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Aggregated chondrocytes as a model system to study cartilage metabolism
440
Preliminary
roles
17. Sanders, B G, Dietert, R R, Kline, K & Dietert, H, Oncodevelopmental biol and med 2 ( 1981)63. 18. Teplitz, R L., Sanders, B G, Brodetsky, A M, Fung, H & Wtley, K L, Cancer res 34 (1974) 1049. 19. Perisic, 0, Biquard, J M, Blanchet, J P, Greenland, T B & Krsmanovic, V, Differentiation 18 (1981) 175. Received November 5, 1981 Revised version received March 16, 1982 Accepted March 18, 1982
Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/82/08044tM4$02.00/0
Aggregated chondrocytes as a model system to study cartilage metabolism G. P. J. van KAMPEN and J. P. VELDHUIJZEN, Department of Oral Cell Biology, School of Dentistry, Vrije Vniversiteit, 1007 MC Amsterdam, The Netherlands Summary. Differentiated chondrocytes, isolated from chick embryo cartilage, were cultured in monolayer, as aggregate or pellet. Aggregation of chondrocytes was accomplished by incubating 2x 105cells in a 5~1 drop of culture medium. Under all three conditions, the cells remained healthy and proliferated during culture. However, matrix production, as indicated by incorporation of [3sS]sulphate into glycosaminoglycans, was greater in aggregated chondrocytes than in monoiayers or pel&s.In addition, aggregates consisting of a well defined number of cells, could easily be maniegates provide pulated for experiments. Therefore a favourable model system to studyT actors modulating the metabolism of chondrocytes.
The intercellular matrix of cartilage consists of several well defined components, such as type II collagen, proteoglycans and hyaluronic acid. Proteoglycans are noncovalently bound to hyaluronic acid in a supramolecular complex. Sulphated glycosaminoglycan chains of chondroitin sulphate and keratan sulphate are attached to the proteoglycan core protein [ 11. For in vitro studies of matrix production and cell proliferation, chondrocytes can be liberated from the matrix by treatment with collagenase and subsequently cultured. In vitro, chondrocytes continue to synthesize matrix products. Many factors, Exp Cell Rcs 140 (1982)
however, influence chondrogenic expression [2]. Several authors reported that interaction between chondrocytes is important in inducing and maintaining matrix production [3-61. Therefore we sought a method with which chondrocytes could be cultured in high density. We now present a simple technique to aggregate a defined number of chondrocytes. Extracellular matrix production and cell division of the aggregated chondrocytes was compared with chondrocytes cultured in monolayers and as pellets. Materials
and Methods
Isolation and culture of the cells. Chondrocytes were isolated from the epiphyses of tibiae of 15-day-old chick embryos (Shaver). The tibiae were aseptically freed of soft tissues and perichondrium. The heads of the tibiae were removed, resulting in exposure of the epiphyseal cartilage. At the proximal and distal ends of the tibiae, slices of the proliferation zone of the epiphyseal cartilage were cut off. These slices were dinested (4x30 min) under gentle stirring with collagenase (CLS II, Worthington Freehold; N.J.), 400 U/ml Hanks Balanced Salt Solution (HBSS. Gibco. Paisley, Scotland). To minimize contamination with libroblasts, the first digest was discarded. Isolated chondrocytes were washed and suspended in culture medium Ml99 (Gibco) supplemented with 250 I.cg L-glutamine and 50 pg ascorbic acid/ml (M199). Aliquots of the cell suspension were used for viability testing (Trypan blue exclusion) and cell counting. The cells were pre-incubated in a humitied (98%) incubator (5 % COPin air) for 20 h at 37°C in monolayers, asaggregates or pellets. For monolayers, 2x 105 cells, suspended in 2 ml medium, were allowed to attach to the wall of a tissue culture tube (Sterilin Ltd, Richmond, Surrey, England). For aggregates, 2x 105cells, suspended in 5 ~1 of medium, were allowed to accumulate in the centre of a drop on the bottom of a plastic Petri dish. For pellets, 2x105 cells, in 2 ml medium, were centrifuged (150 g, 10 min) in a cone-shaped polystyrene tube (Sterilin Ltd.). After the pre-incubation period