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DNA synthesis and the mitotic cycle in frog kidney cells cultivated in vitro
0
1DGS by Academic
Experimental
Press Inc.
Cell Research 45, 277-280
DNA SYNTHESIS KIDNEY
277
(1967)
AND
THE MITOTIC CYCLE IN FROG
CELLS CULTIVATED
IN
VITRO1
D. MALAMUD2 Department
of Biological Sciences,
University of Cincinnati,
Cincinnati,
Ohio, U.S.A.
Received May 23, 19663
IN the course of studies on the LuckC renal adenocarcinoma it became necessary to develop methods for the cultivation of normal cells from the frog (Rana pipiens) kidney. The generation cycle of these cells differs considerably from that of mammalian cells, where the DNA synthetic phase (S) is usually 6 to 8 hr [5, 6, la]. MATERIALS
AND METHODS
Cultures were initiated from adult frog kidneys by a modification of the technique of Auclair [l]. Cells were dispersed in a solution of 0.025 per cent Pronase (Calbiochem) in Ca and Mg free amphibian Ringer’s solution. The cells were plated at 750,000 per ml of Eagle’s Minimum Essential Medium, diluted with distilled water so as to be isotonic for amphibian tissue, and then supplemented with 20 per cent calf serum and 10 per cent whole egg ultrafiltrate (Microbiological Associates). The cells were grown on glass coverslips in disposable tissue culture dishes at 25°C in the presence of 5 per cent CO,. Confluent monolayers formed in 3 to 4 days. In order to determine the lag in DNA synthesis in newly initiated cultures, cells were exposed to 5 PC of tritiated thymidine (3HTdR, Schwarz BioResearch, Spec. act. 1.9 C/mM) per ml of medium for 15 min. Aliquots were removed from the cultures, air dried on microscope slides, fixed in ethanol-acetic acid (3: I), and extracted with 5 per cent cold trichloroacetic acid for 20 min. In order to study the synthesis of rapidly labeled RNA during the lag in DNA synthesis, cultures were exposed to 5 ,uc/ml of either 3H-cytidine (SHCR) or 3Huridine (3HUR), spec. act. 2.0 C/mM, for 5 min. Six- or seven-day-old cultures were pulsed with 0.05 ,uc 3HTdR per ml of medium for 20 min, washed with Ringer’s solution containing an excess of TdR, and chased with fresh medium for predetermined times. Coverslips containing the monolayers were removed from the culture dishes, affixed to microscope slides, and treated as above. In order to determine the cell cycle, slides were scored for the number of labeled metaphases plus anaphases as a per cent of the total number of metaphases plus anaphases. 1 Supported by U.S.P.1I.S. Grant CA-06439 to W. Auclair and American Cancer Sot. Inst. Grant IN-79. 2 Present address: Fels Research Institute, Temple University School of Medicine, Philadelphia, Pa., U.S.A. s Revised version received September 30, 1966. 18 - 671812
Experimental
Cell Research 45
278
Il. Malamud
Autoradiograms were prepared by dipping the slides in diluted NTB-2 (1: 1 with distilled water). The slides were exposed for 2 weeks, developed, and stained through the emulsion with methyl green-pyronin [S]. Cells containing 3 or more silver grains were considered labeled. Control autoradiograms consisted of slides incubated in deoxyribonuclease (DNase, 0.2 mg /ml DNase of 0.2 M phosphate buffer at pH 7,
HOURS
Fig. I.-Incorporation O-O, SHTdR; o-0,
IN TISSUE
CULTURE
of labeled nucleoside at early times after the initiation aHUR; 0. . . 0, 3HCR.
of tissue cultures.
0.05 A4 with MgCl,), ribonuclease (RNase, 0.4 mg RNase per ml of distilled water at pH 6.6), and control blanks which consisted of the buffer or distilled water without the enzymes. Slides were incubated at 37°C for 4 hr.
