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RNA synthesis in cultured chick embryo fibroblasts
Experimental
RNA SYNTHESIS EMBRYO
Cell Research 47, 554-.5G3 (19GY)
IN CULTURED FIBROBLASTS
CHICK
Y. BECKER Department
of Virology,
Hebrew University-Hadassah Jerusalem, Israel
Received
January
Medical
School,
3, 1967
CONTACT inhibition
was defined by Abercrombie [l, 21 as inhibition of the movement of cultured primary chick embryo tibroblasts (CEF) due to cellular surface interactions. Later studies indicated that cultured diploid cells which have a restricted life span in vitro [13] also show a decline in the rate of mitotic activity after monolayer formation [ll]. It was also found that the ability of diploid human hbroblasts to synthesize DNA, RNA and proteins gradually decreases at the stage of monolayer formation. The decline in the rate of protein synthesis was correlated with the disappearance of free polyribosomes [S, 9, 161. Differences between growing and monolayer cells in regard to macromolecular synthesis were thus documented. Nevertheless, the significance of cellular contacts in the formation of monolayers is still obscure. Moreover, the importance of the culture medium for the propagation and maintenance of monolayer cells was not resolved. The decline in the metabolic activity of diploid cells might be explained partly by the depletion from the medium of essential nutrients utilized by the growing cells, although it may also be partly due to internal cellular factors. The present communication deals with the RNA synthesis in chick embryo tibroblasts during their growth in vitro and after monolayer formation. It will be shown that: (a) the ability of chick embryo tibroblasts to synthesize the different RNA species declines after monolayer formation, and only ribosomal RNA is synthesized at a low rate. (h) After addition of fresh growth medium to the confluent monolayers, RNA synthesis markedly increases but again only ribosomal RNA is synthesized and active cellular proliferation does not result. MATERIALS
AND
METHODS
Primary chick embryo fibroblasts.-Ten-day-old chick embryos were minced and trypsinized with several changes of 0.25 per cent trypsin Earle’s buffer. The cell suspensions were filtered through sterile gauze, and resuspended in M 199 medium supplemented with 10 per cent The cells were dispensed into milk dilution and Roux bottles, 10’ and per bottle, respectively. Fifty per cent of the cells seeded attach to Experimental
Cell Research 47
decapitated, solution in centrifuged calf serum. 5 x lo* cells the culture
RNA synthesis
in chick
embryo
w-v 333
fibroblnsts
bottles, enlarge and form monolayer sheets within 24 to 48 hr. The cells were seeded at a high concentration to obtain rapid formation of monolayers [3]. Medium change.-At different time intervals after seeding the cells, the medium was removed and fresh M 199 or Eagle’s medium [7] supplemented with 5 per cent calf serum was added to the cultures. Radioactive isotope.-Each culture in a Roux or milk bottle was labeled with 1.5 jlc or 0.3 plc %-2-uridine, respectively (specific activity, 30 mc/mmole obtained from New England Nuclear Corp., U.S.A.). Analysis of cellular componenfs.-At the end of the labeling period, the CEF were removed from the culture vessels with 0.25 per cent trypsin solution, washed with phosphate buffered saline (PBS) and resuspended in RSB (10m2 n/I KCl, iOm2 &f Tris, pH 7.4, 1.5 x lO-3 M MgZ+ [24]). After 45 min at 4”C, the cells were disrupted in a Dounce glass homogenizer. The nuclei were removed by centrifugation at 1000 rpm for 2 min and the cytoplasmic fraction obtained was analyzed by zone centrifugation in RSB-sucrose gradients (15-30 per cent w/w) at 38,000 rpm for 75 min at 5°C in the SW39 rotor of a Beckman Model L-2 preparative ultracentrifuge. The gradients were fractionated through a Gilford flow cell and the optical density (O.D. mpZ,,) continuously recorded. Samples of whole cells or cytoplasmic fractions were treated with sodium dodecyl sulfate (SDS) buffer (0.01 M Tris, pH 7.4, 0.1 M NaCl, 0.5 per cent SDS [lo]) and the RNA analyzed by zone centrifugation in SDS sucrose gradients (15-30 per cent w/w made in SDS buffer) for 3.5 hr at 38,000 rpm at 25°C. The gradients were collected through a Gilford flow cell and the optical density continuously recorded. Each fraction was precipitated with cold 15 per cent trichloracetic acid (TCA), collected onto Millipore filters, dried and counted in a Nuclear Chicago low background gas flow counter with an end window detector. RESULTS
RXA species synthesized
by growing
and
monolayer
chick
embryo
fibroblasts
Primary chick embryo fibroblasts, in uifro, attach to the glass surface of the culture vessels and start to grow until monolapers are formed within 20 hr. It was thought that labeling the cells with radioactive uridine for the entire growth period would enable the RNA species which accumulate in the growing cells to be studied, in spite of the possibility that short lived RNA molecules would not be detected. For this purpose the growing fibroblasts were incubated in the presence of radioactive uridine for intervals of 15 hr (Table I, Experiment 1) or 40 hr (Table I, Experiment 2). A labeling period of 40 hr was included to determine whether the relationship between the difTerent RNA species synthesized by the growing cells is relatively stable over a long period of time. At the end of the labeling periods, the radioactive RNA was isolated by centrifugation in SDS-sucrose gradients. Table I summarizes the relative amounts of radioactivity found in the different RN,1 species after extraction from whole cells and from the cytoplasm of both growing and monolayer cultures. Experimental
Cell Research 47
Y. Becker
556
It may be seen in Table I (Exp. 1) into the different RNA species during after seeding; sample A). A higher cells were labeled for an equivalent been formed (81-96 hr; sample B). TABLE I. Nature
Sample
Labeling period (hr after seeding)
A
5-20
B
Exp. NO.