and medium change, monolayers, individual aggregates and pellets were cultured in Ml99 with 10% fetal calf serum (FCS) for 22 h. After the addition of FCS, aggregates were fixed in Bouin Hollande for histological purposes, with 4-h intervals. [35S]Sulphate incorporation into glycosaminoglycans. After 22 h of culture the synthesis of sulphated glycosaminoglycans (GAG) was measured by determining the incorporation of [3sS]sulphate into cetylpyridiniumchloride-precipitable glycosaminoglycans. The cells were labelled for 90 min in 2 ml medium with 5 &i NaZ3”S0,/ml (sp. act. 50 mCi/mmol; The Radiochemical Centre, Amersham). Subsequently, the incubation medium was decanted and the culPrinted
m Sweden
Preliminary notes tures were digested in 2 ml fresh medium with papain (Merck) at WC for 16 h [cf 73. To the digests 0.5 ml 10% cetyipy~dinium chloride (CRC, Merck) in 0.2 M NaCl was added. After 24 h at 2PC the precipitate (of CRC and GAG) was washed twice with a solution of 0. I % CRC, a treatment which reduced the activity in tissue blanks to background level. The precipitate was dissolved in 0.5 ml formic acid (26 N) and the amount of ?S was determined with a liquid scintillation counter. [3HjTkymidine incorporation into DNA. DNA synthesis- was measured by determining the incorporation of t3H]thymidine into DNA, according to the method of Ash & Francis [g]. The cells were labelled for 90 min in 2 ml medium containing 0.5 @Zi [3H]thymidinelml (sp. act. 5 Ci/mmol; Radiochemical Centre).
Results
The isolation procedure resulted in suspensions of single cells with a viability of 95% or more. Aggregation of freshly isolated chondrocytes was initiated by placing a 5-~1drop of medium containing 2x 1oJcells on the bottom of a Petri dish (fig. 1A). After 20 h the cells were accumulated in the centre of the drop forming an aggregate (0 2 mm) of cohering cells (fig. 1B). The addition of medium to the Petri dish made the aggregatesfloat. Histological sections showed that they consisted of 3-5 cell layers of chondrocytes separated by a thin layer of intercellular matrix which increased in width with time after the addition of FCS. Polarization microscopy revealed birefringent material in the matrix. The amount of this material, probably collagen, also increased with time. When the chondrocytes were devitalized (56”C, 30 min) or when during the pre-incubation period Hanks Balanced Salt Solution was used instead of M199, the chondrocytes did accumulate in the centre of the drop, but no aggregateswere formed. GAG synthesis and DNA synthesis of chondrocytes cultured in monolayer, as aggregate or pellet, are shown in fig. 2. FCS stimulated matrix production and chondrocytic proliferation in all three conditions.
441
Fig. I. Aggregation of 2xloJ embryonic chondrocy:ss, suspended in 5 ~1 medium. (A) Initial state; (B) aggregate formed after 20 h pre-incubation.
The aggregated chondrocytes deposited in the matrix three times as much GAG as monolayers and six times as much as pellets (fig. 2A). ~3H]Thymidine inco~oration into DNA of aggregated and pelleted cells was 60% of the value found in monolayers (fig. 2B).
Aggregation of embryonic cells in vitro is a well-known phenomenon. Histogenetic linking and association of metazoan cells has recently been reviewed by Garrod & Nicol [9]. Aggregation of embryonic chick cells was used by Ahrens and co-workers, who cultured mesenchymal cells from embryonic chick limb bud of various stages in micromass cultures [lo]. In such cultures a stage-related chondrogenic expression of mesenchymal cells could be shown [ 111. In the present study differentiated chondrocytes were used. They were isolated from 15-day chick embryos and aggregated in a small volume of culture medium on a plastic Petri dish. This procedu,e resulted in floating, high density cultures of a defined number of differentiated chondrocytes. In an ultrastructural and biochemical study, Oakes et al. [5] demonstrated the
442
Preliminary notes matrix
DNA
production
synthesas
m
a
P
Fig. 2. Matrix production (A) and DNA synthesis (B) of monolayers (m), aggregates (a) and pellets (p), 22 h after medium change without 0 or with q FCS. Values are means of six determinations. Bars. SD.
