- Email: [email protected]
The division cycle and RNA-synthesis in diploid human cells at different passage levels in vitro
Experimental
673
Cell Research 42, 673-684 (1966)
THE DIVISION CYCLE AND RNA-SYNTHESIS IN DIPLOID HUMAN CELLS AT DIFFERENT PASSAGE LEVELS IN VITRO1 A. MACIEIRA-COELHO,B Department
J. PONTGN
and L. PHILIPSON
of Cell Biology, Institute of Medical Microbiology, University of Uppsala, Uppsala, Sweden Received January 3, 1966
TISSUEculture
methods permit growth of fibroblast-like somatic cells from normal organs of many different animal species. The explanted cells will multiply rapidly for several generations (phase II) but will then enter a stage of declining growth (phase III) terminating in cell death. Only in some instances will the cells acquire a capacity for indefinite proliferation. Embryonic lung fibroblasts of human origin have been most extensively studied. They remain in phase II during about 40 subcultivations performed at a split ratio of 1:2. Phase III will last about another 10 subcultivations after which complete lysis takes place. Human lung cells have never spontaneously acquired capacity for indefinite growth in vitro [7, 8, 111. The basis for this limitation in proliferative capacity is unknown but does not seem to be related to any lack of essential metabolites in the medium or infection by latent viruses or mycoplasmas [7, 81. It has been suggested that it reflects an intrinsic cell property connected with the aging process [7, 81. The retarded growth of mass cultures of phase III cells could be due to any of the following possibilities: (a) only a small fraction of the cell population in phase III cultures is dividing at an unaltered rate but the majority of the cells are unable to divide, (b) the entire population is uniformly growing slowly possibly because of selective interference with one of the stages in the division cycle, (c) the cell population has become strongly heterogeneous and the cells show a spectrum between the two extremes, i.e., complete inhibition and normal division cycle. The present study aims to 1 This work was supported by grants from the Damon Runyon Memorial Funds, The Swedish Cancer Society and The Swedish 8 Fellow of the Calouste Gulbenkian Foundation.
and the Jane Coffin Childs Medical Research Council.
Experimental
Cell Research 42
674
,4. Macieira-Coelho,
.I. Pontt!n
and I>. Philipson
differentiate between these possibilities by comparing human phase II and phase III cells with respect to cell growth curves, the division cycle and ribonucleic acid (RNA) metabolism studied by autoradiography.
MATERIAL
AND
METHODS
Tissue culture.-The standard human fibroblast strain WI-38 of fetal lung origin was used [7]. Cells were grown in Eagle’s basal medium [6] supplemented with twice the concentration of vitamins and amino acids, 10 per cent calf serum, streptomycin (50 yg/ml) and penicillin (100 U/ml). Sufficient NaHCO, was added to obtain a pH of 7.4. Subcultivation by trypsinization was done as described by Hayflick and Moorhead [8]. The cells were seeded in 60 mm plastic Petri dishes containing 24 x 12 mm coverslips and incubated in a 5 per cent CO, humidified atmosphere at 37°C. Cell numbers were calculated from hemocytometer counts of cells suspended in 0.25 per cent trypsin in phosphate-buffered saline (PBS). Cultures were tested at different times for mycoplasma contamination by streaking cells and supernatant on PPLO agar [9]. Autoradiography.-The technique used for autoradiography was described by Doniach and Pelt [5]. Deoxyribonucleic acid (DNA) synthesis.-Two methods were used for the analysis of the division cycle: with pulse label and continuous label. Pulse label.-Exponentially growing cultures were exposed to 1 &/ml tritium labeled thymidine (3H-TdR) for 15 min. 3H-TdR, as supplied by Schwartz Laboratories, had a specific activity of 1.9 C/mM. After the labeling period the cells were washed twice with culture medium and fresh medium was added supplemented with 10 ,ug/ml (100 x the concentration of 3H-TdR) of non-labeled thymidine (TdR). This was considered zero hr. Duplicate coverslips were removed at different intervals thereafter, washed in PBS and fixed in acetic acid-methanol (1: 3) for 1 hr and dried. The coverslips were glued to a microscopic glass slide by Canada Balsam, dried, dipped in a gelatine-chrome alum solution [2] and coated with film (Kodak ARIO). After a 4 day exposure the preparations were developed for 4 min in Kodak D-19B, kept for 10 min in Kodak Acid Fixer and washed for 10 min in running tap water. Staining was done immediately afterwards in a 0.05 per cent (w/v) solution of toluidine blue for 3-5 min. Film adhering to the back of the slide was scraped off and after drying the preparations were ready for analysis. Continuous label.-This procedure was done as described by Stanners and Till [IO]. 3H-Tdr in a concentration of 0.01 &/ml was added to exponentially growing cultures. Duplicate samples were removed at hourly intervals thereafter and treated as already described. RNA synthesis.-Monolayers in the post-logarithmic resting phase were labeled with 1 &/ml of tritiated uridine (3H-UdR) in culture medium for half an hr. 3HUdR as supplied by Schwartz Laboratories had a specific activity of 2 C/mM. The cultures were then washed twice with culture medium and fresh medium supplemented with IO pg/ml (100 x the concentration of 8H-UdR) of non-labeled urldine (UdR) was added. Duplicate coverslips were removed at zero hr (time of removal of Experimental
Cell Research 42
Nucleic acid synthesis in human fibroblasts
675
radioactivity) and at different intervals thereafter. Samples were washed twice with PBS and fixed for 1 hr in acetic acid-methanol. The coverslips were then broken in half. One fragment was treated with IO pg/ml ribonuclease (RNAase) (Worthington, N.J., U.S.A.) in PBS for 1 hr at 37X, washed twice with PBS and fixed again for 1 hr. The other fragment was left untreated. Both were affixed with Canada Balsam to a microscopic glass slide. The preparations were then treated as described above under DNA synthesis except for an exposure time of 7 rather than 4 days. Analysis of autoradiographs.-The symbols used for the analysis of the division cycle are defined as follows, in agreement with previous literature [3]. Gi = time between end of mitosis and start of DNA synthesis; S = time of DNA synthesis; G2 =time between end of DNA synthesis and beginning of mitosis; tm =mitotic period = time between chromosome condensation of early prophase to separation of two daughter cells in late telophase; generation time = Gl + S + G2 + tm; interphase = Gl + S + G2. A hundred metaphases were counted to determine the percentage of labeled metaphses. One thousand nuclei were observed in the determination of the proportion of labeled interphase nuclei. Samples of 3000 cells were used to determine the mitotic index. Grains were counted over 100 labeled metaphases to determine the peak metaphase grain count. RNA incorporation was measured by counting the grains above the nucleus and in the cytoplasm in 50 cells on both coverslip fragments. The difference between the counts obtained in non-treated and RNAase-treated preparations was considered the specific incorporation into RNA.