RESULTS Fig. 1 shows the results of studies on newly initiated cultures. Note that the lag period prior to DNA synthesis is only slightly longer than that reported for mammalian cells [3, 7, lo]. During the DNA lag phase, the number of cells incorporating 3HCR and 3HUR decreases for the first lo-12 hr, then increases to a maximum at 48 hr. As the cells enter DNA synthesis there is a sharp drop in the number of cells incorporating 3HUR, and a slight drop in the incorporation of 3HCR followed by a subsequent increase in the number of cells incorporating the latter nucleoside. On the basis of extraction with nucleases, it was found that 3HTdR and 3HUR were incorporated only into DNA and RNA, respectively. In studies with 3HCR, all of the label was in RNase digestible material for the first 40 hr, and then approximately 40 per cent of the label was removed with DNase. Eqwimental
Cell Research 45
DNA synthesis in frog kidney cells
250
Fig. 2 shows the results of studies on the generation cycle. From the graph it can be seen that the post-synthetic gap (G2) plus prophase ~7.7 hr, DNA synthesis (S) =22.3 hr, and total intermitotic time (T) =46 hr. The presynthetic gap (Gl) plus telophase was calculated, by subtraction, to be 16 hr.
TIME-
HOURS
Fig. 2.-Generation cycle of adult frog kidney cells in vitro. experiment; o, cultures 7 days old at start of experiment.
l , Cultures
6 days old at start of
DISCUSSION
A lag in DNA synthesis upon the initiation of tissue cultures has been reported by several investigators [3, 7, lo]. Lieberman et al. [7] found an increase in the amount of RNA synthesized during the DNA lag phase. In the present study no change in the number of grains per labeled cell was detected, however, there was an increase in the number of cells incorporating nucleoside into rapidly labeled RNA. It is clear (Fig. 1) that coincident with the start of DNA synthesis there was a sharp drop in the number of cells synthesizing rapidly labeled RNA. This is consistent with reports of a decrease in RNA synthesis associated with the onset of DNA synthesis [15]. It is of interest that only a small percentage of cells was labeled with RNA precursors. This may have been due to the short pulse period. The mammalian cell cycle has been well studied, and although the synthetic phase is usually 6-8 hr, numerous exceptions have been reported [a]. Bresciani [4] has suggested that the 6-8 hr S phase represents a basal level which may be altered by metabolic influences. Can’t Hof [16] found that the length of the S phase in root meristem cells was proportional to the Experimental
Cell Research 45
280
D. Malamud
amount of DNA present. If there is a relationship between the amount of DNA in a cell and the time required for its replication, then the S phase for frog cells should be about 20 hr since the amount of DNA is 2-3 times that present in a mammalian cell [I 11. In addition to the cell cycle described in the present study, a similar cycle with an S phase of 20 hr has been found in frog intestinal epithelium [14] and in hematopoietic nodules in the frog kidney [9]. Recently a cell cycle of 83 days with a synthetic phase of 100 hr has been reported for the lens epithelium of the bullfrog [13]. This suggests that the basal synthetic phase in Rana is 20 hr and this period may be greatly prolonged. SUMMARY
Frog kidney cells in tissue culture exhibit a lag period of about 48 hr before the onset of DNA synthesis. During this time the number of cells incorporating nucleoside into rapidly labeled RNA decreases for lo-12 hr, then increases until 48 hr. Coincident with the start of DNA synthesis there is a sharp drop in the synthesis of rapidly labeled RNA. The generation cycle of frog kidney cells was determined: Gl +telophase =16 hr, S =22.3 hr, G2 +prophase =7.7 hr, T =46 hr.
REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9. IO. Il.
12. 13. 14. 15. 16.
AUCLAIR, W , Nafure 192, 467 (1961) BASERGA, R., Cancer Res. 25, 581 (1965). BENDER, M. A. and PRESCOTT, D. M., Expff Cell Res. 27, 221 (1962). BRESCIANI, F., Science 146, 653 (1964). CAMERON, L., J. Cell Bid. 20, 185 (1964). CLEAVER, J. E., Expfl Cell Res. 39, 697 (1965). LIEBERMAN, I., ABRAMS, R. and OVE, P., J. Bid. Chem. 238, 2141 (1963). LONG, M. E. and TAYLOR, H. C., JR., Ann. N. Y. Acad. Sci. 63, 1095 (1956). MALAMUD, D. and AUCLAIR, W., Unpublished observations. MICHALOWSKI, A., Expff Cell Res. 32, 609 (1963). MIRSKY, -4. E. and RIS, H., .7. Gen. Physiol. 34, 451 (1951). PRESCOTT, D. M. and BENDER, M. A., Expff CeZZRes. 29, 430 (1963). REDDAN, J. R. and ROTHSTEIN, H., J. Cell Physiol. 67, 307 (1966). SCHECHTMAN, L., M.S. Thesis, University of Cincinnati (1966). SISKEN, J. E., Expfl Cell Res. 16, 602 (1959). VAN’T HOF, J., Expff Cell Res. 39, 48 (1965).
