PHlThymidine Incorporation Does Not Correlate with Growth State in Cultured Alveolar Type II Cells Annick Clement, Norbert Riedel, and Jerome S. Brody Pulmonary Center and Departments of Medicine, Boston University School of Medicine, Boston, Massachusetts
Quantitative measurement of (3H]thymidine ((3H]TdR) incorporation into cultured cells is widely used as an indicator of cell proliferation. The observation that adult type II cells are able to incorporate large amounts of (3H]TdR despite the fact that they are not proliferating raised the question of the meaning of (3H]TdR incorporation in these cells. Comparing different systems of proliferating and nonproliferating type II cells and lung fibroblasts, we show that nonproliferating type II cells are able to synthesize some thymidine nucleotides used as immediate precursors for DNA synthesis and that most of the radioactivity incorporated into acid-insoluble material in these cells is actually in DNA. We found that hydroxyurea inhibited (3H]TdR incorporation into DNA, suggesting that nonreplicating type II cells use thymidine for scheduled, i.e., replicative, rather than unscheduled, or repair, DNA synthesis. However, newly synthesized DNA does not appear to be in a stable form, available for replication. These studies demonstrate that, in culture, adult type II cells initiate but are unable to complete scheduled DNA synthesis. They also establish that (3H]TdR incorporation cannot be used as an indicator of cell proliferation in cultured type II cells.
Incorporation of tritiated thymidine ((3H]TdR) into acidinsoluble material is widely used as a marker of DNA synthesis and, by extrapolation, of cell proliferation (1, 2). In fibroblasts and many other cell systems, when cells are allowed to proliferate in the presence of (3H]TdR, the radioactive nucleoside is incorporated into DNA and can be detected in the nuclei of cells. When these cells are growthrrested, the incorporation of (3H]TdR into DNA decreases to low levels and the percentage of cells with labeled nuclei (labeling index) falls. Thus, (3H]TdR incorporation into DNA and labeling index generally correspond to the proportion of cells having an S phase DNA content and that are committed to complete the cell cycle and to divide (2). When alveolar type II cells isolated from adult rats are placed in culture, they do not go through the replication cycle: there is no increase in cell number with time in culture (3-5). However, when these cells are incubated with (3H]TdR, they incorporate large amounts of the radioactive nucleoside. Several investigators have shown that. various Key »Vrds: cell proliferation, DNA synthesis, growth arrest (Received in original form January 4, 1990 and in revised form March 6, 1990)
Address correspondence to: Jerome S. Brody, M.D., Director, Pulmonary Center, Boston University School of Medicine, 80 E. Concord Street, K-603, Boston, MA 02118. Dr. Clement's present address is: Hopital Trousseau, Paris, France. Abbreviations: ethylenediaminetetraacetic acid, EDTA; fetal bovine serum, FBS; hydroxyurea, HU; minimal essential medium, MEM; Nonidet P-40, NP-40; phosphate-buffered saline, PBS; thymidine diphosphate, TOP; thymidine monophosphate, TMP; thymidine triphosphate, TTP. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 159-164, 1990
growth factors, matrix components, or macrophage-derived products can stimulate (3H]TdR incorporation in cultured type II cells, yet actual cell proliferation either does not occur or is minimal despite as much as 50-fold increases in (3H]TdR incorporation (4, 6, 7). We have recently reported that type II cells isolated from neonates are able to go through several rounds of division in culture, and that they can be reversibly arrested by serum deprivation (5). As with adult type II cells, when growth-arrested neonatal type II cells are incubated with (3H]TdR, incorporation into acid-insoluble material remains high. We have also shown that both adult type II cells and serum-deprived neonatal type II cells express growth-related genes associated with the Gl and S phases of the cell cycle, suggesting that the mechanism of growth arrest differs from that described for cultured fibroblasts. These observations leave uncertain the meaning of (3H]TdR incorporation in alveolar type II cells. In the present study, using type II cells isolated from adult rats, we show that despite the fact that the cells do not proliferate in culture, they display sufficient thymidine kinase activity to synthesize some TdR nucleotides used as immediate precursors for DNA synthesis and that (3H]TdR is actually incorporated into DNA. This DNA synthesis likely represents true replicative DNA synthesis since it is completely blocked by hydroxyurea, which has no effect on DNA repair synthesis (8). However, the newly synthesized DNA is not stable and is likely degraded as a result of a block to completion of the cell cycle. It is clear from these studies that (3H]TdR incorporation into acid-insoluble material cannot be equated with cell division in primary cultures of alveolar type II cells.