RESULTS Cell growth Fig. 1 shows the growth curves of phase II and III cells. Cells in phase II showed a lag phase of about 1 day, a logarithmic phase of growth lasting 2-3 days superseded by a post-logarithmic resting phase during which no increase in cell number took place. The doubling time during logarithmic growth was approximately 24 hr. The total increase in cell yield was 350 per cent. Phase III cells differed from phase II cells by having a longer lag phase (1.5 days) and particularly by a short period of only 1 day of slow logarithmic growth. From the slope of the curve a theoretical cell doubling time of about 3 days can be calculated; under the actual conditions, however, no doubling of the input cell number took place, the final cell counts during the resting phase showed a net increase of only about 40 per cent. DNA synthesis The division cycle of cells in early and late passage was studied after a labeling with 3H-TdR applied during the exponential growth phase (Fig. 1). Figs. 2 and 3 show the percentage of labeled metaphases and interphases at Experimental
Cell Research 42
676
A. Macieira-Coelho,
J. Ponttk
and L. Philipson
different times after pulse labeling. The pattern at the 18th passage (Fig. 2) was the same as that gators for the same system [4]. (a) The generation time.-The second wave of 2 an 3 represents cells that have gone through
1
2 Days after
3 rubcultivation
4
obtained for phase II cells described by other investilabeled metaphases of Figs. G2, mitotis, Gl, S, G2 and
5
Fig. l.-Growth curves of WI-38 cells in the 18th ( x - x ) and in 42nd passage (O--O) obtained by hemocytometer cell counting. Each point represents the average of duplicate samples. The arrows indicate the beginning and the end of the experiments done for timing the division cycle.
have entered their second mitosis after a pulse label. Cells with the shortest generation times will register as the first labeled metaphases of the second wave. In phase II this rise occurred 18 hr after labeling (Fig. 2). Since the minimal G2 period was 2 hr (see Fig. 2), the minimal generation time was about 16 hr. The average generation time estimated from the difference between the 50 per cent points on the two ascending limbs of the biphasic curve [3] was approximately 17 hr, a value that shows good correspondence with a previous estimate of 18 hr for the same cells [4]. In phase III cultures the minimal generation time was 21-22 hr (Fig. 3). The average generation time could not be appropriately determined because Experimental
Cell Research 42
Nucleic acid synthesis in human fibroblasts
677
the second wave did no reach the 50 per cent level. It must, however, have lasted more than 25 hr. (b) The S period.-For an asynchronous population the method of Stanners and Till [lo] gives the most accurate estimate. The time interval between the first appearance of labeled metaphases and the point when the number of
5
7
9
11 13 15 Hourr alter labeling
17
19
11 22
Fig. 2.-Distribution of labeled metaphases ( x - x ) and interphases hr after a pulse label with 8H-TdR, of a culture in the 18th passage.
(0 - - - 0) at different
grains over metaphases reaches a plateau during continuous labeling corresponds to the length of the S period. In addition the method also gives information on the time course of DNA synthesis. For the determination of the number of grains over metaphases, the grain counts of 100 labeled metaphases were plotted as histograms. The cumulative percentage of such labeled metaphases plotted against grain counts on probability paper gave a straight line (Fig. 4). The intersection of the straight lines with the 50 per cent line represents the peak values of metaphases grain counts, i.e., the mode of the distribution of grain counts [lo]. The distribution of grain counts did no significantly differ between phase II and phase III cells and was close to a Gaussian distribution. The peak grain counts determined for each hour after addition of 3H-TdR were plotted against time after labeling (Fig. 5). For phase II cultures the S period, i.e., the interval between the first occurrence of detectable label and a plateau, was approximately 6 hr (Fig. 5). A similar plot for phase III cultures gave an S period of about 8 hr (Fig. 5). Experimental
Cell Research 42
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A. Mncieira-Coelho,
J. Pontdn
and I,. Philipson
(c) The G2 period.-Its length was determined as the time interval between addition of label and the point when 50 per cent of the metaphases were labeled. This interval was 4 hr in phase II and phase III cells. However, in the latter (Fig. 3) it takes longer for the percentage labeled metaphases curve to reach a 100 per cent level. 100 90 SO ; 70 2 e t
60 50 40 30 20 10 1
3
5
7
9
11 13 15 Hours after labeling
17
19
21
23
Fig. S.-Distribution of labeled metapbases ( x - x ) and interphases hr after a pulse label with 8H-TdR, of a culture in the 42nd passage.