1
2
of RNA
that radioactive uridine is incorporated the initial period of growth (5-20 hr rate of RN,4 synthesis occurred when period after monolayers had already RNA synthesis declined in cells which
species synthesized
by chick embryo fibroblusts. Per cent CPM in RNA
Cell fractions
species
CPM/culture
28 S
16 S
10 s
4s
Total cells Cytoplasm
36,200 8,200
38.05 24.91
25.70 27.21
18.97 33.98
7.50 11.03
81-96
Total cells Cytoplasm
80,000 36,740
39.29 29.55
27.59 13.06
7.52 9.86
18.09 44.63
c
145-160
Total cells Cytoplasm
47,200 20,970
30.47 31.16
39.03 32.20
9.26 15.11
9.07 21.53
A
5545
Total cells Cytoplasm
158,760 56,700
36.86 26.94
21.96 21.95
20.77 38.67
19.71 11.26
B
120&160
Total cells Cytoplasm
77,400 11,000
31.02 53.11
35.13 35.08
16.84 9.94
10.35 2.0
Chick embryo fibroblasts in Roux bottles were labeled for 15 (Exp. 1) and 40 hr (Exp. 2) with 1 ,uC %-2-uridine per culture at different intervals after seeding. Cells were harvested and a portion (10 X) was treated with SDS-buffer. The cytoplasmic fraction obtained from the cells was treated with SDS and centrifuged into SDS-sucrose gradients (15-30 % w/w). The radioactivity in each fraction and the distribution of the different RNA species in the gradients were determined. The percentage of the radioactivity present in the different RNA species was calculated.
were maintained in vitro for 160 hr (sample C) without medium replenishment. This was also found when cultures were labeled for 40 hr intervals (Table I, Exp. 2, samples A and B and Fig. 1). The synthesis of ribosomal precursor RNA was not detected in CEF labeled for 15 to 40 hr with radioactive uridine. Experiments in which shorter labeling periods (1 to 3 hr) were used resulted in the appearance of high molecular weight RNA, 45s and 35s RNA species, which are the precursors of ribosomal RNA. However, under these conditions the distribution of radioactivity in the 28S, 16s and 10s RNA species was not clearly resolved. In order to compare the behaviour of growing cells with that of aging cells, similar labeling periods were used. Such long labeling periods enhanced the possibility of detecting RN,4 species which might be synthesized at a low rate. A change in the relative amount of the 10s RNA species in monolayer Experimental
Cell Research 47
RNA synthesis in chick embryo fibroblasts
557
cells as compared to growing cells was observed. Growing cells (Table I, Exp. 1, sample A and Exp. 2, sample A) synthesize more 10s RNA (18.97 and 20.77 per cent) than nongrowing cells (7.52 and 9.26 per cent) (Table I, Exp. 1, samples B and C). Similarly, a higher concentration of radioactive 10s RNA was found in the cytoplasm of growing cells, 33.98 and 38.67 per cent after 15 and 40 hr, respectively (Exp. 1, sample A; Exp. 2, sample A) than in monolayer cells (Exp. 1, samples B and C; Exp. 2, sample B). Similar changes regarding 4s RNA were also recorded. ,4s indicated in Table I, most of the RNA synthesized by CEF at any time during their in vitro existence is ribosomal RNA. To determine the
Fig. 1. Fig. l.-RNA species in the cytoplasm of growing chick embryo fibroblasts. Five hr after transfer of CEF to in uifro conditions, 1 /AC 14C-2-uridine was added and the cultures were further incubated for 40 hr. Cells were harvested, washed, resuspended in RSB and the cytoplasmic fraction was prepared by Dounce homogenization. SDS (final concentration 0.5 %) was immediately addedand the homogenate was analyzed in an SDS-sucrose (15-30 % w/w) gradient. The percentage of radioactivity in the different RNA species is indicated in the graph. ---, O.D.scomp; O---O, CPM.
Fig. 2.-Distribution of RNA in the cytoplasm of growing chick embryo fibroblasts. Five hr after transfer of CEF to cultural conditions, 1.5 PC lPC-2-uridine was added to each culture and the cells were incubated for an additional 15 hr period at 37°C. The cells were released from the glass by trypsinization for 2 min, washed in phosphate buffered saline (PBS), resuspended in RSB (6 x 10’ cells/ml) and disrupted with a Dounce glass homogenizer. Nuclei were removed by centrifugation for 2 min at 1000 rpm at 4°C and the cytoplasmic extract was centrifuged into a RSB-sucrose (15-30 % w/w) gradient for 75 min at 38,000 rpm (A). The pellet obtained in gradient A was recovered, dissolved in SDS buffer and analyzed in SDS-sucrose gradient (B). The figures in the graphs indicate the per cent radioactivity present in the different RNA species. ----, O.D.zso m,r; l --- l , CPM. Experimental
Cell Research 47
558
Y. Becker
localization of the labeled RNA, the cytoplasmic extract of growing CEF, labeled for 15 hr with radioactive uridine, was centrifuged in RSB sucrose gradients and the ribosomal fraction was examined (Fig. 2A). It was found that 31.07 per cent of the radioactivity was associated with the 74 S ribosomal region of the gradient, while 11.84 per cent was associated with the region
Fig. 3.-Distribution of radioactive RNA in the cytoplasm of “crowded” (monolayer) cultures. CEF in monolayer cultures were labeled with 14C-2-uridine (1.5 PC per 3 x 10’ cells) for 40 hr starting at 120 hr after seeding to in vitro conditions. At the end of the labeling period the cells were removed from the glass by brief trypsinization, washed with PBS and resuspended in RSB. After disruption of the cells in a Dounce homogenizer, the cytoplasmic fraction was obtained, half of which was analyzed in a RSB-sucrose (15-30 % w/w) gradient (A) and the rest dissolved in SDS buffer (final concentration 0.5 %), and analyzed in a SDS-sucrose gradient (B). -~-, 0. D.zso mU; o-p- l , CPM.