Experimental
Cell Research 45
1DGS by Academic
Experimental
Press Inc.
Cell Research 45, 277-280
DNA SYNTHESIS KIDNEY
277
(1967)
AND
THE MITOTIC CYCLE IN FROG
CELLS CULTIVATED
IN
VITRO1
D. MALAMUD2 Department
of Biological Sciences,
University of Cincinnati,
Cincinnati,
Ohio, U.S.A.
Received May 23, 19663
IN the course of studies on the LuckC renal adenocarcinoma it became necessary to develop methods for the cultivation of normal cells from the frog (Rana pipiens) kidney. The generation cycle of these cells differs considerably from that of mammalian cells, where the DNA synthetic phase (S) is usually 6 to 8 hr [5, 6, la]. MATERIALS
AND METHODS
Cultures were initiated from adult frog kidneys by a modification of the technique of Auclair [l]. Cells were dispersed in a solution of 0.025 per cent Pronase (Calbiochem) in Ca and Mg free amphibian Ringer’s solution. The cells were plated at 750,000 per ml of Eagle’s Minimum Essential Medium, diluted with distilled water so as to be isotonic for amphibian tissue, and then supplemented with 20 per cent calf serum and 10 per cent whole egg ultrafiltrate (Microbiological Associates). The cells were grown on glass coverslips in disposable tissue culture dishes at 25°C in the presence of 5 per cent CO,. Confluent monolayers formed in 3 to 4 days. In order to determine the lag in DNA synthesis in newly initiated cultures, cells were exposed to 5 PC of tritiated thymidine (3HTdR, Schwarz BioResearch, Spec. act. 1.9 C/mM) per ml of medium for 15 min. Aliquots were removed from the cultures, air dried on microscope slides, fixed in ethanol-acetic acid (3: I), and extracted with 5 per cent cold trichloroacetic acid for 20 min. In order to study the synthesis of rapidly labeled RNA during the lag in DNA synthesis, cultures were exposed to 5 ,uc/ml of either 3H-cytidine (SHCR) or 3Huridine (3HUR), spec. act. 2.0 C/mM, for 5 min. Six- or seven-day-old cultures were pulsed with 0.05 ,uc 3HTdR per ml of medium for 20 min, washed with Ringer’s solution containing an excess of TdR, and chased with fresh medium for predetermined times. Coverslips containing the monolayers were removed from the culture dishes, affixed to microscope slides, and treated as above. In order to determine the cell cycle, slides were scored for the number of labeled metaphases plus anaphases as a per cent of the total number of metaphases plus anaphases. 1 Supported by U.S.P.1I.S. Grant CA-06439 to W. Auclair and American Cancer Sot. Inst. Grant IN-79. 2 Present address: Fels Research Institute, Temple University School of Medicine, Philadelphia, Pa., U.S.A. s Revised version received September 30, 1966. 18 - 671812
Experimental
Cell Research 45
278
Il. Malamud
Autoradiograms were prepared by dipping the slides in diluted NTB-2 (1: 1 with distilled water). The slides were exposed for 2 weeks, developed, and stained through the emulsion with methyl green-pyronin [S]. Cells containing 3 or more silver grains were considered labeled. Control autoradiograms consisted of slides incubated in deoxyribonuclease (DNase, 0.2 mg /ml DNase of 0.2 M phosphate buffer at pH 7,
HOURS
Fig. I.-Incorporation O-O, SHTdR; o-0,
IN TISSUE
CULTURE
of labeled nucleoside at early times after the initiation aHUR; 0. . . 0, 3HCR.
of tissue cultures.
0.05 A4 with MgCl,), ribonuclease (RNase, 0.4 mg RNase per ml of distilled water at pH 6.6), and control blanks which consisted of the buffer or distilled water without the enzymes. Slides were incubated at 37°C for 4 hr.
RESULTS Fig. 1 shows the results of studies on newly initiated cultures. Note that the lag period prior to DNA synthesis is only slightly longer than that reported for mammalian cells [3, 7, lo]. During the DNA lag phase, the number of cells incorporating 3HCR and 3HUR decreases for the first lo-12 hr, then increases to a maximum at 48 hr. As the cells enter DNA synthesis there is a sharp drop in the number of cells incorporating 3HUR, and a slight drop in the incorporation of 3HCR followed by a subsequent increase in the number of cells incorporating the latter nucleoside. On the basis of extraction with nucleases, it was found that 3HTdR and 3HUR were incorporated only into DNA and RNA, respectively. In studies with 3HCR, all of the label was in RNase digestible material for the first 40 hr, and then approximately 40 per cent of the label was removed with DNase. Eqwimental
Cell Research 45
DNA synthesis in frog kidney cells
250
Fig. 2 shows the results of studies on the generation cycle. From the graph it can be seen that the post-synthetic gap (G2) plus prophase ~7.7 hr, DNA synthesis (S) =22.3 hr, and total intermitotic time (T) =46 hr. The presynthetic gap (Gl) plus telophase was calculated, by subtraction, to be 16 hr.