25
27
29
( 0 - - - 0 ) at different
99.99 El W.5 99 9s 95 ?O z B" 10 50 io Lo IO
i; 0 g $ ;
10
;
IO 5 2 1 0.5 0.2 0.1 0.05 0.01 25
50 75 100 125 Number of grains
150
Fig. I.-Cumulative percentage of labeled metaphases sample of phase II cells, after continuous labeling.
Experimental Cell Research 42
plotted
against grain counts for the 8 hr
Nucleic acid synthesis in human fibroblasts
679
In the continuous labeling experiment (Fig. 6) it can also be seen that many cells in late passage have a very long G2 period. While in early passage all metaphases are labeled from the 5th hr on, in late passage 100 per cent labeled metaphases are found only in the 12 hr sample. In the 13 and 14 hr samples unlabeled metaphases were again observed. Since these cells did not take up thymidine, they must have been for at least 13 and 14 hr in the post-DNA-synthesis period (G2). (d) Mitotic period.-The mitotic index of phase II cells in exponential growth was 3.5 per cent which corresponds to a division time of 50 min as calculated from the formula tm =MT/0.693 given by Stanners and Till [lo] for an exponentially growing culture. In the formula tm is the time spent in mitosis,
Hourr
Fig. 5.-Distribution of the peak metaphase with SH-TdR, of 20th ( 0 - 0 ) and 46th ( x -
Hours
after
labeling
grain counts at each hr after continuous x ) passage cells.
after
labeling
labeling
Fig. 6.-Distribution of the percentage labeled metaphases at each hr after continuous with ‘H-TdR, of 20th ( 0 - 0 ) and 46th ( x - x ) passage cells. Experimental
labeling
Cell Research 42
A. Macieira-Coelho,
680
J. Pontin
and L. Philipson
M X 100 is the mitotic index and T is the generation time. As the average generation time of phase III cells could not be determined it was also impossible to calculate accurately their mitotic period, but a minimal value of 30 min can be estimated assuming 21 hr for the generation time and the observed mitotic index of 1.6 per cent.
Fig. 7.-Specific incorporation into RNA after a pulse label with %H-UdR, in the cytoplasm ( 0 - -- 0 ) and nucleus ( 0 - 0 ) of 18th passage cells and in the cytoplasm ( o - - -0 ) and nucleus ( O-O ) of 46th passage cells.
(e) Gl period.-The duration of S +G2 + tm subtracted from the generation time gives the duration of the Gl period. It would thus have a length of 6 hr for phase II cells. Only a minimal of about 8 hr can be estimated in phase III cells for the same reasons mentioned above. The curve representing percentage of labeled interphases after a pulse label in phase II cells (Fig. 2) followed the pattern described by Taylor [la] for hamster cells. It decreased during the first hr due to division of nonlabeled cells; afterwards the curve rose because of division of labeled cells, to go down again to low values after a complete growth cycle. This variation in the percentage of labeled interphases is not so evident in phase III cells (Fig. 3) because less cells take up label during a short pulse as compared with phase II cells. In fact 15 per cent and 62 per cent labeled interphases are found after a 15 min exposure to 3H-TdR in the respective cultures. RNA synthesis The incoporation of a labeled precursor into RNA was studied in two different cultures by pulse labeling at the 18th and 46th passage. Growth Experimental
Cell Research 42
Nucleic acid synthesis in human fibroblasts
681
curves had shown that both had entered the post-logarithmic resting period period at the time when labeled uridine was added. Fig. 7 demonstrates that total RNA synthesis was considerably more active in phase II than phase III cells as reflected by the higher grain counts. Specific uridine incorporation in the cytoplasm was significantly higher in early than late passage cells between l-24 hr after the pulse label. Early as well as late passage cells showed an early peak of nuclear uptake which was higher in the former type of culture. In the 8 and 24 hr samples no difference was found between nuclei of phase II and phase III cells. Fig. 8 represents the distribution of
boric,,, 25
25
50
75- loo
so 75 Numberof
125
150
loo 125 cells
150
Fig. S.-Distribution of grain counts in cytoplasm and (b) 46th passage cells.
,;.
25
(x-
50 75 Numberof
100 125 cells
x ) and nucleus ( 0 - - - 0 ) of (a) 18th
Experimental
Cell Research 42
682
A. Macieira-Coelho,
J. Ponttk
and I,. Philipson
grain counts over 50 labeled nuclei and cytoplasm at different hours after labeling. It is seen that the distributions are similar in early and late passages and that all phase Ill cells show incorporation into their RNA.