heavier than ribosomes and 22.75 per cent was found at the bottom of the centrifuge tube. The rest banded in a region lighter than the ribosomes. The radioactive fraction found at the bottom of the centrifuge tube was dissolved in SDS buffer and analyzed by zone centrifugation in SDS-sucrose gradients. As shown in Fig. 2 B, both 28 S and 16 S RNAspecies could beidentified by their absorbance at 260 rnp and radioactivity, indicating that newly labeled cytoplasmic ribosomes are bound to large structures, probably membranes, and thus sediment to the bottom of the centrifuge tube. It is of interest that the 10 S radioactive RNA is also found to be associated with the above-mentioned structures. The intracellular localization of RNA, synthesized during a period of 40 hr, 120 hr after seeding the cells, was similarly studied. A marked decrease in RNA synthesis was found (Fig. 3A). The distribution of this RNA in the cytoplasm resembled that of growing cells (Fig. 2 A). Further analysis (Fig. 3B) showed that mostly ribosomal RNA (28s and 16s) but very little 10s RNA is present in the cytoplasm of the monolayer cells. Experimental
Cell Research 47
RNA synthesis in chick embryo fibroblasts T.WLE II.
359
of RLVA synthesis in primary Effect of medium on the stimulation chick embryo fibroblasts.
Medium Unchanged 11 199 ivI 199 ii1 199 Eagle’s
Per cent calf serum
0 2.5 5 5
CPM in RNA 7-Exp. 1 Exp. 2 600 7000 7000 7000 2400
750 5100 5700 6000 2120
Monolayer cultures (5 days old) of chick embryo fibroblasts in milk dilution bottles (5 x lo6 cells/culture) were used. The medium was changed and 14C-2-uridine was added to each culture. Duplicate cultures were removed 6 hr later, the cells were dissolved in SDS buffer and samples were taken for the determination of radioactivity. The figures represent the average of the duplicate cultures examined.
Effect of fresh medium on R,VA synthesis of CEF
in “crowded”
(monolayer)
cultures
The experiments described in the previous section were done with CEF kept in the initial culture medium. In the present series of experiments, the ability of the cells to incorporate 14C-2-uridine into RNA was determined in aging CEF cultures which had received fresh Ml99 or Eagle’s medium, supplemented with 5 per cent calf serum, after five days. Monolayers which were left in the initial culture medium served as controls. ,4 typical experiment is presented in Fig. 4. It may be seen that the incorporation of radioactive uridine is markedly stimulated (6 to 10 fold) after the addition of fresh ,\I199 medium. The results presented in Table II demonstrate, that the stimulation of RNA synthesis in monolayer cells is not due to the addition of serum and is more marked with Ml99 medium than with Eagle’s medium. Following this finding, monolayer cells which received fresh medium were analyzed in regard to the nature and distribution of the RNA in the cell cytoplasm. The cells were labeled with radioactive uridine for 6 hr starting immediately after medium replenishment, the cytoplasmic fraction was prepared and analyzed in sucrose gradients. The results shown in Fig. 5A indicate that most of the radioactive RNA is associated \vith large cytoplasmic structures which sediment to the bottom of the sucrose gradient, with the 74 S ribosome peak and with the subribosomal units. Essentially, the distribution of radioactive RNA is similar to the distribution of RN;A in cells which did Experimental
Cell Research 47
Y. Becker
560
not receive fresh medium. Analysis of the cytoplasmic fractions with regard to the nature of the RNA species synthesized by CEF before or after medium replenishment (Fig. 5B) showed that mostly 28 S and 16 S RNA species were synthesized in the cells which were stimulated by fresh medium. EfJ‘ct of medium cultures of CEF
change on cellular
proliferation
in “crowded”
monolayer
The effect of fresh medium on the proliferation of CEF in monolayer cultures was studied by determining the number of cells in duplicate cultures with or without medium replenishment. In three identical experiments, no cell proliferation was detectable in the cultures after addition of fresh medium (Fig. 6). DISCUSSION
The present experiments were performed by labeling the newly synthesized RNA species in primary chick embryo fibroblasts during the entire growth period following their transfer from in uiuo to in vitro conditions. Essentially, only the labeled RNA molecules present at the end of the labeling periods were detected in the sucrose gradient analysis. It was found that the growing CEF synthesize 28 S, 16 S, 10 S and 4 S RNA species, similar to HeLa cells [6, 20, 211 and developing chick embryos [15]. Due to the long labeling periods used in the present experiments, ribosomal precursor RNA species were not detectable. An analysis of RNA species synthesized by cultured mouse embryo pancreas cells [25] also demonstrated the absence of ribosomal precursor RNA species when long labeling periods were used. The synthesis of ribosomal precursor RNA was therefore studied by labeling CEF with radioactive uridine for shorter time periods (1 to 3 hr). Under these conditions 45 S and 35 S ribosomal precursor RNA were detected in the growing CEF similarly to HeLa cells [‘LO]. Attention is drawn to the 10 S and 4 S RNA species which were found to be synthesized to a greater extent by growing than by monolayer cells. A 10 S RNA species was identified as messenger RNA in HeLa cells [19] and was also demonstrated in developing embryos of sea urchins [17, 181, Arbacia punctulata [la] and the South African clawed toad [4, 51. analysis of the distribution of the RNA species synthesized by CEF demonstrated the presence of 10 S RNA molecules in membrane-bound ribosomes which sediment through the sucrose gradient to the bottom of the centrifuge tube (Fig. 2s). Incorporation of amino acids in these cells occurs on such structures (ref. [22]; Becker, unpublished results). Experimental
Cell Research 47
561
R;t’A synthesis in chick embryo fibroblasts
;‘I 0
,
,
,
,
10
20
30
LO TI
Fig. 4.
, 50 ME
,
60
70
, 00
90
100
, 110 120
IHOURS)
Fig. 6.
