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Regulation of cartilage enzymes in cultured chondrocytes and the effect of 5-bromodeoxyuridine
DEVELOPMENTAL
BIOLOGY
Regulation
27, 20-26 (1972)
of Cartilage
Enzymes
in Cultured
Chondrocytes
and the Effect of 5-Bromodeoxyuridine’ GIOVANNI MARZULLO Department
of Medical
Genetics, School of Medicine, University Philadelphia, Pennsylvania 19104 Accepted
September
of Pennsylvania,
17, 1971
When sternal cartilage from 15-day chick embryos is dissociated and cultured in normal media, the activity of the two cartilage enzymes, UDPG-epimerase and UDPG-dehydrogenase, falls to a level of about 25% of the original value within 3 days, but remains stable at this level thereafter. No change, on the other hand, occurs in the activity of the non-tissue-specific enzyme, phosphoglucose isomerase. The specific downward regulation of the cartilage enzymes seems to be attributable to the mitotic activity induced by the culture conditions. The addition of low concentrations of 5-bromodeoxyuridine to the culture medium, while having no effect on the isomerase or on cell division, causes a further drop in the activity of the cartilage enzymes from the 25% level to a value about 5%, but not further than this. In the case of the epimerase the latter low level is ubiquitously found in all tissues at all stages, indicating that only the cartilage-specific excess of this enzyme is susceptible of suppression by low concentrations of bromodeoxyuridine. The reduction of isomerase activity caused by higher concentrations of the analog is concomitant with a reduction of cell division and seems to be attributable to cell death. A scheme is presented summarizing the results and interpretations of the present paper and of a previous one.
INTRODUCTION
ter cessation of mitotic activity (maturation phase). The present experiments examine this hypothesis by testing for a specific downward regulation of the cartilage enzymes upon reinduction of cell division under culture conditions. Furthermore, the specific effect of the thymidine analog 5-bromodeoxyuridine (BUDR) on the level of the cartilage enzyme is investigated. This analog has been shown to block the expression of the differentiated phenotype in cultures of pancreatic tissue (Wessells, 1964; Rutter et al., 1968) and of myoblasts (Stockdale et al., 1964) as well as chondrocytes (Abbott and Holtzer, 1968). Moreover, BUDR has been found to reduce the level of three cartilage enzymes in cultured chondrocytes (Holthausen et al., 1969). A similar effect has been described recently in hepatoma cell cultures, where in the presence of BUDR the liver enzyme tyrosineaminotransferase is reduced considerably while total protein is affected only slightly (Stellwagen and Tomkins 1971). The re-
Previous experiments have shown that two enzymes involved in the synthesis of cartilaginous matrix, UDPG-4-epimerase and UDPG-dehydrogenase, are present in X-day chick embryo cartilage at levels 20-30 times higher than in nonchondrogenie tissues, while the glycolytic enzyme phosphoglucose isomerase is uniformly distributed in all tissues (Marzullo and Desiderio, 1972). The present work is a study of the fate of the two “cartilage enzymes,” viz. UDPG-epimerase and dehydrogenase, relative to the “ubiquitous” isomerase in cell cultures of sternal cartilage. Some evidence was presented previously suggesting that full development of the cartilage enzymes may occur in two stages, a first relatively small increase occurring in dividing chondroblasts (differentiation proper) being followed by a second larger increase occurring af‘Supported by Grant Institutes of Health.
AM-12076
from the National 20
Copyright
0 1972 by Academc
Press. Inc.