DISCUSSION
The autoradiographic analysis showed profound differences in the nucleic acid metabolism of phase 11 cells and cells in phase Ill approaching the end of their finite life span in vitro. The experiments for the timing of the division cycle show a great increase in the heterogeneity of the late passage populations. A comparison between the percentage of labeled interphases shows that an initial figure of 62 per cent was obtained for early passage cultures (Fig. 2) as compared to 15 per cent for late passage cells (Fig. 3). This shows that only a small proportion of phase Ill cells are synthesizing DNA during a short period. The generation time is altered in phase Ill cells. Their average generation time could not be determined because the second wave of labeled metaphases did not reach the 50 per cent level (Fig. 3). A minimum value of 21 hr could, however, be determined but the main population is further delayed. The average and the minimum generation of phase 11 cells was 17 and 16 hr, respectively. The duration of DNA synthesis (S period) was 6 hr in phase 11 cells and 8 hr in phase Ill cells. It can be seen that the three methods used in general in the literature for determination of the S period [3, 4] may be inexact when compared with the more accurate method of Stanners and Till [IO]. The disadvantage is that they are very sensitive to any synchronization within the culture [lo]. If the length of the S period in phase II cells would be calculated from the proportion of the labeled interphases after a short period of labeling or from the distance between the 50 per cent points in the first wave of labeled metaphases, a value of 10 hr would be obtained; however, if it would be measured as the time when 90-100 per cent metaphases are labeled a value of 7 hr would be obtained. The average S period of phase Ill cells would be 11 hr if measured as the difference between the 50 per cent points and 4 hr if measured as the time during which 90-100 per cent metaphases are labeled. The average G2 period (4 hr) was the same in phase 11 and phase Ill cells. However, in late passage there was a fraction of cells with a prolonged G2. This can be seen after a pulse label (Fig. 3) when the time required for the percentage labeled metaphases to reach a plateau was prolonged; it can Experimental
Cell Research 42
hkcleic
acid synthesis in human fibroblasts
683
also be seen in the presence of a continuous label since unlabeled metaphases were still present 13 hr after the addition of label. The mitotic period in phase 11 cells was estimated to 50 min but this period could not be accurately determined in phase Ill cells. A minimal value of 30 min was, however, obtained and since most human cells have mitotic periods lasting for less than 1 hr [l] and the mitotic index was found to be lower, it appears unlikely that this period should be extensively prolonged in phase Ill cells. Thus the major differences between phase 11 and phase Ill cells, which may possibly account for the prolonged generation time of the latter cells, reside in the G2 and Gl periods. It is possible that the fraction of cells with a prolonged G2 demonstrated above, could explain the delay in the average generation time, but a delay in the Gl period should also be considered. The pattern of RNA synthesis in nucleus and cytoplasm was found to be similar (Fig. 7) although a slower rate of RNA synthesis was observed in phase Ill cells. Of the three possibilities outlined in the introduction to explain the retarded growth in phase Ill, the data presented favour alternative (c). Less cells are capable of entering division at any given time and those that initiate their cycle are heterogeneous and appear to have prolonged Gl and G2 periods. The emergence of cells with a different growth cycle may be due to damage of the whole population during the life in vitro or to selection of certain types of cells with different growth patterns. This may well explain the long lag phase and the slow growth seen in a mass culture of phase Ill cells. Those features cannot, however, explain why phase Ill cultures stop growing at a cell density much lower than that of phase 11 cells. The postlogarithmic resting phase is generally thought to be an effect of mutual cell contacts inhibiting cell duplication. If this applies also to phase Ill cells, these cells must either be more sensitive to cell crowding or exert a stronger inhibiting influence on neighboring cells.
SUMMARY
The division cycle and RNA synthesis were analysed in early and late passage cells of a human fibroblastic strain (WI-38). Cells were exposed to tritium labeled thymidine or uridine. Cultures after the 40th passage were found to be very heterogeneous regarding the length of the division cycle. The fraction of cells involved in DNA synthesis after a short pulse was smaller and the generation time was prolonged in late passage cells. Evidence Experimental
Cell Research 42
684
A. Macieira-Coelho,
J. PontPn and II. Philipson
is presented that the cause for a prolonged generation time was mainy due to retardation in the Gl and G2 periods. The rate of synthesis of RNA was found to be decreased in late passage cells. However, the pattern of incorporation of labeled precursor was the same in both types of cells and active RNA synthesis occurred in 100 per cent of the phase III cells. The authors are thankful for valuable help and advice from Drs C. M. Lundmark and B. Santesson. REFERENCES 1. ALTMAN, L. P. and DITTMER, D. S. (eds.), in Growth, p. 137, Fed. Am. Sot. Washington, 1962. 2. ATKINS, L., GUSTAVSON, K.-H. and HANSSON, 0.. Cytooenetics 2, 208 (1960). - ” 3. BASER~A, fi., Cancer R&. 25, 581 (1965). . 4. DEFENDI, V. and MANSON, L. A., Nature 198, 359 (1963). 5. DONIACH, I. and PELC, S. R., Brit. .7. Radiation 23, 184 (1950). 6. EAGLE, H., J. Exptl Med. 102, 595 (1955). 7. HAYFLICK,. L., Eipptl Cell Res..37, 6i4 (1465). 8. HAYFLICK, L. and MOORHEAD, P. S., ibid. 25, 585 (1961). 9. MADOFF, S., Ann. N.Y. Acad. SC. 79, 383 (1960). 10. STANNERS, C. P. and TILL, J. E., Biochim. Biophys. Acta 37, 406 (1960). 11. SWIM, H. E. and PARKER, R. T., Am. J. Hyg. 66, 235 (1957). 12. TAYLOR, J. H., J. Biophys. Biochem. Cytol. 7, 455 (1960).