possibility of maintaining chondrocytes in a differentiated state by using high density cultures. In the present study routine histology of the aggregates revealed the presence of rapidly increasing, Alcian-blue stainable matrix, indicating well differentiated chondrocytes. It is shown that accumulation of the cells in the centre of the drop is a passive phenomenon. The accumulation of the cells .!T.rp Cell Res IdO (1982)
after devitalization of the chondrocytes support this. The actual aggregation probably requires surface proteins or glycoproteins [cf 93. When HBSS instead of Ml99 was used during pre-incubation, the cells did not cohere. This supports the notion that medium factors are needed to synthesize new intercellular ligands. The enzymatic steps involved in proteoglycan synthesis are well defined [ 11. Since sulphation is a late stage in proteoglycan biosynthesis, sulphate incorporation is a convenient means to study the synthesis of sulphated glycosaminoglycan. The incorporation of C”5S]sulphate into GAG clearly demonstrates that maximum matrix deposition occurs when chondrocytes are cultured as aggregates. On the other hand, cell proliferation was lower compared with monolayers. An inverse relationship between matrix production and cell division has been reported by Miller et al. [12]. It is noteworthy that aggregated chondrocytes deposit much GAG in the matrix, even if they are maintained in culture medium without FCS. Solursh [ 131 suggested that conditioning of the culture medium might contribute to rapid acceleration of the expression of differentiation in culture, and that accumulated matrix might provide a micro-environment that itself promotes better expression of the cartilage phenotype. Perhaps the small (5 ~1) volume during pre-incubation enables the chondrocytes to provide such a micro-environment. In this paper a simple way of aggregating chondrocytes is described. The aggregates are equal in cell number and easy to manipulate in experiments. Aggregated chondrocytes deposit considerable amounts of GAG in the intercellular matrix, so changes can easily be observed. It is concluded that aggregates provide a favourable model system to study factors
Preliminary
modulating cytes.
the metabolism of chondro-
References 1. Stockwell, R A, Biology of cartilage cells (ed R J Harrison & R M H McMinn). Cambridge University Press Cambridge (1979). 2. Sokoloff, L, Malemud, C J, Srivastava, V M L & Morgan, M D, Fed proc 32 (1973) 1499. 3. Abbott, J & Holtzer, H, J cell biol 28 (1966) 473. 4. Lavietes, B B, Dev biol 21 (1970) 584. 5. Oakes, B W, Handley, C J, Lisner, F & Lowther, D A, J embryo1 exp morph01 38 (1977) 239. 6. Yasumoto, S, Kato, Y, Oguri, K, Yamagata, S & Yamagata, T, Dev growth differ 22 (1980) 445. 7. Scott, J E, Methods biochem anal 8 (1960) 145. 8. Ash, P & Francis, M J 0, J endocrinol 66 (1975) 9. &rod D R 62 Nicol A Biol rev 56 (1981) 199 10. Ahrens: P B, Solursh, M Jr Reiter, R S, Dev biol 60 (1977) 69. 11. Solursh, M. Ahrens, P B & Reiter, R S, In vitro 14 (1978) 51. 12. Miller, R P, Husain, M & Lohim, S, J cell physiol 100 (1979) 63. 13. Solursh, M & Meier, S, J exp zoo1 187 (1974) 311. Received December 8, 1981 Revised version received April 7, 1982 Accepted April 15, 1982
Copyright IQ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
0014-4827/82/080143~S~2.~/0
Reevaluation of DNA chain elongation rate in human diploid fibroblasts N. HASEGAWA,’ F. HANAOKA,’ T. HORP and M. YAMADA,’ ‘Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113, and 2Division of Genetics, National Institute of Radiological Sciences, 4-9-l Anagawa, Chiba 260, Japan Summary. The rate of DNA chain elongation in human diploid fibroblasts (IMR90) of different ages was examined by DNA fiber autoradiography and alkaline sucrose density gradient centrifugation. There was no difference in chain elongation rate in various population doubling level cells.