Regulatic m of Cartilage
MARZLLLO
pression of cartilage enzymes by BUDR is studied here anew in view of the extensive information presently available concerning the normal regulation of the UDPG-enzymes and in order to test the specificity of this effect by quantitating the extent to which non-tissue-specific enzymes are also affected. MATERIALS
AND
Sternal tissue from E-day White Leghorn chick embryos was dissociated and cultured in Ham’s F-12 medium with bovine serum albumin and fetal calf serum as described previously (Marzullo and Lash, 1970). An appropriate number of
---
1
2
3
4
5
DAYS
21
cells (from 500 to 105) was plated in loo-mm dishes to yield a uniformly high cell harvest on the day of collection. The cultures were fed by replacing one-third of the medium on alternate days. 5-Bromodeoxyuridine (grade A, from CalBiothem) was added in most cases on the day following plating in order to reduce possible effects on plating efficiency. The cells were collected by scraping after rinsing repeatedly with Hanks’ basal salt solution. The collected cells were resuspended in an appropriate volume of Tris buffer (0.1 M, pH 8.0) and broken by sonication. After centrifugation at 1500 g, the extracts were assayed for protein and for en-
METHODS
0
Enzymes
EPIMERASE ISOMERASE
6
7
9
9
1011
(umtslmg) (units/o I mg)
12 13 14
IN CULTURE
FIG. 1. UDPG-4-epimerase and phosphoglucoisomerase in chondrocytes cultured with and without 5-bromodeoxyuridine. 0, 0, no BUDR; +, 1.2 x 10m6 M BUDR; A, A, 4 x 10e6 M BUDR; W, 0, 1.2 x 10m5 M BUDR; 6, 4 x lo-@ M BUDR added on day 7. Average values from three or more experiments are shown. In order to indicate the variability, various sets of experiments are shown for epimerase in cultures without BUDR.
DEVELOPMENTAL BIOLOGY
22
VOLUME27, 1972
I-
O
“““““““’
0
1
2
3
4
5
6
7
DAYS
IN
CULTURE
FIG. 2. UDPG-dehydrogenase in chondrocytes cultured BUDR;+, 1.2 x W6MBUDR; A, 4 x 10-6MBUDR; ‘:4x
zymes as described previously (Marzullo and Desiderio, 1972). To test of the effect of BUDR on cell division the number of cells per clone in sparsely plated cultures was counted under phase microscopy using an ocular grid. RESULTS
Single-cell suspensions of freshly liberated chondrocytes obtained from 14-day embryos were divided into two parts. One part was homogenized and assayed for the three enzymes and one part was cultured. An appropriate number of cells (from 5 x lo5 to 500) was added to each culture dish to yield a uniform cell number per plate on the day of harvesting. The cell density was kept sparse and well below
9
with
9
10 11 12 13 14
and
without
5-bromodeoxyuridine
0,
no
10-6MBUDRaddedonday7.
confluency in the short-term cultures. Figure 1 shows the results of UDPG-4-epimerase and phosphoglucose isomerase determinations on freshly liberated cells and on cells cultured in the presence and the absence of various concentrations of BUDR. There is no change in the isomerase level during the entire culture period either in the absence or presence of moderate concentrations of BUDR. The epimerase however falls rapidly in the cultured cells. In the absence of BUDR, this fall stops after about 2 days and the enzyme levels off at a value of about 1.3 units per milligram, or about 25% of the “mature-cartilage level” (MCL, set at 5.0 units/mg). In cultures containing BUDR
Regulation
MARZULLO
the epimerase fall proceeds past this level gradually leveling off at a value of about 0.2-0.3 unit/mg, or 5% MCL. The time required for reduction to this level appears to be directly related to the concentration of BUDR. At 1.2 x lo-” M BUDR the 5% level is attained in about 4 days, at 4 x 10m6 M in about 10 days, while at 1.2 x 10m6 only a reduction to about 7% is attained by day 14. The epimerase suppression does not proceed appreciably further than the 5% level, even at BUDR concentrations (1.2 x 10m5 M) that begin to affect the isomerase as well. In one experiment 4 x lo- 6 M BUDR was added after 7 days of culture in normal medium. The time-course of epimerase reduction in this case was similar to that observed when the analog was added on the first day. q -
of Cartilage
Enzymes
23
Figure 2 shows the results of the UDPGdehydrogenase assays. Since most of the determinations were performed on the same extracts used for Fig. 1, the isomerase curve is not shown here again. The dehydrogenase also falls in the normal cultures from the 100% MCL (0.41 unit/ mg) to a 27% level (0.11 unit/mg) within 3 days, but stays unchanged thereafter. In the presence of 4 x 10m6 M BUDR the fall proceeds to about 7% (0.03 unit/mg). At 1.2 x 10m6 M BUDR the same degree of suppression is attained in a longer period. Since this level is close to the lower limit of the assay for this reaction, nothing can be said about a lower bound of suppressibility of this enzyme by BUDR, as is the case for the epimerase. As discussed below, the observed drop
CONlIIOL
o-
1.1
x
10-s
.-
4.0
x
10-6
M
BUDI
nm
. . .