160
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
Materials and Methods Cell Isolation and Culture Type II cells were isolated from pathogen-free, SpragueDawley rats, as previously described in detail (9) according to the method of Dobbs and colleagues (10). Adult male animals were 2 mo old; neonatal animals were 10 to 12 d old. For enzymatic dispersement of lung cells, we used 180 U/adult lung and 36 Vlneonatallung of intratracheal elastase. After panning the cells on IgG-coated plates, unadhered cells were collected by centrifugation and replated onto tissue culture dishes at 2 to 3 x 1
Quantitative measurement of (3H]thymidine ((3H]TdR) incorporation into cultured cells is widely used as an indicator of cell proliferation. The observation that adult type II cells are able to incorporate large amounts of (3H]TdR despite the fact that they are not proliferating raised the question of the meaning of (3H]TdR incorporation in these cells. Comparing different systems of proliferating and nonproliferating type II cells and lung fibroblasts, we show that nonproliferating type II cells are able to synthesize some thymidine nucleotides used as immediate precursors for DNA synthesis and that most of the radioactivity incorporated into acid-insoluble material in these cells is actually in DNA. We found that hydroxyurea inhibited (3H]TdR incorporation into DNA, suggesting that nonreplicating type II cells use thymidine for scheduled, i.e., replicative, rather than unscheduled, or repair, DNA synthesis. However, newly synthesized DNA does not appear to be in a stable form, available for replication. These studies demonstrate that, in culture, adult type II cells initiate but are unable to complete scheduled DNA synthesis. They also establish that (3H]TdR incorporation cannot be used as an indicator of cell proliferation in cultured type II cells.
Incorporation of tritiated thymidine ((3H]TdR) into acidinsoluble material is widely used as a marker of DNA synthesis and, by extrapolation, of cell proliferation (1, 2). In fibroblasts and many other cell systems, when cells are allowed to proliferate in the presence of (3H]TdR, the radioactive nucleoside is incorporated into DNA and can be detected in the nuclei of cells. When these cells are growthrrested, the incorporation of (3H]TdR into DNA decreases to low levels and the percentage of cells with labeled nuclei (labeling index) falls. Thus, (3H]TdR incorporation into DNA and labeling index generally correspond to the proportion of cells having an S phase DNA content and that are committed to complete the cell cycle and to divide (2). When alveolar type II cells isolated from adult rats are placed in culture, they do not go through the replication cycle: there is no increase in cell number with time in culture (3-5). However, when these cells are incubated with (3H]TdR, they incorporate large amounts of the radioactive nucleoside. Several investigators have shown that. various Key »Vrds: cell proliferation, DNA synthesis, growth arrest (Received in original form January 4, 1990 and in revised form March 6, 1990)
Address correspondence to: Jerome S. Brody, M.D., Director, Pulmonary Center, Boston University School of Medicine, 80 E. Concord Street, K-603, Boston, MA 02118. Dr. Clement's present address is: Hopital Trousseau, Paris, France. Abbreviations: ethylenediaminetetraacetic acid, EDTA; fetal bovine serum, FBS; hydroxyurea, HU; minimal essential medium, MEM; Nonidet P-40, NP-40; phosphate-buffered saline, PBS; thymidine diphosphate, TOP; thymidine monophosphate, TMP; thymidine triphosphate, TTP. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 159-164, 1990
growth factors, matrix components, or macrophage-derived products can stimulate (3H]TdR incorporation in cultured type II cells, yet actual cell proliferation either does not occur or is minimal despite as much as 50-fold increases in (3H]TdR incorporation (4, 6, 7). We have recently reported that type II cells isolated from neonates are able to go through several rounds of division in culture, and that they can be reversibly arrested by serum deprivation (5). As with adult type II cells, when growth-arrested neonatal type II cells are incubated with (3H]TdR, incorporation into acid-insoluble material remains high. We have also shown that both adult type II cells and serum-deprived neonatal type II cells express growth-related genes associated with the Gl and S phases of the cell cycle, suggesting that the mechanism of growth arrest differs from that described for cultured fibroblasts. These observations leave uncertain the meaning of (3H]TdR incorporation in alveolar type II cells. In the present study, using type II cells isolated from adult rats, we show that despite the fact that the cells do not proliferate in culture, they display sufficient thymidine kinase activity to synthesize some TdR nucleotides used as immediate precursors for DNA synthesis and that (3H]TdR is actually incorporated into DNA. This DNA synthesis likely represents true replicative DNA synthesis since it is completely blocked by hydroxyurea, which has no effect on DNA repair synthesis (8). However, the newly synthesized DNA is not stable and is likely degraded as a result of a block to completion of the cell cycle. It is clear from these studies that (3H]TdR incorporation into acid-insoluble material cannot be equated with cell division in primary cultures of alveolar type II cells.
160
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
Materials and Methods Cell Isolation and Culture Type II cells were isolated from pathogen-free, SpragueDawley rats, as previously described in detail (9) according to the method of Dobbs and colleagues (10). Adult male animals were 2 mo old; neonatal animals were 10 to 12 d old. For enzymatic dispersement of lung cells, we used 180 U/adult lung and 36 Vlneonatallung of intratracheal elastase. After panning the cells on IgG-coated plates, unadhered cells were collected by centrifugation and replated onto tissue culture dishes at 2 to 3 x 1