TIME-
HOURS
Fig. 2.-Generation cycle of adult frog kidney cells in vitro. experiment; o, cultures 7 days old at start of experiment.
l , Cultures
6 days old at start of
DISCUSSION
A lag in DNA synthesis upon the initiation of tissue cultures has been reported by several investigators [3, 7, lo]. Lieberman et al. [7] found an increase in the amount of RNA synthesized during the DNA lag phase. In the present study no change in the number of grains per labeled cell was detected, however, there was an increase in the number of cells incorporating nucleoside into rapidly labeled RNA. It is clear (Fig. 1) that coincident with the start of DNA synthesis there was a sharp drop in the number of cells synthesizing rapidly labeled RNA. This is consistent with reports of a decrease in RNA synthesis associated with the onset of DNA synthesis [15]. It is of interest that only a small percentage of cells was labeled with RNA precursors. This may have been due to the short pulse period. The mammalian cell cycle has been well studied, and although the synthetic phase is usually 6-8 hr, numerous exceptions have been reported [a]. Bresciani [4] has suggested that the 6-8 hr S phase represents a basal level which may be altered by metabolic influences. Can’t Hof [16] found that the length of the S phase in root meristem cells was proportional to the Experimental
Cell Research 45
280
D. Malamud
amount of DNA present. If there is a relationship between the amount of DNA in a cell and the time required for its replication, then the S phase for frog cells should be about 20 hr since the amount of DNA is 2-3 times that present in a mammalian cell [I 11. In addition to the cell cycle described in the present study, a similar cycle with an S phase of 20 hr has been found in frog intestinal epithelium [14] and in hematopoietic nodules in the frog kidney [9]. Recently a cell cycle of 83 days with a synthetic phase of 100 hr has been reported for the lens epithelium of the bullfrog [13]. This suggests that the basal synthetic phase in Rana is 20 hr and this period may be greatly prolonged. SUMMARY
Frog kidney cells in tissue culture exhibit a lag period of about 48 hr before the onset of DNA synthesis. During this time the number of cells incorporating nucleoside into rapidly labeled RNA decreases for lo-12 hr, then increases until 48 hr. Coincident with the start of DNA synthesis there is a sharp drop in the synthesis of rapidly labeled RNA. The generation cycle of frog kidney cells was determined: Gl +telophase =16 hr, S =22.3 hr, G2 +prophase =7.7 hr, T =46 hr.
REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9. IO. Il.
12. 13. 14. 15. 16.
AUCLAIR, W , Nafure 192, 467 (1961) BASERGA, R., Cancer Res. 25, 581 (1965). BENDER, M. A. and PRESCOTT, D. M., Expff Cell Res. 27, 221 (1962). BRESCIANI, F., Science 146, 653 (1964). CAMERON, L., J. Cell Bid. 20, 185 (1964). CLEAVER, J. E., Expfl Cell Res. 39, 697 (1965). LIEBERMAN, I., ABRAMS, R. and OVE, P., J. Bid. Chem. 238, 2141 (1963). LONG, M. E. and TAYLOR, H. C., JR., Ann. N. Y. Acad. Sci. 63, 1095 (1956). MALAMUD, D. and AUCLAIR, W., Unpublished observations. MICHALOWSKI, A., Expff Cell Res. 32, 609 (1963). MIRSKY, -4. E. and RIS, H., .7. Gen. Physiol. 34, 451 (1951). PRESCOTT, D. M. and BENDER, M. A., Expff CeZZRes. 29, 430 (1963). REDDAN, J. R. and ROTHSTEIN, H., J. Cell Physiol. 67, 307 (1966). SCHECHTMAN, L., M.S. Thesis, University of Cincinnati (1966). SISKEN, J. E., Expfl Cell Res. 16, 602 (1959). VAN’T HOF, J., Expff Cell Res. 39, 48 (1965).
Experimental
Cell Research 45