1.1 3.6
x x
10-5 10-S
IS IB
10 -
IO88-
1
2
FIG. 3. Effect of 5-bromodeoxyuridine cells per clone from 15 or more clones.
3 DAYS
4 5 IN CULTURE
on cell division.
6
(The values shown represent
average counts of
24
DEVELOPMENTAL
BIOLOGY
in epimerase and dehydrogenase in the normal cultures may be accountable in terms of the division activity stimulated by the culture conditions. Accordingly, it seemed possible that BUDR acted by accelerating the rate of cell division. The effect of various concentrations of the analog on cell division was tested by counting the cells in each clone under the phase microscope at daily intervals. The results are shown in Fig. 3. There is no effect on cell division with concentrations up to 4 x 10e6 M. At concentrations of 1.2 x 10m5 M and higher, the number of cells per clone is reduced. In these clones many cells appear necrotic and in a process of disintegration. Thus the lowered cell count may result from the breakdown of some cells, rather than from a decrease in the division rate per se. Similarly, the reduction in the isomerase level observed with 1.2 x lo- 5 M BUDR may be due to the assay of dead cells, rather than to a direct effect of the analog on this enzyme. DISCUSSION
Normal regulation. The present results show the specific downward regulation of the two cartilage enzymes, UDPG-epimerase and UDPG-dehydrogenase from the 100% mature-cartilage level of fresh chondrocytes to about a 25% stable level in cultured cells. For both enzymes the latter level is clearly distinguishable from that found in early tissues before differentiation or in noncartilage tissues. Insofar as the cultured chondrocytes are differentiated, matrix-synthesizing cells (as shown previously, Coon and Marzullo, 1967; Marzullo and Lash, 1970), their cartilageenzyme level may be termed Differentiated I, and the level before culturing may be termed Differentiated II. It seems likely that during normal chondrogenesis differentiating cells progress from the undifferentiated state through the Differentiated I condition to the Differentiated II state. In fact the fall from Differentiated II to Differentiated I observed in vitro is likely to represent the reversal of the
VOLUME
27. 1972
“maturation process” described in the preceding paper. According to this view, the chondrocytes of g-day sternal plates on the average would appear to be in the Differentiated I state, since they contain about the same epimerase and dehydrogenase level as the cultured chondrocytes. Unfortunately, it is not presently possible to test for the presence of a Differentiated I plateau in the epimerase development curve because pure chondrogenic tissue earlier than g-day cannot be obtained in suficient amounts. Microscale reduction of the enzyme assay systems may render this feasible in the future. Since there is little cell division in the intact E-day sternum, while the cultured chondrocytes are rapidly dividing, it seems reasonable to attribute the Differentiated II enzyme level to the postmitotic state of the cells and the Differentiated I level to the mitotic condition. The above results and suggestions are summarized schematically in the model shown below. This scheme is analogous in many respects to that put forth by Rutter et al. (1968) based on the development of exocrine enzymes in the rat pancreas. The stages here termed Differentiated I and Differentiated II seem to correspond, respectively, to the “protodifferentiated” and the “differentiated” states of Rutter et al. In both systems the transition from the lower to the higher stage is concomitant with the cessation of cell division. In the present case, however, it seems less appropriate to refer to lower stage as anything less than differentiated since these cells are surrounded by abundant cartilaginous matrix both in viva and in vitro. Thus the term Differentiated I is used. The pancreatic system, being heterogeneous with respect to cell type and degree of differentiation, is open to the criticism that inflections in the enzyme development curve may reflect the differentiation of different groups of cells at various times, rather than staged events in the same group. The cartilage system, on the other
MARZULLO
25
Regulation of Cartilage Enzymes DIFmTIAm
11 (PC~~T-MITOTICI
100
:mDucEC CELL DIVISION
?