Experimenfal
Cell Research 42
Exptl
Biol.,
673
Cell Research 42, 673-684 (1966)
THE DIVISION CYCLE AND RNA-SYNTHESIS IN DIPLOID HUMAN CELLS AT DIFFERENT PASSAGE LEVELS IN VITRO1 A. MACIEIRA-COELHO,B Department
J. PONTGN
and L. PHILIPSON
of Cell Biology, Institute of Medical Microbiology, University of Uppsala, Uppsala, Sweden Received January 3, 1966
TISSUEculture
methods permit growth of fibroblast-like somatic cells from normal organs of many different animal species. The explanted cells will multiply rapidly for several generations (phase II) but will then enter a stage of declining growth (phase III) terminating in cell death. Only in some instances will the cells acquire a capacity for indefinite proliferation. Embryonic lung fibroblasts of human origin have been most extensively studied. They remain in phase II during about 40 subcultivations performed at a split ratio of 1:2. Phase III will last about another 10 subcultivations after which complete lysis takes place. Human lung cells have never spontaneously acquired capacity for indefinite growth in vitro [7, 8, 111. The basis for this limitation in proliferative capacity is unknown but does not seem to be related to any lack of essential metabolites in the medium or infection by latent viruses or mycoplasmas [7, 81. It has been suggested that it reflects an intrinsic cell property connected with the aging process [7, 81. The retarded growth of mass cultures of phase III cells could be due to any of the following possibilities: (a) only a small fraction of the cell population in phase III cultures is dividing at an unaltered rate but the majority of the cells are unable to divide, (b) the entire population is uniformly growing slowly possibly because of selective interference with one of the stages in the division cycle, (c) the cell population has become strongly heterogeneous and the cells show a spectrum between the two extremes, i.e., complete inhibition and normal division cycle. The present study aims to 1 This work was supported by grants from the Damon Runyon Memorial Funds, The Swedish Cancer Society and The Swedish 8 Fellow of the Calouste Gulbenkian Foundation.
and the Jane Coffin Childs Medical Research Council.
Experimental
Cell Research 42
674
,4. Macieira-Coelho,
.I. Pontt!n
and I>. Philipson
differentiate between these possibilities by comparing human phase II and phase III cells with respect to cell growth curves, the division cycle and ribonucleic acid (RNA) metabolism studied by autoradiography.
MATERIAL
AND
METHODS
Tissue culture.-The standard human fibroblast strain WI-38 of fetal lung origin was used [7]. Cells were grown in Eagle’s basal medium [6] supplemented with twice the concentration of vitamins and amino acids, 10 per cent calf serum, streptomycin (50 yg/ml) and penicillin (100 U/ml). Sufficient NaHCO, was added to obtain a pH of 7.4. Subcultivation by trypsinization was done as described by Hayflick and Moorhead [8]. The cells were seeded in 60 mm plastic Petri dishes containing 24 x 12 mm coverslips and incubated in a 5 per cent CO, humidified atmosphere at 37°C. Cell numbers were calculated from hemocytometer counts of cells suspended in 0.25 per cent trypsin in phosphate-buffered saline (PBS). Cultures were tested at different times for mycoplasma contamination by streaking cells and supernatant on PPLO agar [9]. Autoradiography.-The technique used for autoradiography was described by Doniach and Pelt [5]. Deoxyribonucleic acid (DNA) synthesis.-Two methods were used for the analysis of the division cycle: with pulse label and continuous label. Pulse label.-Exponentially growing cultures were exposed to 1 &/ml tritium labeled thymidine (3H-TdR) for 15 min. 3H-TdR, as supplied by Schwartz Laboratories, had a specific activity of 1.9 C/mM. After the labeling period the cells were washed twice with culture medium and fresh medium was added supplemented with 10 ,ug/ml (100 x the concentration of 3H-TdR) of non-labeled thymidine (TdR). This was considered zero hr. Duplicate coverslips were removed at different intervals thereafter, washed in PBS and fixed in acetic acid-methanol (1: 3) for 1 hr and dried. The coverslips were glued to a microscopic glass slide by Canada Balsam, dried, dipped in a gelatine-chrome alum solution [2] and coated with film (Kodak ARIO). After a 4 day exposure the preparations were developed for 4 min in Kodak D-19B, kept for 10 min in Kodak Acid Fixer and washed for 10 min in running tap water. Staining was done immediately afterwards in a 0.05 per cent (w/v) solution of toluidine blue for 3-5 min. Film adhering to the back of the slide was scraped off and after drying the preparations were ready for analysis. Continuous label.-This procedure was done as described by Stanners and Till [IO]. 3H-Tdr in a concentration of 0.01 &/ml was added to exponentially growing cultures. Duplicate samples were removed at hourly intervals thereafter and treated as already described. RNA synthesis.-Monolayers in the post-logarithmic resting phase were labeled with 1 &/ml of tritiated uridine (3H-UdR) in culture medium for half an hr. 3HUdR as supplied by Schwartz Laboratories had a specific activity of 2 C/mM. The cultures were then washed twice with culture medium and fresh medium supplemented with IO pg/ml (100 x the concentration of 8H-UdR) of non-labeled urldine (UdR) was added. Duplicate coverslips were removed at zero hr (time of removal of Experimental
Cell Research 42
Nucleic acid synthesis in human fibroblasts
675