Fig. 4.-Effect of medium change on RNA synthesis in primary chick embryo fibroblasts. Monolayer cultures (five days old) were used. One series of the cultures was left in the initial medium, and to the other one fresh medium was added after removal of the medium. Immediately, 1 ,UC i4C-2-uridine was added to all cultures and duplicates were removed at different intervals. The amount of radioactive RNA in each culture was determined. O-O, cultures with unchanged medium; O--O, cultures with fresh medium. Fig. B.-The effect of medium change on the proliferation of “crowded” (monolayer) chick embryonic fibroblasts. After monolayer formation one series of cultures received fresh medium while the other one was left in the initial medium. At different time intervals, duplicate cultures were removed from each series and the number of cells was determined microscopically. O----O, cultures incubated in the growth medium; O-O, cultures incubated in fresh medium.
(monolayer) fibroblasts. MonoFig. 5.-Nature of the RNA species synthesized in “crowded” layer cultures with or without medium replenishment were labeled with %-2-uridine. At different time intervals duplicate samples were removed and the cytoplasmic fractions were prepared and analyzed in RSB-sucrose gradients (A) and in SDS sucrose gradients (B). The amount of radioactivity in the pellet at the bottom of the centrifuge tubes and in each fraction was also CPM in CEF in unchanged medium; 0-0, CPM determined. __ , O.D.zse mp; O--O, in CEF after medium replenishment. Experimental
Cell Research 47
Y. Becker
562
After monolayer formation, CEF were left in the initial growth medium. To be able to compare RNA synthesis in these aging cells with the experiments performed on actively growing cells, long labeling periods were also used. This procedure enabled RK;A molecules which accumulated slowly in the cells to be detected. It was found that the aging cells synthesize 28 S and 16 S ribosomal RNA at a lower rate than actively growing cells. Shorter labeling periods (1 to 3 hr) enabled the detection of the ribosomal precursor RNA (45 S and 35 S) but the distribution of the radioactivity in other RNA species was not clearly resolved. It is possible that prolonged maintenance of CEF in the culture medium leads to the depletion of essential nutrients, thus causing a decline in the ability of the cells to synthesize ribosomal RNA. The addition of fresh medium to monolayers of chick embryo fibroblasts caused a marked stimulaof 14C-2tion of ribosomal RNA synthesis. In contrast, the incorporation thymidine into DNA was stimulated only slightly (Becker, unpublished results). From the results presented in Table II it is evident that the serum used for cultivation of cells was not responsible for the stimulation of RN-4 synthesis. After it had been found that mainly ribosomal RNA molecules are synthesized by aging cells after medium replenishment, the kinetics of this process were determined using shorter labeling periods (1 to 6 hr, Fig. 4). It was found that ribosomal RNA is synthesized and appears as ribosomal subunits or ribosomes in the cytoplasm of the cells. Chick embryo libroblasts differ in this respect from primary mouse diploid cells in which serum stimulates the proliferation of some cells and the synthesis of all RNA species [23,. The results of the present experiments indicate the existence of a coordinated synthesis of the two ribosomal RNA species. This supports the idea that the two ribosomal RNA species are regulated to the same extent and may have a common genetic origin as suggested by Drown and Littna [a, 5:. Independent regulation of ribosomal and transfer RNA species has also been described in bacterial cells [ 141. The inability of the cells to synthesize the 10 S and 4 S RNA species after medium replenishment, in contrast to the stimulation of ribosomal RNA synthesis, might indicate that non-ribosomal RNA synthesis is regulated by a process different from that of ribosomal RNA. This may indicate that additional or new mechanisms appear in cells at or after the formation of monolayers, which prevent the synthesis of certain RNA species. This can be overcome when monolayer cells are dispersed, reseeded to new cultures and cellular proliferation is restored. It is tempting to speculate that the very limited synthesis of nonribosomal RNA in monolayer cells restricts protein synthesis mainly to those species coded for by Experimental
Cell Research 47
RNA synthesis in chick embryo fibroblusfs
563
messenger RNA molecules synthesized during the initial growth period of the fibroblasts and still exist in the cytoplasm after monolayer formation. Cellular proliferation is thus inevitably restricted, although ribosomes continued to be synthesized by the aging monolayer cells. The nature of the regulatory mechanisms which are responsible for the inhibition of the synthesis of the nonribosomal RNA and their relationship to the phenomenon of contact inhibition in cultured cells are still unknown. SUMMARY The in uifro behaviour of primary chick embryo fibroblasts (CEF) in respect to RNA synthesis was studied. It was found that actively growing CEF synthesize all RNA species. After monolayer formation, the cells synthesize mainly ribosomal RNA, though at a lower rate than actively growing cells. After the addition of fresh medium to monolayer cultures, cellular proliferation was not detectable, but the synthesis of mainly ribosomal RXA was stimulated. This work was aided in part by grant 5 R05 TW00152-1 and 2 from the National Institutes of Health, Bethesda, Md, U.S.A. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24. 25.
