been contaminated since significant 1I \ enzymatic activity was also detectable in this fraction although levels l0 / \ were much lower in both 5- and 7// t. t BUd R day samples. After 48-hr exposure 0 2 to BUdR, values are 10% or less of 6 12 2 4 8 10 control figures, although never zero. DAYS

cell interactions, but the relationship between the production of these materials and the mutual repulsion of the BUdR-altered cells is uncertain. The polysaccharide material isolated from BUdR-altered cells is, within the limits of detection, substantially identical in chemical structure to that produced by normal cells. The sulfate ester groups are on the four position of the galactosamine moiety, and the characteristic components D-glucuronic acid and Dgalactosamine are present. The selective nature of the BUdR-suppression on the biosynthetic activities of chondrocytes is shown in Table 2. The phosphatase and cytochrome oxidase activities were the same in both groups over the entire assay period. Spot checks of hexokinase activity have shown no difference between BUdR and control cells. The inhibitory action of BUdR on differentiating chondrocytes might not be due to the incorporation of the analogue into DNA. For example, the analogue might be incorporated into abnormal nucleotides which secondarily could interfere with the synthesis of glycoprotein components of the plasma membranes. The acid-soluble nucleotides from control cells and cells grown in the presence of 3H-BUdR were fractionated on Dowex-1 formate form resin, according TABLE 2. Specific activities (units per mg of protein) of mitochondrial and lysosomal marker enzymes after exposure of cultured chondrocytes to BUdM. . -Cy Time

(1 unit

=

eCytochrome Oxidase AA65o of 0.001/min/mg protein)

Acid Phosphatase (1 unit = 1 sum/min) Control BUdR

Control BUdR (days) 1 3.7 3.4 0.09 0.11 2 2.9 2.8 0.07 0.08 4.1 4 3.3 0.07 0.08 2.7 6 3.1 0.10 0.14 4.2 3.8 0.12 8 0.09 Values are substantially identical for both groups. Assays were performed according to stan-

dard procedure.20-22

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to the procedures of Smith and Wheat.22 The major components were identified by their elution properties and, afterpooling peak tubes, bypaper chromatography. There was no significant difference between the two groups of cells nor was any significant radioactivity incorporated into the nucleoside diphosphohexose fractions. The major tritium-containing fraction from the BUdR-grown cells had elution properties consistent with those expected for the monophosphate derivative, but there was insufficient material present to permit definitive characterization. The expected uridine-diphospho sugars were found in approximately normal amounts, and the major nucleotide present was adenosine triphosphate. It would appear that the effects exerted by BUdR are apparently at the level of transcription or translation, since BUdR itself does not inhibit the enzymes assayed and their activity clearly declined quite strikingly. There are no data on the relative lifetime of these enzymes in the replicating cells and, of course, no information on the stability on the messenger RNA's associated with the synthesis of the individual proteins. Experiments have been performed with chondrocytes by using actinomycin D, but this agent is cytotoxic in a concentra;tion-dependent fashion and the results are difficult to interpret. It may be suggested that the substitution of thymine by BUdR results in little or no effect on phenotypic expression in some systems, since infectious OX 174 phage can be synthesized when 5-bromouracil is substituted for thymine.'9 However, it is clear that the incorporation of BUdR into the DNA of embryonic cells may lead to relatively profound changes, at least with respect to certain classes of molecules. One interesting parameter, for which no information is as yet available, is the effect of BUdR on the production of the protein core of the extracellular matrix. Since we do not have specific techniques for the isolation and identification of the protein component alone, this question cannot be answered at this time. An assessment of the role of the enzymes assayed in the over-all functioning of the cell can, however, be made. The product of the UDP-glucose dehydrogenase, UDP-D-glucuronic acid, apparently is required only for the synthesis of the extracellular matrix polysaccharides, since glucuronic acid is not a normal constituent of cell membrane material or glycoproteins that may be associated with the cell surface. Similarly, the ester sulfate moiety is believed to be restricted to the characteristic polysaccharides produced by connective tissues and is not known to be associated with normal cellular organelles. However, sulfated polysaccharides have been reported to be produced by cell types in vitro other than connective tissue cells.23 The maintenance of most cellular functions associated with energy production, protein and nucleic acid synthesis, and cell division suggests that the synthesis of "essential" molecules is either less tightly regulated or less subject to perturbations than the synthesis of those "luxury" molecules' which serve as phenotypic markers of specialized cells. If the BUdR effect is due to its incorporation into discrete genes, it might suggest that there is a redundancy of genes associated with essential cellular functions in contrast to single loci controlling the production of luxury molecules. Alternatively, specific transcription effects may be involved.