radioactivity) and at different intervals thereafter. Samples were washed twice with PBS and fixed for 1 hr in acetic acid-methanol. The coverslips were then broken in half. One fragment was treated with IO pg/ml ribonuclease (RNAase) (Worthington, N.J., U.S.A.) in PBS for 1 hr at 37X, washed twice with PBS and fixed again for 1 hr. The other fragment was left untreated. Both were affixed with Canada Balsam to a microscopic glass slide. The preparations were then treated as described above under DNA synthesis except for an exposure time of 7 rather than 4 days. Analysis of autoradiographs.-The symbols used for the analysis of the division cycle are defined as follows, in agreement with previous literature [3]. Gi = time between end of mitosis and start of DNA synthesis; S = time of DNA synthesis; G2 =time between end of DNA synthesis and beginning of mitosis; tm =mitotic period = time between chromosome condensation of early prophase to separation of two daughter cells in late telophase; generation time = Gl + S + G2 + tm; interphase = Gl + S + G2. A hundred metaphases were counted to determine the percentage of labeled metaphses. One thousand nuclei were observed in the determination of the proportion of labeled interphase nuclei. Samples of 3000 cells were used to determine the mitotic index. Grains were counted over 100 labeled metaphases to determine the peak metaphase grain count. RNA incorporation was measured by counting the grains above the nucleus and in the cytoplasm in 50 cells on both coverslip fragments. The difference between the counts obtained in non-treated and RNAase-treated preparations was considered the specific incorporation into RNA.
RESULTS Cell growth Fig. 1 shows the growth curves of phase II and III cells. Cells in phase II showed a lag phase of about 1 day, a logarithmic phase of growth lasting 2-3 days superseded by a post-logarithmic resting phase during which no increase in cell number took place. The doubling time during logarithmic growth was approximately 24 hr. The total increase in cell yield was 350 per cent. Phase III cells differed from phase II cells by having a longer lag phase (1.5 days) and particularly by a short period of only 1 day of slow logarithmic growth. From the slope of the curve a theoretical cell doubling time of about 3 days can be calculated; under the actual conditions, however, no doubling of the input cell number took place, the final cell counts during the resting phase showed a net increase of only about 40 per cent. DNA synthesis The division cycle of cells in early and late passage was studied after a labeling with 3H-TdR applied during the exponential growth phase (Fig. 1). Figs. 2 and 3 show the percentage of labeled metaphases and interphases at Experimental
Cell Research 42
676
A. Macieira-Coelho,
J. Ponttk
and L. Philipson
different times after pulse labeling. The pattern at the 18th passage (Fig. 2) was the same as that gators for the same system [4]. (a) The generation time.-The second wave of 2 an 3 represents cells that have gone through
1
2 Days after
3 rubcultivation
4
obtained for phase II cells described by other investilabeled metaphases of Figs. G2, mitotis, Gl, S, G2 and
5
Fig. l.-Growth curves of WI-38 cells in the 18th ( x - x ) and in 42nd passage (O--O) obtained by hemocytometer cell counting. Each point represents the average of duplicate samples. The arrows indicate the beginning and the end of the experiments done for timing the division cycle.
have entered their second mitosis after a pulse label. Cells with the shortest generation times will register as the first labeled metaphases of the second wave. In phase II this rise occurred 18 hr after labeling (Fig. 2). Since the minimal G2 period was 2 hr (see Fig. 2), the minimal generation time was about 16 hr. The average generation time estimated from the difference between the 50 per cent points on the two ascending limbs of the biphasic curve [3] was approximately 17 hr, a value that shows good correspondence with a previous estimate of 18 hr for the same cells [4]. In phase III cultures the minimal generation time was 21-22 hr (Fig. 3). The average generation time could not be appropriately determined because Experimental
Cell Research 42
Nucleic acid synthesis in human fibroblasts
677
the second wave did no reach the 50 per cent level. It must, however, have lasted more than 25 hr. (b) The S period.-For an asynchronous population the method of Stanners and Till [lo] gives the most accurate estimate. The time interval between the first appearance of labeled metaphases and the point when the number of
5
7
9
11 13 15 Hourr alter labeling
17
19
11 22
Fig. 2.-Distribution of labeled metaphases ( x - x ) and interphases hr after a pulse label with 8H-TdR, of a culture in the 18th passage.
(0 - - - 0) at different
grains over metaphases reaches a plateau during continuous labeling corresponds to the length of the S period. In addition the method also gives information on the time course of DNA synthesis. For the determination of the number of grains over metaphases, the grain counts of 100 labeled metaphases were plotted as histograms. The cumulative percentage of such labeled metaphases plotted against grain counts on probability paper gave a straight line (Fig. 4). The intersection of the straight lines with the 50 per cent line represents the peak values of metaphases grain counts, i.e., the mode of the distribution of grain counts [lo]. The distribution of grain counts did no significantly differ between phase II and phase III cells and was close to a Gaussian distribution. The peak grain counts determined for each hour after addition of 3H-TdR were plotted against time after labeling (Fig. 5). For phase II cultures the S period, i.e., the interval between the first occurrence of detectable label and a plateau, was approximately 6 hr (Fig. 5). A similar plot for phase III cultures gave an S period of about 8 hr (Fig. 5). Experimental
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(c) The G2 period.-Its length was determined as the time interval between addition of label and the point when 50 per cent of the metaphases were labeled. This interval was 4 hr in phase II and phase III cells. However, in the latter (Fig. 3) it takes longer for the percentage labeled metaphases curve to reach a 100 per cent level. 100 90 SO ; 70 2 e t
60 50 40 30 20 10 1
3
5
7
9
11 13 15 Hours after labeling
17
19
21
23
Fig. S.-Distribution of labeled metapbases ( x - x ) and interphases hr after a pulse label with 8H-TdR, of a culture in the 42nd passage.