It is well established that human diploid fibroblasts in culture have a limited lifespan, which is inversely correlated with the age of the donor [ 1, 23. The decrease in proliferative capacity was found to result Printed
m Sweden
notes
443
from the lengthening duration of the cell cycle [3, 43 and from a decreasing proportion of cycling cells [5-71. Autoradiographic analysis revealed that this retarded progression of the cell cycle is not the result of a prolongation of the average S phase, but rather is a result of slowed progression through, or permanent arrest in the Gl phase [8, 93. These observations suggest that the DNA replication rate in most of the cycling cells remains constant. On the other hand, Petes et al. [lo] reported from their autoradiographic data that the rate of DNA chain growth in MRCS fibroblasts is slower in senescent than in non-senescent culture. The report is referred to frequently as a distinct characteristic of senescent cells, but, to our knowledge, is the sole report which describes the decreasing rate of DNA chain growth in aged cells. When their data, expressed in figures, were plotted by mean track length vs labelling time, the slopes of young and old cells, from 10 to 30 min, became the same, indicating that their chain elongation rates did not differ from one another. The only difference was the initial rate (from 0 to 10 min) of the chain elongation. For these reasons we re-examined the rate of DNA chain elongation in various PDL (population doubling level) cells by two different methods: DNA fiber-autoradiography and alkaline sucrose density gradient centrifugation. Materials
and Methods
All studies were done with IMR90 cells [7] which were initially obtained through the Institute for Medical Research, Camden, N.J. Cells were grown in Eagle’s MEM containing 10% fetal bovine serum (FBS) and antibiotics in a water-saturated atmosphere of5 % COZ in air. Cultures were split 1 : 4 at earlier passage and 1: 2 at later passage. The line of IMR90 which we used here possessed a limited lifespan of 56 PDL in our culture conditions. The cells were mycoplasma-free, as tested by the method described by Todaro et al.
[ill. Erp Cell Rcs 140 (19821
Preliminary
roles
17. Sanders, B G, Dietert, R R, Kline, K & Dietert, H, Oncodevelopmental biol and med 2 ( 1981)63. 18. Teplitz, R L., Sanders, B G, Brodetsky, A M, Fung, H & Wtley, K L, Cancer res 34 (1974) 1049. 19. Perisic, 0, Biquard, J M, Blanchet, J P, Greenland, T B & Krsmanovic, V, Differentiation 18 (1981) 175. Received November 5, 1981 Revised version received March 16, 1982 Accepted March 18, 1982
Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/82/08044tM4$02.00/0
Aggregated chondrocytes as a model system to study cartilage metabolism G. P. J. van KAMPEN and J. P. VELDHUIJZEN, Department of Oral Cell Biology, School of Dentistry, Vrije Vniversiteit, 1007 MC Amsterdam, The Netherlands Summary. Differentiated chondrocytes, isolated from chick embryo cartilage, were cultured in monolayer, as aggregate or pellet. Aggregation of chondrocytes was accomplished by incubating 2x 105cells in a 5~1 drop of culture medium. Under all three conditions, the cells remained healthy and proliferated during culture. However, matrix production, as indicated by incorporation of [3sS]sulphate into glycosaminoglycans, was greater in aggregated chondrocytes than in monoiayers or pel&s.In addition, aggregates consisting of a well defined number of cells, could easily be maniegates provide pulated for experiments. Therefore a favourable model system to studyT actors modulating the metabolism of chondrocytes.