CARTILAGESPECIFI FXCESS BUDR-S!XSITIVE:
/ (CETSATIONOF INTERSTITIAL CELL DIVISlON'
I
I I I x UBIQUITOUSL!JJEX ,,,
(BUDR-RESISTANTi
In vitro
In vivoFIG. 4. Scheme summarizing
results and interpretations
hand, obviates these difficulties because it is homogeneous in cell type and considerably synchronous with respect to degree of differentiation of the cells. Thus the present results reinforce the claim already made for a two main-step process to full differentiation. Effect of bromodeoxyuridine. The results shown here confirm the report by Holthausen et al. (1969) that certain cartilage enzymes are suppressed by concentrations of BUDR that do not inhibit cell division. The present evidence further shows that this effect is specific insofar as a ubiquitously distributed enzyme is not similarly affected. The isomerase reduction observed with high concentrations of the analog may be accounted for in terms of the presence of dead cells. Thus it is possible that only tissue-specific enzymes are affected directly.
-B
on epimerase development
and control
It is of interest that only the epimerase level in excess of the ubiquitous 0.3 unit/mg is sensitive to BUDR. This may indicate that two epimerases are involved, one cartilage-specific and BUDR-sensitive and the other ubiquitous and BUDRresistant. Alternatively, there may be two superimposed modes of regulation of the same gene, BUDR being able to interfere with the higher mode, but not the lower. Work is in progress to test these hypotheses. REFERENCES ABBOTT, J., and HOLTZER, H. (1968). Effect of 5 bromodeoxyuridine on chondrocytes. Proc. Nat. Acad. Sci. U.S. 59, 1144-1151. COON, H., and MARZULLO, G. (1968). Effect of a factor from embryo extract on cloned chondrocytes,
Znt. Congr. Biochem. 7th Tokyo, Japan 1967. H. S., CHACKO, S., DAVIDSON, E. A., and HOLTZER, H. (1969). Effect of 5-bromodeoxy-
HOLTHAUSEN,
26
DEVELOPMENTALBIOLOGY
uridine on expression of cultured chondrocytes grown in vitro. Proc. Nat. Amid. Sci. U.S. 63, 864870. MARZULLO, G., and DESIDERIO, E. (1972). Tissue distribution and development of some cartilage enzymes. Deuelop. Biol. 27, 13-19. MARZULLO, G., and LASH, J. W. (1970). Control of phenotypic expression in cultured chondrocytes: Investigation on the mechanism. Deuelop. Biol. 22,638-654. PONTIS, H. G., and LELOIR, L. F. (1962). Measurement of UDP-enzyme systems. Methods Biochem. Ad. 10, 107-136. RKJITER, W. J., KEMP, J. D., BF~ADSHAW,W. S., CLARK, W. R., RONZIO, R. A., and SANDERS, T. G.