ABERCROMBIE, iIf., Cold Spring EZarb. Symp. Quant. Biol. 27, 427 (1962). ABERCROMBIE, M. and HEAYSMAN, J. E. M., Exptt Cell Res. 6, 293 (1954). BECKER, Y. and CHEN, Y., Zsrael J. Med. Sci. 2, 417 (1966). BROWN, D. D. and LITTNA, E.. J. Mol. Biol. 8, 669 (1964). \ I -ibid. 8, 688 (1964). DARNELL, J. E., PENMAN, S., SCHERRER, K. and BECKER, Y., Cold Spring Harb. Symp. Quant. Biol. 28, 211 (1963). EAGLE, H., Science 130, 432 (1959). --ibid. 148, 42 (1965). -Israel J. Med. Sci. 1, 1220 (1965). GILBERT, W., J. Mol. Biol. 6, 389 (1963). GOLDE, A., Virology 16, 9 (1962). GROSS, P. R., MALKIN, L. I. and MEYER, W. A., Proc. Xatl Acad. Sci. U.S. 51. 407 (1964). HAYFLICK, L., Exptl Cell. Res. 37, 614 (1965). KJELDGAARD, N. 0. and KURLAND, C. G., J. Mol. Biol. 6. 341 (1963). LERNER, A. M., BELL, E. and DARRELL, J. E., Science 14i, 1187 (1963). LEVINR, E. M., BECKER, Y., BOONE, C. 1%‘. and EAGLE, H., Proc. Natl Acad. Sci. 1T.S. 53, 350 (1965). NEMER, M., I’roc. Nat1 Acad. Sci. U.S. 50, 230 (1963). NEMER, M. and INFANTE, A. A., Science 150, 217 (1965). PEXX~X, S., SCHERRER, K., BECKER, Y. and DARNELL, J. E., Z’roc. Nat1 Acad. Sci. U.S. 49, 654 (1963). SCHEKRER, K. and DARNELL, J. IL, Biochem. Biophys. Res. Comm. 7, 486 (1962). SCHERRER, K., LATHAM, M. and DARNELL, J. E., Proc. Natt Acad. Sci. U.S. 49, 240 (1963). SOEIRO, R. and AMOS, H., Science 154, 662 (1966). TOD~RO, G., LMAR, G. and GREEN, H., J. Cell Camp. Physiot. 66, 325 (1965). \VAHSER. J. R., KNOPF, P. and RICH, A., Z’roc. Natl Acad. Sci. U.S. 49, 122 (1963). VESSELS, N. K. and WILT, F. H., J. 1Mo2. Biot. 13, 767 (1965).
Experimental
Cell Research 47
RNA SYNTHESIS EMBRYO
Cell Research 47, 554-.5G3 (19GY)
IN CULTURED FIBROBLASTS
CHICK
Y. BECKER Department
of Virology,
Hebrew University-Hadassah Jerusalem, Israel
Received
January
Medical
School,
3, 1967
CONTACT inhibition
was defined by Abercrombie [l, 21 as inhibition of the movement of cultured primary chick embryo tibroblasts (CEF) due to cellular surface interactions. Later studies indicated that cultured diploid cells which have a restricted life span in vitro [13] also show a decline in the rate of mitotic activity after monolayer formation [ll]. It was also found that the ability of diploid human hbroblasts to synthesize DNA, RNA and proteins gradually decreases at the stage of monolayer formation. The decline in the rate of protein synthesis was correlated with the disappearance of free polyribosomes [S, 9, 161. Differences between growing and monolayer cells in regard to macromolecular synthesis were thus documented. Nevertheless, the significance of cellular contacts in the formation of monolayers is still obscure. Moreover, the importance of the culture medium for the propagation and maintenance of monolayer cells was not resolved. The decline in the metabolic activity of diploid cells might be explained partly by the depletion from the medium of essential nutrients utilized by the growing cells, although it may also be partly due to internal cellular factors. The present communication deals with the RNA synthesis in chick embryo tibroblasts during their growth in vitro and after monolayer formation. It will be shown that: (a) the ability of chick embryo tibroblasts to synthesize the different RNA species declines after monolayer formation, and only ribosomal RNA is synthesized at a low rate. (h) After addition of fresh growth medium to the confluent monolayers, RNA synthesis markedly increases but again only ribosomal RNA is synthesized and active cellular proliferation does not result. MATERIALS
AND
METHODS
Primary chick embryo fibroblasts.-Ten-day-old chick embryos were minced and trypsinized with several changes of 0.25 per cent trypsin Earle’s buffer. The cell suspensions were filtered through sterile gauze, and resuspended in M 199 medium supplemented with 10 per cent The cells were dispensed into milk dilution and Roux bottles, 10’ and per bottle, respectively. Fifty per cent of the cells seeded attach to Experimental
Cell Research 47
decapitated, solution in centrifuged calf serum. 5 x lo* cells the culture
RNA synthesis
in chick
embryo
w-v 333
fibroblnsts
bottles, enlarge and form monolayer sheets within 24 to 48 hr. The cells were seeded at a high concentration to obtain rapid formation of monolayers [3]. Medium change.-At different time intervals after seeding the cells, the medium was removed and fresh M 199 or Eagle’s medium [7] supplemented with 5 per cent calf serum was added to the cultures. Radioactive isotope.-Each culture in a Roux or milk bottle was labeled with 1.5 jlc or 0.3 plc %-2-uridine, respectively (specific activity, 30 mc/mmole obtained from New England Nuclear Corp., U.S.A.). Analysis of cellular componenfs.-At the end of the labeling period, the CEF were removed from the culture vessels with 0.25 per cent trypsin solution, washed with phosphate buffered saline (PBS) and resuspended in RSB (10m2 n/I KCl, iOm2 &f Tris, pH 7.4, 1.5 x lO-3 M MgZ+ [24]). After 45 min at 4”C, the cells were disrupted in a Dounce glass homogenizer. The nuclei were removed by centrifugation at 1000 rpm for 2 min and the cytoplasmic fraction obtained was analyzed by zone centrifugation in RSB-sucrose gradients (15-30 per cent w/w) at 38,000 rpm for 75 min at 5°C in the SW39 rotor of a Beckman Model L-2 preparative ultracentrifuge. The gradients were fractionated through a Gilford flow cell and the optical density (O.D. mpZ,,) continuously recorded. Samples of whole cells or cytoplasmic fractions were treated with sodium dodecyl sulfate (SDS) buffer (0.01 M Tris, pH 7.4, 0.1 M NaCl, 0.5 per cent SDS [lo]) and the RNA analyzed by zone centrifugation in SDS sucrose gradients (15-30 per cent w/w made in SDS buffer) for 3.5 hr at 38,000 rpm at 25°C. The gradients were collected through a Gilford flow cell and the optical density continuously recorded. Each fraction was precipitated with cold 15 per cent trichloracetic acid (TCA), collected onto Millipore filters, dried and counted in a Nuclear Chicago low background gas flow counter with an end window detector. RESULTS
RXA species synthesized
by growing
and
monolayer
chick
embryo
fibroblasts
Primary chick embryo fibroblasts, in uifro, attach to the glass surface of the culture vessels and start to grow until monolapers are formed within 20 hr. It was thought that labeling the cells with radioactive uridine for the entire growth period would enable the RNA species which accumulate in the growing cells to be studied, in spite of the possibility that short lived RNA molecules would not be detected. For this purpose the growing fibroblasts were incubated in the presence of radioactive uridine for intervals of 15 hr (Table I, Experiment 1) or 40 hr (Table I, Experiment 2). A labeling period of 40 hr was included to determine whether the relationship between the difTerent RNA species synthesized by the growing cells is relatively stable over a long period of time. At the end of the labeling periods, the radioactive RNA was isolated by centrifugation in SDS-sucrose gradients. Table I summarizes the relative amounts of radioactivity found in the different RN,1 species after extraction from whole cells and from the cytoplasm of both growing and monolayer cultures. Experimental
Cell Research 47
Y. Becker
556
It may be seen in Table I (Exp. 1) into the different RNA species during after seeding; sample A). A higher cells were labeled for an equivalent been formed (81-96 hr; sample B). TABLE I. Nature
Sample
Labeling period (hr after seeding)
A
5-20
B
Exp. NO.