The intercellular matrix of cartilage consists of several well defined components, such as type II collagen, proteoglycans and hyaluronic acid. Proteoglycans are noncovalently bound to hyaluronic acid in a supramolecular complex. Sulphated glycosaminoglycan chains of chondroitin sulphate and keratan sulphate are attached to the proteoglycan core protein [ 11. For in vitro studies of matrix production and cell proliferation, chondrocytes can be liberated from the matrix by treatment with collagenase and subsequently cultured. In vitro, chondrocytes continue to synthesize matrix products. Many factors, Exp Cell Rcs 140 (1982)
however, influence chondrogenic expression [2]. Several authors reported that interaction between chondrocytes is important in inducing and maintaining matrix production [3-61. Therefore we sought a method with which chondrocytes could be cultured in high density. We now present a simple technique to aggregate a defined number of chondrocytes. Extracellular matrix production and cell division of the aggregated chondrocytes was compared with chondrocytes cultured in monolayers and as pellets. Materials
and Methods
Isolation and culture of the cells. Chondrocytes were isolated from the epiphyses of tibiae of 15-day-old chick embryos (Shaver). The tibiae were aseptically freed of soft tissues and perichondrium. The heads of the tibiae were removed, resulting in exposure of the epiphyseal cartilage. At the proximal and distal ends of the tibiae, slices of the proliferation zone of the epiphyseal cartilage were cut off. These slices were dinested (4x30 min) under gentle stirring with collagenase (CLS II, Worthington Freehold; N.J.), 400 U/ml Hanks Balanced Salt Solution (HBSS. Gibco. Paisley, Scotland). To minimize contamination with libroblasts, the first digest was discarded. Isolated chondrocytes were washed and suspended in culture medium Ml99 (Gibco) supplemented with 250 I.cg L-glutamine and 50 pg ascorbic acid/ml (M199). Aliquots of the cell suspension were used for viability testing (Trypan blue exclusion) and cell counting. The cells were pre-incubated in a humitied (98%) incubator (5 % COPin air) for 20 h at 37°C in monolayers, asaggregates or pellets. For monolayers, 2x 105 cells, suspended in 2 ml medium, were allowed to attach to the wall of a tissue culture tube (Sterilin Ltd, Richmond, Surrey, England). For aggregates, 2x 105cells, suspended in 5 ~1 of medium, were allowed to accumulate in the centre of a drop on the bottom of a plastic Petri dish. For pellets, 2x105 cells, in 2 ml medium, were centrifuged (150 g, 10 min) in a cone-shaped polystyrene tube (Sterilin Ltd.). After the pre-incubation period and medium change, monolayers, individual aggregates and pellets were cultured in Ml99 with 10% fetal calf serum (FCS) for 22 h. After the addition of FCS, aggregates were fixed in Bouin Hollande for histological purposes, with 4-h intervals. [35S]Sulphate incorporation into glycosaminoglycans. After 22 h of culture the synthesis of sulphated glycosaminoglycans (GAG) was measured by determining the incorporation of [3sS]sulphate into cetylpyridiniumchloride-precipitable glycosaminoglycans. The cells were labelled for 90 min in 2 ml medium with 5 &i NaZ3”S0,/ml (sp. act. 50 mCi/mmol; The Radiochemical Centre, Amersham). Subsequently, the incubation medium was decanted and the culPrinted
m Sweden
Preliminary notes tures were digested in 2 ml fresh medium with papain (Merck) at WC for 16 h [cf 73. To the digests 0.5 ml 10% cetyipy~dinium chloride (CRC, Merck) in 0.2 M NaCl was added. After 24 h at 2PC the precipitate (of CRC and GAG) was washed twice with a solution of 0. I % CRC, a treatment which reduced the activity in tissue blanks to background level. The precipitate was dissolved in 0.5 ml formic acid (26 N) and the amount of ?S was determined with a liquid scintillation counter. [3HjTkymidine incorporation into DNA. DNA synthesis- was measured by determining the incorporation of t3H]thymidine into DNA, according to the method of Ash & Francis [g]. The cells were labelled for 90 min in 2 ml medium containing 0.5 @Zi [3H]thymidinelml (sp. act. 5 Ci/mmol; Radiochemical Centre).