VOLUME 27, 1972
(1968). Regulation of specific protein synthesis in cytodifferentiation. J. Cell Physiol. 72, Suppl. 1, l-18. STELLWAGEN,R. H., and TOMKINS, G. M. (1971). Preferential inhibition by 5-bromodeoxyuridine of the synthesis of tyrosine aminotransferase in hepatoma cell cultures. J. Mol. Biol. 56, 167-182. STOCKDALE, F., ABBOTT, J., HOLTZER, S., and HOLTZER, T. (1964). The loss of phenotypic traits by differentiated cells. II. Behavior of chondrocytes and their progeny in uitro. Deuelop. Biol.7,293-302. WESSELLS, N. K. (1964). DNA synthesis, mitosis and differentiation of pancreatic acinar cells in vitro. J. Cell Biol. 20, 415-433.
BIOLOGY
Regulation
27, 20-26 (1972)
of Cartilage
Enzymes
in Cultured
Chondrocytes
and the Effect of 5-Bromodeoxyuridine’ GIOVANNI MARZULLO Department
of Medical
Genetics, School of Medicine, University Philadelphia, Pennsylvania 19104 Accepted
September
of Pennsylvania,
17, 1971
When sternal cartilage from 15-day chick embryos is dissociated and cultured in normal media, the activity of the two cartilage enzymes, UDPG-epimerase and UDPG-dehydrogenase, falls to a level of about 25% of the original value within 3 days, but remains stable at this level thereafter. No change, on the other hand, occurs in the activity of the non-tissue-specific enzyme, phosphoglucose isomerase. The specific downward regulation of the cartilage enzymes seems to be attributable to the mitotic activity induced by the culture conditions. The addition of low concentrations of 5-bromodeoxyuridine to the culture medium, while having no effect on the isomerase or on cell division, causes a further drop in the activity of the cartilage enzymes from the 25% level to a value about 5%, but not further than this. In the case of the epimerase the latter low level is ubiquitously found in all tissues at all stages, indicating that only the cartilage-specific excess of this enzyme is susceptible of suppression by low concentrations of bromodeoxyuridine. The reduction of isomerase activity caused by higher concentrations of the analog is concomitant with a reduction of cell division and seems to be attributable to cell death. A scheme is presented summarizing the results and interpretations of the present paper and of a previous one.
INTRODUCTION
ter cessation of mitotic activity (maturation phase). The present experiments examine this hypothesis by testing for a specific downward regulation of the cartilage enzymes upon reinduction of cell division under culture conditions. Furthermore, the specific effect of the thymidine analog 5-bromodeoxyuridine (BUDR) on the level of the cartilage enzyme is investigated. This analog has been shown to block the expression of the differentiated phenotype in cultures of pancreatic tissue (Wessells, 1964; Rutter et al., 1968) and of myoblasts (Stockdale et al., 1964) as well as chondrocytes (Abbott and Holtzer, 1968). Moreover, BUDR has been found to reduce the level of three cartilage enzymes in cultured chondrocytes (Holthausen et al., 1969). A similar effect has been described recently in hepatoma cell cultures, where in the presence of BUDR the liver enzyme tyrosineaminotransferase is reduced considerably while total protein is affected only slightly (Stellwagen and Tomkins 1971). The re-
Previous experiments have shown that two enzymes involved in the synthesis of cartilaginous matrix, UDPG-4-epimerase and UDPG-dehydrogenase, are present in X-day chick embryo cartilage at levels 20-30 times higher than in nonchondrogenie tissues, while the glycolytic enzyme phosphoglucose isomerase is uniformly distributed in all tissues (Marzullo and Desiderio, 1972). The present work is a study of the fate of the two “cartilage enzymes,” viz. UDPG-epimerase and dehydrogenase, relative to the “ubiquitous” isomerase in cell cultures of sternal cartilage. Some evidence was presented previously suggesting that full development of the cartilage enzymes may occur in two stages, a first relatively small increase occurring in dividing chondroblasts (differentiation proper) being followed by a second larger increase occurring af‘Supported by Grant Institutes of Health.
AM-12076
from the National 20
Copyright
0 1972 by Academc
Press. Inc.