1
2
of RNA
that radioactive uridine is incorporated the initial period of growth (5-20 hr rate of RN,4 synthesis occurred when period after monolayers had already RNA synthesis declined in cells which
species synthesized
by chick embryo fibroblusts. Per cent CPM in RNA
Cell fractions
species
CPM/culture
28 S
16 S
10 s
4s
Total cells Cytoplasm
36,200 8,200
38.05 24.91
25.70 27.21
18.97 33.98
7.50 11.03
81-96
Total cells Cytoplasm
80,000 36,740
39.29 29.55
27.59 13.06
7.52 9.86
18.09 44.63
c
145-160
Total cells Cytoplasm
47,200 20,970
30.47 31.16
39.03 32.20
9.26 15.11
9.07 21.53
A
5545
Total cells Cytoplasm
158,760 56,700
36.86 26.94
21.96 21.95
20.77 38.67
19.71 11.26
B
120&160
Total cells Cytoplasm
77,400 11,000
31.02 53.11
35.13 35.08
16.84 9.94
10.35 2.0
Chick embryo fibroblasts in Roux bottles were labeled for 15 (Exp. 1) and 40 hr (Exp. 2) with 1 ,uC %-2-uridine per culture at different intervals after seeding. Cells were harvested and a portion (10 X) was treated with SDS-buffer. The cytoplasmic fraction obtained from the cells was treated with SDS and centrifuged into SDS-sucrose gradients (15-30 % w/w). The radioactivity in each fraction and the distribution of the different RNA species in the gradients were determined. The percentage of the radioactivity present in the different RNA species was calculated.
were maintained in vitro for 160 hr (sample C) without medium replenishment. This was also found when cultures were labeled for 40 hr intervals (Table I, Exp. 2, samples A and B and Fig. 1). The synthesis of ribosomal precursor RNA was not detected in CEF labeled for 15 to 40 hr with radioactive uridine. Experiments in which shorter labeling periods (1 to 3 hr) were used resulted in the appearance of high molecular weight RNA, 45s and 35s RNA species, which are the precursors of ribosomal RNA. However, under these conditions the distribution of radioactivity in the 28S, 16s and 10s RNA species was not clearly resolved. In order to compare the behaviour of growing cells with that of aging cells, similar labeling periods were used. Such long labeling periods enhanced the possibility of detecting RN,4 species which might be synthesized at a low rate. A change in the relative amount of the 10s RNA species in monolayer Experimental
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RNA synthesis in chick embryo fibroblasts
557
cells as compared to growing cells was observed. Growing cells (Table I, Exp. 1, sample A and Exp. 2, sample A) synthesize more 10s RNA (18.97 and 20.77 per cent) than nongrowing cells (7.52 and 9.26 per cent) (Table I, Exp. 1, samples B and C). Similarly, a higher concentration of radioactive 10s RNA was found in the cytoplasm of growing cells, 33.98 and 38.67 per cent after 15 and 40 hr, respectively (Exp. 1, sample A; Exp. 2, sample A) than in monolayer cells (Exp. 1, samples B and C; Exp. 2, sample B). Similar changes regarding 4s RNA were also recorded. ,4s indicated in Table I, most of the RNA synthesized by CEF at any time during their in vitro existence is ribosomal RNA. To determine the
Fig. 1. Fig. l.-RNA species in the cytoplasm of growing chick embryo fibroblasts. Five hr after transfer of CEF to in uifro conditions, 1 /AC 14C-2-uridine was added and the cultures were further incubated for 40 hr. Cells were harvested, washed, resuspended in RSB and the cytoplasmic fraction was prepared by Dounce homogenization. SDS (final concentration 0.5 %) was immediately addedand the homogenate was analyzed in an SDS-sucrose (15-30 % w/w) gradient. The percentage of radioactivity in the different RNA species is indicated in the graph. ---, O.D.scomp; O---O, CPM.