Results
The isolation procedure resulted in suspensions of single cells with a viability of 95% or more. Aggregation of freshly isolated chondrocytes was initiated by placing a 5-~1drop of medium containing 2x 1oJcells on the bottom of a Petri dish (fig. 1A). After 20 h the cells were accumulated in the centre of the drop forming an aggregate (0 2 mm) of cohering cells (fig. 1B). The addition of medium to the Petri dish made the aggregatesfloat. Histological sections showed that they consisted of 3-5 cell layers of chondrocytes separated by a thin layer of intercellular matrix which increased in width with time after the addition of FCS. Polarization microscopy revealed birefringent material in the matrix. The amount of this material, probably collagen, also increased with time. When the chondrocytes were devitalized (56”C, 30 min) or when during the pre-incubation period Hanks Balanced Salt Solution was used instead of M199, the chondrocytes did accumulate in the centre of the drop, but no aggregateswere formed. GAG synthesis and DNA synthesis of chondrocytes cultured in monolayer, as aggregate or pellet, are shown in fig. 2. FCS stimulated matrix production and chondrocytic proliferation in all three conditions.
441
Fig. I. Aggregation of 2xloJ embryonic chondrocy:ss, suspended in 5 ~1 medium. (A) Initial state; (B) aggregate formed after 20 h pre-incubation.
The aggregated chondrocytes deposited in the matrix three times as much GAG as monolayers and six times as much as pellets (fig. 2A). ~3H]Thymidine inco~oration into DNA of aggregated and pelleted cells was 60% of the value found in monolayers (fig. 2B).
Aggregation of embryonic cells in vitro is a well-known phenomenon. Histogenetic linking and association of metazoan cells has recently been reviewed by Garrod & Nicol [9]. Aggregation of embryonic chick cells was used by Ahrens and co-workers, who cultured mesenchymal cells from embryonic chick limb bud of various stages in micromass cultures [lo]. In such cultures a stage-related chondrogenic expression of mesenchymal cells could be shown [ 111. In the present study differentiated chondrocytes were used. They were isolated from 15-day chick embryos and aggregated in a small volume of culture medium on a plastic Petri dish. This procedu,e resulted in floating, high density cultures of a defined number of differentiated chondrocytes. In an ultrastructural and biochemical study, Oakes et al. [5] demonstrated the
442
Preliminary notes matrix
DNA
production
synthesas
m
a
P
Fig. 2. Matrix production (A) and DNA synthesis (B) of monolayers (m), aggregates (a) and pellets (p), 22 h after medium change without 0 or with q FCS. Values are means of six determinations. Bars. SD.
possibility of maintaining chondrocytes in a differentiated state by using high density cultures. In the present study routine histology of the aggregates revealed the presence of rapidly increasing, Alcian-blue stainable matrix, indicating well differentiated chondrocytes. It is shown that accumulation of the cells in the centre of the drop is a passive phenomenon. The accumulation of the cells .!T.rp Cell Res IdO (1982)
after devitalization of the chondrocytes support this. The actual aggregation probably requires surface proteins or glycoproteins [cf 93. When HBSS instead of Ml99 was used during pre-incubation, the cells did not cohere. This supports the notion that medium factors are needed to synthesize new intercellular ligands. The enzymatic steps involved in proteoglycan synthesis are well defined [ 11. Since sulphation is a late stage in proteoglycan biosynthesis, sulphate incorporation is a convenient means to study the synthesis of sulphated glycosaminoglycan. The incorporation of C”5S]sulphate into GAG clearly demonstrates that maximum matrix deposition occurs when chondrocytes are cultured as aggregates. On the other hand, cell proliferation was lower compared with monolayers. An inverse relationship between matrix production and cell division has been reported by Miller et al. [12]. It is noteworthy that aggregated chondrocytes deposit much GAG in the matrix, even if they are maintained in culture medium without FCS. Solursh [ 131 suggested that conditioning of the culture medium might contribute to rapid acceleration of the expression of differentiation in culture, and that accumulated matrix might provide a micro-environment that itself promotes better expression of the cartilage phenotype. Perhaps the small (5 ~1) volume during pre-incubation enables the chondrocytes to provide such a micro-environment. In this paper a simple way of aggregating chondrocytes is described. The aggregates are equal in cell number and easy to manipulate in experiments. Aggregated chondrocytes deposit considerable amounts of GAG in the intercellular matrix, so changes can easily be observed. It is concluded that aggregates provide a favourable model system to study factors
Preliminary
modulating cytes.