Regulatic m of Cartilage
MARZLLLO
pression of cartilage enzymes by BUDR is studied here anew in view of the extensive information presently available concerning the normal regulation of the UDPG-enzymes and in order to test the specificity of this effect by quantitating the extent to which non-tissue-specific enzymes are also affected. MATERIALS
AND
Sternal tissue from E-day White Leghorn chick embryos was dissociated and cultured in Ham’s F-12 medium with bovine serum albumin and fetal calf serum as described previously (Marzullo and Lash, 1970). An appropriate number of
---
1
2
3
4
5
DAYS
21
cells (from 500 to 105) was plated in loo-mm dishes to yield a uniformly high cell harvest on the day of collection. The cultures were fed by replacing one-third of the medium on alternate days. 5-Bromodeoxyuridine (grade A, from CalBiothem) was added in most cases on the day following plating in order to reduce possible effects on plating efficiency. The cells were collected by scraping after rinsing repeatedly with Hanks’ basal salt solution. The collected cells were resuspended in an appropriate volume of Tris buffer (0.1 M, pH 8.0) and broken by sonication. After centrifugation at 1500 g, the extracts were assayed for protein and for en-
METHODS
0
Enzymes
EPIMERASE ISOMERASE
6
7
9
9
1011
(umtslmg) (units/o I mg)
12 13 14
IN CULTURE
FIG. 1. UDPG-4-epimerase and phosphoglucoisomerase in chondrocytes cultured with and without 5-bromodeoxyuridine. 0, 0, no BUDR; +, 1.2 x 10m6 M BUDR; A, A, 4 x 10e6 M BUDR; W, 0, 1.2 x 10m5 M BUDR; 6, 4 x lo-@ M BUDR added on day 7. Average values from three or more experiments are shown. In order to indicate the variability, various sets of experiments are shown for epimerase in cultures without BUDR.
DEVELOPMENTAL BIOLOGY
22
VOLUME27, 1972
I-
O
“““““““’
0
1
2
3
4
5
6
7
DAYS
IN
CULTURE
FIG. 2. UDPG-dehydrogenase in chondrocytes cultured BUDR;+, 1.2 x W6MBUDR; A, 4 x 10-6MBUDR; ‘:4x
zymes as described previously (Marzullo and Desiderio, 1972). To test of the effect of BUDR on cell division the number of cells per clone in sparsely plated cultures was counted under phase microscopy using an ocular grid. RESULTS
Single-cell suspensions of freshly liberated chondrocytes obtained from 14-day embryos were divided into two parts. One part was homogenized and assayed for the three enzymes and one part was cultured. An appropriate number of cells (from 5 x lo5 to 500) was added to each culture dish to yield a uniform cell number per plate on the day of harvesting. The cell density was kept sparse and well below
9
with
9
10 11 12 13 14
and
without
5-bromodeoxyuridine
0,
no
10-6MBUDRaddedonday7.