25
27
29
( 0 - - - 0 ) at different
99.99 El W.5 99 9s 95 ?O z B" 10 50 io Lo IO
i; 0 g $ ;
10
;
IO 5 2 1 0.5 0.2 0.1 0.05 0.01 25
50 75 100 125 Number of grains
150
Fig. I.-Cumulative percentage of labeled metaphases sample of phase II cells, after continuous labeling.
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plotted
against grain counts for the 8 hr
Nucleic acid synthesis in human fibroblasts
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In the continuous labeling experiment (Fig. 6) it can also be seen that many cells in late passage have a very long G2 period. While in early passage all metaphases are labeled from the 5th hr on, in late passage 100 per cent labeled metaphases are found only in the 12 hr sample. In the 13 and 14 hr samples unlabeled metaphases were again observed. Since these cells did not take up thymidine, they must have been for at least 13 and 14 hr in the post-DNA-synthesis period (G2). (d) Mitotic period.-The mitotic index of phase II cells in exponential growth was 3.5 per cent which corresponds to a division time of 50 min as calculated from the formula tm =MT/0.693 given by Stanners and Till [lo] for an exponentially growing culture. In the formula tm is the time spent in mitosis,
Hourr
Fig. 5.-Distribution of the peak metaphase with SH-TdR, of 20th ( 0 - 0 ) and 46th ( x -
Hours
after
labeling
grain counts at each hr after continuous x ) passage cells.
after
labeling
labeling
Fig. 6.-Distribution of the percentage labeled metaphases at each hr after continuous with ‘H-TdR, of 20th ( 0 - 0 ) and 46th ( x - x ) passage cells. Experimental
labeling
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M X 100 is the mitotic index and T is the generation time. As the average generation time of phase III cells could not be determined it was also impossible to calculate accurately their mitotic period, but a minimal value of 30 min can be estimated assuming 21 hr for the generation time and the observed mitotic index of 1.6 per cent.
Fig. 7.-Specific incorporation into RNA after a pulse label with %H-UdR, in the cytoplasm ( 0 - -- 0 ) and nucleus ( 0 - 0 ) of 18th passage cells and in the cytoplasm ( o - - -0 ) and nucleus ( O-O ) of 46th passage cells.
(e) Gl period.-The duration of S +G2 + tm subtracted from the generation time gives the duration of the Gl period. It would thus have a length of 6 hr for phase II cells. Only a minimal of about 8 hr can be estimated in phase III cells for the same reasons mentioned above. The curve representing percentage of labeled interphases after a pulse label in phase II cells (Fig. 2) followed the pattern described by Taylor [la] for hamster cells. It decreased during the first hr due to division of nonlabeled cells; afterwards the curve rose because of division of labeled cells, to go down again to low values after a complete growth cycle. This variation in the percentage of labeled interphases is not so evident in phase III cells (Fig. 3) because less cells take up label during a short pulse as compared with phase II cells. In fact 15 per cent and 62 per cent labeled interphases are found after a 15 min exposure to 3H-TdR in the respective cultures. RNA synthesis The incoporation of a labeled precursor into RNA was studied in two different cultures by pulse labeling at the 18th and 46th passage. Growth Experimental
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curves had shown that both had entered the post-logarithmic resting period period at the time when labeled uridine was added. Fig. 7 demonstrates that total RNA synthesis was considerably more active in phase II than phase III cells as reflected by the higher grain counts. Specific uridine incorporation in the cytoplasm was significantly higher in early than late passage cells between l-24 hr after the pulse label. Early as well as late passage cells showed an early peak of nuclear uptake which was higher in the former type of culture. In the 8 and 24 hr samples no difference was found between nuclei of phase II and phase III cells. Fig. 8 represents the distribution of
boric,,, 25
25
50
75- loo
so 75 Numberof
125
150
loo 125 cells
150
Fig. S.-Distribution of grain counts in cytoplasm and (b) 46th passage cells.
,;.
25
(x-
50 75 Numberof
100 125 cells
x ) and nucleus ( 0 - - - 0 ) of (a) 18th
Experimental
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grain counts over 50 labeled nuclei and cytoplasm at different hours after labeling. It is seen that the distributions are similar in early and late passages and that all phase Ill cells show incorporation into their RNA.