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PROC. N. A. S.

In summary, when cartilage cells are exposed to BUdR, they rapidly lose their ability to synthesize the chondrotin 4-sulfate-protein complex characteristic of the extracellular matrix of differentiated cartilage. This loss of gross functional activity as exemplified by the marked reduction in the amount of proteinpolysaccharide synthesized correlates very well with the progressive decline of three of the enzymes which must be involved in the biosynthesis of this macromolecule. A small amount of residual polysaccharide or enzymatic activity may be due to the presence, in the culture, of cells which have not undergone a sufficient number of divisions, although it is impossible to document this completely. Spot checks of other cellular functions, particularly thosec oncerned with cell division and mitochondrial activity, appear to be normal. However, there are marked morphological changes which may be associated with plasma membrane components or the synthesis of the protein-polysaccharide complex. * Studies were supported by the National Institutes of Health (AM-12074 and HD-00189) and the National Science Foundation. S. C. is a USPHS trainee (GM-1400); H. H. is a Career Developmental Awardee (5-K3-HD-2970) from the U.S. Public Health Service. t Present address: Department of Biological Chemistry, The Milton S. Hershey Medical Center of the Pennsylvania State University, Hershey, Pa. 17033. t Present address: Department of Pathology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pa. 19104. § Present address: Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19104. 1 Holtzer, H., and J. Abbott, in Stability of the Differentiated State, ed. H. Ursprung (Berlin, New York: Springer-Verlag, 1968). 20kazaki, K., and H. Holtzer, J. Histochem. Cytochem., 131, 726 (1965). 3Chacko, S., S. Holtzer, and H. Holtzer, Biochem. Biophys. Res. Commun., 34, 183 (1969). 4Bischoff, R., and H. Holtzer, J. Cell Biol., 40, 943 (1969). 5 Bischoff, R., and H. Holtzer, J. Comp. Phyciol., in press. 6 Coleman, J., and A. Coleman, J. Cell Biol., 31, 22 (1966). 7Ishikawa, H., R. Bischoff, and H. Holtzer, J. Cell Biol., 38, 538 (1968). 8 Abbott, J., and H. Holtzer, these PROCEEDINGS, 59, 1144 (1968). 9 Bischoff, R., and H. Holtzer, Anat. Rec., 160, 317 (1968). 10 Abbott, J., and H. Holtzer, J. Cell Biol., 28, 473 (1966). l1 Nameroff, M., and H. Holtzer, Develop. Biol., 16, 250 (1967). 12 Shulman, H., and K. Meyer, J. Exptl. Med., 128, 1353 (1968). 13 Marler, E., and E. A. Davidson, these PROCEEDINGS, 54, 648 (1965). 14 Woodward, C., and E. A. Davidson, these PROCEEDINGS, 60, 20 (1968). 15 Ham, R., these PROCEEDINGS, 53, 288 (1965). 16 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 17Robbins, P. W., in Methods in Enzymology, ed. S. P. Colowick and N. 0. Kaplan (New York: Academic Press, 1962), vol. 5, p. 964. 18 Meezan, E., and E. A. Davidson, J. Biol. Chem., 242, 4956 (1967). 19 Goulian, M., A. Kornberg, and R. L. Sinsheimer, these PROCEEDINGS, 58, 2321 (1967). 20 Bessey, 0. A., 0. H. Lowry, and M. J. Brock, J. Biol. Chem., 164, 321 (1946). 21 S. P. Colowick and N. 0. Kaplan, ed., Methods in Enzymology (New York: Academic Press, 1957), vol. 2, p. 735. 22 Smith, E. J., and R. W. Wheat, Arch. Biochem. Biophys., 86, 267 (1960). 23 Holtzer, H., in Epithelial-Mesenchymal Interactions, ed. R. Fleischmajer (Baltimore: Williams & Wilkins Co., 1968).