the metabolism of chondro-
References 1. Stockwell, R A, Biology of cartilage cells (ed R J Harrison & R M H McMinn). Cambridge University Press Cambridge (1979). 2. Sokoloff, L, Malemud, C J, Srivastava, V M L & Morgan, M D, Fed proc 32 (1973) 1499. 3. Abbott, J & Holtzer, H, J cell biol 28 (1966) 473. 4. Lavietes, B B, Dev biol 21 (1970) 584. 5. Oakes, B W, Handley, C J, Lisner, F & Lowther, D A, J embryo1 exp morph01 38 (1977) 239. 6. Yasumoto, S, Kato, Y, Oguri, K, Yamagata, S & Yamagata, T, Dev growth differ 22 (1980) 445. 7. Scott, J E, Methods biochem anal 8 (1960) 145. 8. Ash, P & Francis, M J 0, J endocrinol 66 (1975) 9. &rod D R 62 Nicol A Biol rev 56 (1981) 199 10. Ahrens: P B, Solursh, M Jr Reiter, R S, Dev biol 60 (1977) 69. 11. Solursh, M. Ahrens, P B & Reiter, R S, In vitro 14 (1978) 51. 12. Miller, R P, Husain, M & Lohim, S, J cell physiol 100 (1979) 63. 13. Solursh, M & Meier, S, J exp zoo1 187 (1974) 311. Received December 8, 1981 Revised version received April 7, 1982 Accepted April 15, 1982
Copyright IQ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
0014-4827/82/080143~S~2.~/0
Reevaluation of DNA chain elongation rate in human diploid fibroblasts N. HASEGAWA,’ F. HANAOKA,’ T. HORP and M. YAMADA,’ ‘Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113, and 2Division of Genetics, National Institute of Radiological Sciences, 4-9-l Anagawa, Chiba 260, Japan Summary. The rate of DNA chain elongation in human diploid fibroblasts (IMR90) of different ages was examined by DNA fiber autoradiography and alkaline sucrose density gradient centrifugation. There was no difference in chain elongation rate in various population doubling level cells.
It is well established that human diploid fibroblasts in culture have a limited lifespan, which is inversely correlated with the age of the donor [ 1, 23. The decrease in proliferative capacity was found to result Printed
m Sweden
notes
443
from the lengthening duration of the cell cycle [3, 43 and from a decreasing proportion of cycling cells [5-71. Autoradiographic analysis revealed that this retarded progression of the cell cycle is not the result of a prolongation of the average S phase, but rather is a result of slowed progression through, or permanent arrest in the Gl phase [8, 93. These observations suggest that the DNA replication rate in most of the cycling cells remains constant. On the other hand, Petes et al. [lo] reported from their autoradiographic data that the rate of DNA chain growth in MRCS fibroblasts is slower in senescent than in non-senescent culture. The report is referred to frequently as a distinct characteristic of senescent cells, but, to our knowledge, is the sole report which describes the decreasing rate of DNA chain growth in aged cells. When their data, expressed in figures, were plotted by mean track length vs labelling time, the slopes of young and old cells, from 10 to 30 min, became the same, indicating that their chain elongation rates did not differ from one another. The only difference was the initial rate (from 0 to 10 min) of the chain elongation. For these reasons we re-examined the rate of DNA chain elongation in various PDL (population doubling level) cells by two different methods: DNA fiber-autoradiography and alkaline sucrose density gradient centrifugation. Materials
and Methods
All studies were done with IMR90 cells [7] which were initially obtained through the Institute for Medical Research, Camden, N.J. Cells were grown in Eagle’s MEM containing 10% fetal bovine serum (FBS) and antibiotics in a water-saturated atmosphere of5 % COZ in air. Cultures were split 1 : 4 at earlier passage and 1: 2 at later passage. The line of IMR90 which we used here possessed a limited lifespan of 56 PDL in our culture conditions. The cells were mycoplasma-free, as tested by the method described by Todaro et al.
[ill. Erp Cell Rcs 140 (19821