confluency in the short-term cultures. Figure 1 shows the results of UDPG-4-epimerase and phosphoglucose isomerase determinations on freshly liberated cells and on cells cultured in the presence and the absence of various concentrations of BUDR. There is no change in the isomerase level during the entire culture period either in the absence or presence of moderate concentrations of BUDR. The epimerase however falls rapidly in the cultured cells. In the absence of BUDR, this fall stops after about 2 days and the enzyme levels off at a value of about 1.3 units per milligram, or about 25% of the “mature-cartilage level” (MCL, set at 5.0 units/mg). In cultures containing BUDR
Regulation
MARZULLO
the epimerase fall proceeds past this level gradually leveling off at a value of about 0.2-0.3 unit/mg, or 5% MCL. The time required for reduction to this level appears to be directly related to the concentration of BUDR. At 1.2 x lo-” M BUDR the 5% level is attained in about 4 days, at 4 x 10m6 M in about 10 days, while at 1.2 x 10m6 only a reduction to about 7% is attained by day 14. The epimerase suppression does not proceed appreciably further than the 5% level, even at BUDR concentrations (1.2 x 10m5 M) that begin to affect the isomerase as well. In one experiment 4 x lo- 6 M BUDR was added after 7 days of culture in normal medium. The time-course of epimerase reduction in this case was similar to that observed when the analog was added on the first day. q -
of Cartilage
Enzymes
23
Figure 2 shows the results of the UDPGdehydrogenase assays. Since most of the determinations were performed on the same extracts used for Fig. 1, the isomerase curve is not shown here again. The dehydrogenase also falls in the normal cultures from the 100% MCL (0.41 unit/ mg) to a 27% level (0.11 unit/mg) within 3 days, but stays unchanged thereafter. In the presence of 4 x 10m6 M BUDR the fall proceeds to about 7% (0.03 unit/mg). At 1.2 x 10m6 M BUDR the same degree of suppression is attained in a longer period. Since this level is close to the lower limit of the assay for this reaction, nothing can be said about a lower bound of suppressibility of this enzyme by BUDR, as is the case for the epimerase. As discussed below, the observed drop
CONlIIOL
o-
1.1
x
10-s
.-
4.0
x
10-6
M
BUDI
nm
. . .
1.1 3.6
x x
10-5 10-S
IS IB
10 -
IO88-
1
2
FIG. 3. Effect of 5-bromodeoxyuridine cells per clone from 15 or more clones.
3 DAYS
4 5 IN CULTURE
on cell division.
6
(The values shown represent
average counts of
24
DEVELOPMENTAL
BIOLOGY
in epimerase and dehydrogenase in the normal cultures may be accountable in terms of the division activity stimulated by the culture conditions. Accordingly, it seemed possible that BUDR acted by accelerating the rate of cell division. The effect of various concentrations of the analog on cell division was tested by counting the cells in each clone under the phase microscope at daily intervals. The results are shown in Fig. 3. There is no effect on cell division with concentrations up to 4 x 10e6 M. At concentrations of 1.2 x 10m5 M and higher, the number of cells per clone is reduced. In these clones many cells appear necrotic and in a process of disintegration. Thus the lowered cell count may result from the breakdown of some cells, rather than from a decrease in the division rate per se. Similarly, the reduction in the isomerase level observed with 1.2 x lo- 5 M BUDR may be due to the assay of dead cells, rather than to a direct effect of the analog on this enzyme. DISCUSSION
Normal regulation. The present results show the specific downward regulation of the two cartilage enzymes, UDPG-epimerase and UDPG-dehydrogenase from the 100% mature-cartilage level of fresh chondrocytes to about a 25% stable level in cultured cells. For both enzymes the latter level is clearly distinguishable from that found in early tissues before differentiation or in noncartilage tissues. Insofar as the cultured chondrocytes are differentiated, matrix-synthesizing cells (as shown previously, Coon and Marzullo, 1967; Marzullo and Lash, 1970), their cartilageenzyme level may be termed Differentiated I, and the level before culturing may be termed Differentiated II. It seems likely that during normal chondrogenesis differentiating cells progress from the undifferentiated state through the Differentiated I condition to the Differentiated II state. In fact the fall from Differentiated II to Differentiated I observed in vitro is likely to represent the reversal of the
VOLUME
27. 1972
“maturation process” described in the preceding paper. According to this view, the chondrocytes of g-day sternal plates on the average would appear to be in the Differentiated I state, since they contain about the same epimerase and dehydrogenase level as the cultured chondrocytes. Unfortunately, it is not presently possible to test for the presence of a Differentiated I plateau in the epimerase development curve because pure chondrogenic tissue earlier than g-day cannot be obtained in suficient amounts. Microscale reduction of the enzyme assay systems may render this feasible in the future. Since there is little cell division in the intact E-day sternum, while the cultured chondrocytes are rapidly dividing, it seems reasonable to attribute the Differentiated II enzyme level to the postmitotic state of the cells and the Differentiated I level to the mitotic condition. The above results and suggestions are summarized schematically in the model shown below. This scheme is analogous in many respects to that put forth by Rutter et al. (1968) based on the development of exocrine enzymes in the rat pancreas. The stages here termed Differentiated I and Differentiated II seem to correspond, respectively, to the “protodifferentiated” and the “differentiated” states of Rutter et al. In both systems the transition from the lower to the higher stage is concomitant with the cessation of cell division. In the present case, however, it seems less appropriate to refer to lower stage as anything less than differentiated since these cells are surrounded by abundant cartilaginous matrix both in viva and in vitro. Thus the term Differentiated I is used. The pancreatic system, being heterogeneous with respect to cell type and degree of differentiation, is open to the criticism that inflections in the enzyme development curve may reflect the differentiation of different groups of cells at various times, rather than staged events in the same group. The cartilage system, on the other
MARZULLO
25
Regulation of Cartilage Enzymes DIFmTIAm
11 (PC~~T-MITOTICI
100
:mDucEC CELL DIVISION
?