Fig. 2.-Distribution of RNA in the cytoplasm of growing chick embryo fibroblasts. Five hr after transfer of CEF to cultural conditions, 1.5 PC lPC-2-uridine was added to each culture and the cells were incubated for an additional 15 hr period at 37°C. The cells were released from the glass by trypsinization for 2 min, washed in phosphate buffered saline (PBS), resuspended in RSB (6 x 10’ cells/ml) and disrupted with a Dounce glass homogenizer. Nuclei were removed by centrifugation for 2 min at 1000 rpm at 4°C and the cytoplasmic extract was centrifuged into a RSB-sucrose (15-30 % w/w) gradient for 75 min at 38,000 rpm (A). The pellet obtained in gradient A was recovered, dissolved in SDS buffer and analyzed in SDS-sucrose gradient (B). The figures in the graphs indicate the per cent radioactivity present in the different RNA species. ----, O.D.zso m,r; l --- l , CPM. Experimental
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Y. Becker
localization of the labeled RNA, the cytoplasmic extract of growing CEF, labeled for 15 hr with radioactive uridine, was centrifuged in RSB sucrose gradients and the ribosomal fraction was examined (Fig. 2A). It was found that 31.07 per cent of the radioactivity was associated with the 74 S ribosomal region of the gradient, while 11.84 per cent was associated with the region
Fig. 3.-Distribution of radioactive RNA in the cytoplasm of “crowded” (monolayer) cultures. CEF in monolayer cultures were labeled with 14C-2-uridine (1.5 PC per 3 x 10’ cells) for 40 hr starting at 120 hr after seeding to in vitro conditions. At the end of the labeling period the cells were removed from the glass by brief trypsinization, washed with PBS and resuspended in RSB. After disruption of the cells in a Dounce homogenizer, the cytoplasmic fraction was obtained, half of which was analyzed in a RSB-sucrose (15-30 % w/w) gradient (A) and the rest dissolved in SDS buffer (final concentration 0.5 %), and analyzed in a SDS-sucrose gradient (B). -~-, 0. D.zso mU; o-p- l , CPM.
heavier than ribosomes and 22.75 per cent was found at the bottom of the centrifuge tube. The rest banded in a region lighter than the ribosomes. The radioactive fraction found at the bottom of the centrifuge tube was dissolved in SDS buffer and analyzed by zone centrifugation in SDS-sucrose gradients. As shown in Fig. 2 B, both 28 S and 16 S RNAspecies could beidentified by their absorbance at 260 rnp and radioactivity, indicating that newly labeled cytoplasmic ribosomes are bound to large structures, probably membranes, and thus sediment to the bottom of the centrifuge tube. It is of interest that the 10 S radioactive RNA is also found to be associated with the above-mentioned structures. The intracellular localization of RNA, synthesized during a period of 40 hr, 120 hr after seeding the cells, was similarly studied. A marked decrease in RNA synthesis was found (Fig. 3A). The distribution of this RNA in the cytoplasm resembled that of growing cells (Fig. 2 A). Further analysis (Fig. 3B) showed that mostly ribosomal RNA (28s and 16s) but very little 10s RNA is present in the cytoplasm of the monolayer cells. Experimental
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RNA synthesis in chick embryo fibroblasts T.WLE II.
359
of RLVA synthesis in primary Effect of medium on the stimulation chick embryo fibroblasts.
Medium Unchanged 11 199 ivI 199 ii1 199 Eagle’s
Per cent calf serum
0 2.5 5 5
CPM in RNA 7-Exp. 1 Exp. 2 600 7000 7000 7000 2400
750 5100 5700 6000 2120
Monolayer cultures (5 days old) of chick embryo fibroblasts in milk dilution bottles (5 x lo6 cells/culture) were used. The medium was changed and 14C-2-uridine was added to each culture. Duplicate cultures were removed 6 hr later, the cells were dissolved in SDS buffer and samples were taken for the determination of radioactivity. The figures represent the average of the duplicate cultures examined.
Effect of fresh medium on R,VA synthesis of CEF
in “crowded”
(monolayer)
cultures
The experiments described in the previous section were done with CEF kept in the initial culture medium. In the present series of experiments, the ability of the cells to incorporate 14C-2-uridine into RNA was determined in aging CEF cultures which had received fresh Ml99 or Eagle’s medium, supplemented with 5 per cent calf serum, after five days. Monolayers which were left in the initial culture medium served as controls. ,4 typical experiment is presented in Fig. 4. It may be seen that the incorporation of radioactive uridine is markedly stimulated (6 to 10 fold) after the addition of fresh ,\I199 medium. The results presented in Table II demonstrate, that the stimulation of RNA synthesis in monolayer cells is not due to the addition of serum and is more marked with Ml99 medium than with Eagle’s medium. Following this finding, monolayer cells which received fresh medium were analyzed in regard to the nature and distribution of the RNA in the cell cytoplasm. The cells were labeled with radioactive uridine for 6 hr starting immediately after medium replenishment, the cytoplasmic fraction was prepared and analyzed in sucrose gradients. The results shown in Fig. 5A indicate that most of the radioactive RNA is associated \vith large cytoplasmic structures which sediment to the bottom of the sucrose gradient, with the 74 S ribosome peak and with the subribosomal units. Essentially, the distribution of radioactive RNA is similar to the distribution of RN;A in cells which did Experimental
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560
not receive fresh medium. Analysis of the cytoplasmic fractions with regard to the nature of the RNA species synthesized by CEF before or after medium replenishment (Fig. 5B) showed that mostly 28 S and 16 S RNA species were synthesized in the cells which were stimulated by fresh medium. EfJ‘ct of medium cultures of CEF
change on cellular
proliferation
in “crowded”
monolayer
The effect of fresh medium on the proliferation of CEF in monolayer cultures was studied by determining the number of cells in duplicate cultures with or without medium replenishment. In three identical experiments, no cell proliferation was detectable in the cultures after addition of fresh medium (Fig. 6). DISCUSSION
The present experiments were performed by labeling the newly synthesized RNA species in primary chick embryo fibroblasts during the entire growth period following their transfer from in uiuo to in vitro conditions. Essentially, only the labeled RNA molecules present at the end of the labeling periods were detected in the sucrose gradient analysis. It was found that the growing CEF synthesize 28 S, 16 S, 10 S and 4 S RNA species, similar to HeLa cells [6, 20, 211 and developing chick embryos [15]. Due to the long labeling periods used in the present experiments, ribosomal precursor RNA species were not detectable. An analysis of RNA species synthesized by cultured mouse embryo pancreas cells [25] also demonstrated the absence of ribosomal precursor RNA species when long labeling periods were used. The synthesis of ribosomal precursor RNA was therefore studied by labeling CEF with radioactive uridine for shorter time periods (1 to 3 hr). Under these conditions 45 S and 35 S ribosomal precursor RNA were detected in the growing CEF similarly to HeLa cells [‘LO]. Attention is drawn to the 10 S and 4 S RNA species which were found to be synthesized to a greater extent by growing than by monolayer cells. A 10 S RNA species was identified as messenger RNA in HeLa cells [19] and was also demonstrated in developing embryos of sea urchins [17, 181, Arbacia punctulata [la] and the South African clawed toad [4, 51. analysis of the distribution of the RNA species synthesized by CEF demonstrated the presence of 10 S RNA molecules in membrane-bound ribosomes which sediment through the sucrose gradient to the bottom of the centrifuge tube (Fig. 2s). Incorporation of amino acids in these cells occurs on such structures (ref. [22]; Becker, unpublished results). Experimental
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R;t’A synthesis in chick embryo fibroblasts
;‘I 0
,
,
,
,
10
20
30
LO TI
Fig. 4.