DISCUSSION
The autoradiographic analysis showed profound differences in the nucleic acid metabolism of phase 11 cells and cells in phase Ill approaching the end of their finite life span in vitro. The experiments for the timing of the division cycle show a great increase in the heterogeneity of the late passage populations. A comparison between the percentage of labeled interphases shows that an initial figure of 62 per cent was obtained for early passage cultures (Fig. 2) as compared to 15 per cent for late passage cells (Fig. 3). This shows that only a small proportion of phase Ill cells are synthesizing DNA during a short period. The generation time is altered in phase Ill cells. Their average generation time could not be determined because the second wave of labeled metaphases did not reach the 50 per cent level (Fig. 3). A minimum value of 21 hr could, however, be determined but the main population is further delayed. The average and the minimum generation of phase 11 cells was 17 and 16 hr, respectively. The duration of DNA synthesis (S period) was 6 hr in phase 11 cells and 8 hr in phase Ill cells. It can be seen that the three methods used in general in the literature for determination of the S period [3, 4] may be inexact when compared with the more accurate method of Stanners and Till [IO]. The disadvantage is that they are very sensitive to any synchronization within the culture [lo]. If the length of the S period in phase II cells would be calculated from the proportion of the labeled interphases after a short period of labeling or from the distance between the 50 per cent points in the first wave of labeled metaphases, a value of 10 hr would be obtained; however, if it would be measured as the time when 90-100 per cent metaphases are labeled a value of 7 hr would be obtained. The average S period of phase Ill cells would be 11 hr if measured as the difference between the 50 per cent points and 4 hr if measured as the time during which 90-100 per cent metaphases are labeled. The average G2 period (4 hr) was the same in phase 11 and phase Ill cells. However, in late passage there was a fraction of cells with a prolonged G2. This can be seen after a pulse label (Fig. 3) when the time required for the percentage labeled metaphases to reach a plateau was prolonged; it can Experimental
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acid synthesis in human fibroblasts
683
also be seen in the presence of a continuous label since unlabeled metaphases were still present 13 hr after the addition of label. The mitotic period in phase 11 cells was estimated to 50 min but this period could not be accurately determined in phase Ill cells. A minimal value of 30 min was, however, obtained and since most human cells have mitotic periods lasting for less than 1 hr [l] and the mitotic index was found to be lower, it appears unlikely that this period should be extensively prolonged in phase Ill cells. Thus the major differences between phase 11 and phase Ill cells, which may possibly account for the prolonged generation time of the latter cells, reside in the G2 and Gl periods. It is possible that the fraction of cells with a prolonged G2 demonstrated above, could explain the delay in the average generation time, but a delay in the Gl period should also be considered. The pattern of RNA synthesis in nucleus and cytoplasm was found to be similar (Fig. 7) although a slower rate of RNA synthesis was observed in phase Ill cells. Of the three possibilities outlined in the introduction to explain the retarded growth in phase Ill, the data presented favour alternative (c). Less cells are capable of entering division at any given time and those that initiate their cycle are heterogeneous and appear to have prolonged Gl and G2 periods. The emergence of cells with a different growth cycle may be due to damage of the whole population during the life in vitro or to selection of certain types of cells with different growth patterns. This may well explain the long lag phase and the slow growth seen in a mass culture of phase Ill cells. Those features cannot, however, explain why phase Ill cultures stop growing at a cell density much lower than that of phase 11 cells. The postlogarithmic resting phase is generally thought to be an effect of mutual cell contacts inhibiting cell duplication. If this applies also to phase Ill cells, these cells must either be more sensitive to cell crowding or exert a stronger inhibiting influence on neighboring cells.
SUMMARY
The division cycle and RNA synthesis were analysed in early and late passage cells of a human fibroblastic strain (WI-38). Cells were exposed to tritium labeled thymidine or uridine. Cultures after the 40th passage were found to be very heterogeneous regarding the length of the division cycle. The fraction of cells involved in DNA synthesis after a short pulse was smaller and the generation time was prolonged in late passage cells. Evidence Experimental
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is presented that the cause for a prolonged generation time was mainy due to retardation in the Gl and G2 periods. The rate of synthesis of RNA was found to be decreased in late passage cells. However, the pattern of incorporation of labeled precursor was the same in both types of cells and active RNA synthesis occurred in 100 per cent of the phase III cells. The authors are thankful for valuable help and advice from Drs C. M. Lundmark and B. Santesson. REFERENCES 1. ALTMAN, L. P. and DITTMER, D. S. (eds.), in Growth, p. 137, Fed. Am. Sot. Washington, 1962. 2. ATKINS, L., GUSTAVSON, K.-H. and HANSSON, 0.. Cytooenetics 2, 208 (1960). - ” 3. BASER~A, fi., Cancer R&. 25, 581 (1965). . 4. DEFENDI, V. and MANSON, L. A., Nature 198, 359 (1963). 5. DONIACH, I. and PELC, S. R., Brit. .7. Radiation 23, 184 (1950). 6. EAGLE, H., J. Exptl Med. 102, 595 (1955). 7. HAYFLICK,. L., Eipptl Cell Res..37, 6i4 (1465). 8. HAYFLICK, L. and MOORHEAD, P. S., ibid. 25, 585 (1961). 9. MADOFF, S., Ann. N.Y. Acad. SC. 79, 383 (1960). 10. STANNERS, C. P. and TILL, J. E., Biochim. Biophys. Acta 37, 406 (1960). 11. SWIM, H. E. and PARKER, R. T., Am. J. Hyg. 66, 235 (1957). 12. TAYLOR, J. H., J. Biophys. Biochem. Cytol. 7, 455 (1960).
Experimenfal
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Biol.,