CARTILAGESPECIFI FXCESS BUDR-S!XSITIVE:
/ (CETSATIONOF INTERSTITIAL CELL DIVISlON'
I
I I I x UBIQUITOUSL!JJEX ,,,
(BUDR-RESISTANTi
In vitro
In vivoFIG. 4. Scheme summarizing
results and interpretations
hand, obviates these difficulties because it is homogeneous in cell type and considerably synchronous with respect to degree of differentiation of the cells. Thus the present results reinforce the claim already made for a two main-step process to full differentiation. Effect of bromodeoxyuridine. The results shown here confirm the report by Holthausen et al. (1969) that certain cartilage enzymes are suppressed by concentrations of BUDR that do not inhibit cell division. The present evidence further shows that this effect is specific insofar as a ubiquitously distributed enzyme is not similarly affected. The isomerase reduction observed with high concentrations of the analog may be accounted for in terms of the presence of dead cells. Thus it is possible that only tissue-specific enzymes are affected directly.
-B
on epimerase development
and control
It is of interest that only the epimerase level in excess of the ubiquitous 0.3 unit/mg is sensitive to BUDR. This may indicate that two epimerases are involved, one cartilage-specific and BUDR-sensitive and the other ubiquitous and BUDRresistant. Alternatively, there may be two superimposed modes of regulation of the same gene, BUDR being able to interfere with the higher mode, but not the lower. Work is in progress to test these hypotheses. REFERENCES ABBOTT, J., and HOLTZER, H. (1968). Effect of 5 bromodeoxyuridine on chondrocytes. Proc. Nat. Acad. Sci. U.S. 59, 1144-1151. COON, H., and MARZULLO, G. (1968). Effect of a factor from embryo extract on cloned chondrocytes,
Znt. Congr. Biochem. 7th Tokyo, Japan 1967. H. S., CHACKO, S., DAVIDSON, E. A., and HOLTZER, H. (1969). Effect of 5-bromodeoxy-
HOLTHAUSEN,
26
DEVELOPMENTALBIOLOGY
uridine on expression of cultured chondrocytes grown in vitro. Proc. Nat. Amid. Sci. U.S. 63, 864870. MARZULLO, G., and DESIDERIO, E. (1972). Tissue distribution and development of some cartilage enzymes. Deuelop. Biol. 27, 13-19. MARZULLO, G., and LASH, J. W. (1970). Control of phenotypic expression in cultured chondrocytes: Investigation on the mechanism. Deuelop. Biol. 22,638-654. PONTIS, H. G., and LELOIR, L. F. (1962). Measurement of UDP-enzyme systems. Methods Biochem. Ad. 10, 107-136. RKJITER, W. J., KEMP, J. D., BF~ADSHAW,W. S., CLARK, W. R., RONZIO, R. A., and SANDERS, T. G.
VOLUME 27, 1972
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