, 50 ME
,
60
70
, 00
90
100
, 110 120
IHOURS)
Fig. 6.
Fig. 4.-Effect of medium change on RNA synthesis in primary chick embryo fibroblasts. Monolayer cultures (five days old) were used. One series of the cultures was left in the initial medium, and to the other one fresh medium was added after removal of the medium. Immediately, 1 ,UC i4C-2-uridine was added to all cultures and duplicates were removed at different intervals. The amount of radioactive RNA in each culture was determined. O-O, cultures with unchanged medium; O--O, cultures with fresh medium. Fig. B.-The effect of medium change on the proliferation of “crowded” (monolayer) chick embryonic fibroblasts. After monolayer formation one series of cultures received fresh medium while the other one was left in the initial medium. At different time intervals, duplicate cultures were removed from each series and the number of cells was determined microscopically. O----O, cultures incubated in the growth medium; O-O, cultures incubated in fresh medium.
(monolayer) fibroblasts. MonoFig. 5.-Nature of the RNA species synthesized in “crowded” layer cultures with or without medium replenishment were labeled with %-2-uridine. At different time intervals duplicate samples were removed and the cytoplasmic fractions were prepared and analyzed in RSB-sucrose gradients (A) and in SDS sucrose gradients (B). The amount of radioactivity in the pellet at the bottom of the centrifuge tubes and in each fraction was also CPM in CEF in unchanged medium; 0-0, CPM determined. __ , O.D.zse mp; O--O, in CEF after medium replenishment. Experimental
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After monolayer formation, CEF were left in the initial growth medium. To be able to compare RNA synthesis in these aging cells with the experiments performed on actively growing cells, long labeling periods were also used. This procedure enabled RK;A molecules which accumulated slowly in the cells to be detected. It was found that the aging cells synthesize 28 S and 16 S ribosomal RNA at a lower rate than actively growing cells. Shorter labeling periods (1 to 3 hr) enabled the detection of the ribosomal precursor RNA (45 S and 35 S) but the distribution of the radioactivity in other RNA species was not clearly resolved. It is possible that prolonged maintenance of CEF in the culture medium leads to the depletion of essential nutrients, thus causing a decline in the ability of the cells to synthesize ribosomal RNA. The addition of fresh medium to monolayers of chick embryo fibroblasts caused a marked stimulaof 14C-2tion of ribosomal RNA synthesis. In contrast, the incorporation thymidine into DNA was stimulated only slightly (Becker, unpublished results). From the results presented in Table II it is evident that the serum used for cultivation of cells was not responsible for the stimulation of RN-4 synthesis. After it had been found that mainly ribosomal RNA molecules are synthesized by aging cells after medium replenishment, the kinetics of this process were determined using shorter labeling periods (1 to 6 hr, Fig. 4). It was found that ribosomal RNA is synthesized and appears as ribosomal subunits or ribosomes in the cytoplasm of the cells. Chick embryo libroblasts differ in this respect from primary mouse diploid cells in which serum stimulates the proliferation of some cells and the synthesis of all RNA species [23,. The results of the present experiments indicate the existence of a coordinated synthesis of the two ribosomal RNA species. This supports the idea that the two ribosomal RNA species are regulated to the same extent and may have a common genetic origin as suggested by Drown and Littna [a, 5:. Independent regulation of ribosomal and transfer RNA species has also been described in bacterial cells [ 141. The inability of the cells to synthesize the 10 S and 4 S RNA species after medium replenishment, in contrast to the stimulation of ribosomal RNA synthesis, might indicate that non-ribosomal RNA synthesis is regulated by a process different from that of ribosomal RNA. This may indicate that additional or new mechanisms appear in cells at or after the formation of monolayers, which prevent the synthesis of certain RNA species. This can be overcome when monolayer cells are dispersed, reseeded to new cultures and cellular proliferation is restored. It is tempting to speculate that the very limited synthesis of nonribosomal RNA in monolayer cells restricts protein synthesis mainly to those species coded for by Experimental
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RNA synthesis in chick embryo fibroblusfs
563
messenger RNA molecules synthesized during the initial growth period of the fibroblasts and still exist in the cytoplasm after monolayer formation. Cellular proliferation is thus inevitably restricted, although ribosomes continued to be synthesized by the aging monolayer cells. The nature of the regulatory mechanisms which are responsible for the inhibition of the synthesis of the nonribosomal RNA and their relationship to the phenomenon of contact inhibition in cultured cells are still unknown. SUMMARY The in uifro behaviour of primary chick embryo fibroblasts (CEF) in respect to RNA synthesis was studied. It was found that actively growing CEF synthesize all RNA species. After monolayer formation, the cells synthesize mainly ribosomal RNA, though at a lower rate than actively growing cells. After the addition of fresh medium to monolayer cultures, cellular proliferation was not detectable, but the synthesis of mainly ribosomal RXA was stimulated. This work was aided in part by grant 5 R05 TW00152-1 and 2 from the National Institutes of Health, Bethesda, Md, U.S.A. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24. 25.
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