Cell, Vol.
13, 121-127,
January
1978, Copyright
0 1978 by MIT
Differences in rRNA Metabolism of Primary SV40-Transformed Human Fibroblasts
Stephen A. Liebhaber, Stanley Wolf and David Schlessinger Department of Microbiology and immunology Washington University School of Medicine St. Louis, Missouri 63110
Summary rRNA metabolism was compared in primary human diploid fibroblasts (WI-38) and in SV40-transformed cultures derived from WI-38 (VA-13 and SV-C52). Both in actively growing and nondividing diploid fibroblasts, mature rRNA, labeled with 3Huridine or 3H-methyl-methionine, decayed with a half-life of 72 f 4 hr. In contrast, in growing SV40-transformed cells, as in HeLa and other established lines, rRNA was stable, and only began to show a half-life shorter than 700 hr as the cells reached maximum density. To determine the relative importance of degradation and synthesis in maintaining the cellular content of rRNA, the net synthesis of rRNA was evaluated by measurements of the transcription, processing and wastage of pre-rRNA. Accumulation of 3H-methyl-methionine into precursors and mature rRNA was quantitated by an established computer program (Wolf and Schlessinger, 1977). The relative synthetic rates of 45s pre-rRNA increased by 50% or less in the transformed cells. In all cases, the rates of processing were identical and little if any nuclear wastage was observed: all of the 45s pre-rRNA gave rise to mature 28s and 18s rRNA. In growing normal cells, the net level of RNA could therefore be accounted for by the balance of the transcription of 45s pre-rRNA and cytoplasmic decay of 18s and 28s rRNA. In growing transformed cells, 18s and 28s rRNA were stable, and hence the net formation of rRNA was determined only by the rate of synthesis. This qualitative difference could give transformed cells a selective advantage. Introduction The rate of cell proliferation is dependent upon coordinated synthesis of ribosomes and protein (Brachet, 1941; Caspersson, 1941; Koch, 1971). Thus changes in growth state-for example, from nondividing to exponentially growing cells-necessitate parallel adjustments in the net accumulation of ribosomes (Daskal and Sinclair, 1975). This close relationship between cell growth and ribosome production suggests that changes in RNA metabolism are important in the regulation of cell growth and might accompany the transition of normal primary diploid cells to longer-lived viral transformed cells.
and
Possible mechanisms for the regulation of eucaryotic cell ribosome content include the following: the rate of rRNA transcription (Maden et al., 1969; Emerson, 1971) could vary as is seen in procaryotes (Kjeldgaard and Gausing, 1974); the time required to process rRNA precursors could vary widely with growth rate [as is observed in bacteria (Schlessinger, 1974)]; a variable fraction of newly formed i-RNA could be discarded (“wastage”) before leaving the nucleus in completed ribosomes (Cooper, 1973); and the rate of breakdown of mature cytoplasmic ribosomes could vary (Hirsch and Hiatt, 1966; Weber, 1972; Abelson et al., 1974). Thus to determine how rRNA levels are regulated, measurements of synthesis, nuclear wastage and cytoplasmic turnover are required. Studies with cultured cells have examined one or two of these parameters. Here we assess and compare quantitatively all of these possible control points of net rRNA accumulation in exponentially growing primary human diploid fibroblasts and in these fibroblasts transformed by simian virus 40 (SV40). The critical control points are inferred to be the rates of synthesis and cytoplasmic turnover. One major difference was found: the normal cells showed cytoplasmic rRNA turnover, whereas transformed cells did not. Results How important are rates of cytoplasmic turnover compared to nuclear events in maintaining the rRNA content of fibroblasts? Independent measures of the rates of decay and synthesis are required to answer this question. rRNA Decays in Growing Primary Fibroblasts The turnover rate of rRNA in diploid fibroblasts was measured by labeling RNA with 3H-uridine or 3H-methyl-methionine and monitoring decay over a chase period of approximately 1 week. Chases of both uridine and methyl label are highly efficient (Weber, 1972; Murphy and Attardi, 1973). The initial labeling period (18-24 hr) was prolonged to ensure the labeling of all cells. A low level of 14C-thymidine was added during the labeling period to permit corrections of the decay rate for cell loss during manipulations (in general these were ~1% per day). Once labeled, cells were dispersed into 60 mm petri dishes, and medium was changed every 48 hr. RNA was extracted from plates on subsequent days and its specific radioactivity was measured. For some experiments, the radioactivity in individual RNA species was estimated after they were separated by polyacrylamide gel electrophoresis. Figure 1 displays the resultant decay curves of labeled RNA in exponentially growing fibroblasts
Cell
122
2
t
It .2 IO 51
2
I
L
2
11
4
6
2
4
DAYS Figure
1. Decay
of %H-Labeled
6 OF
a 10
I
1
I
,
2
4
6
a
CHASE
RNA from
Growing
WI-38 Cells
Exponentially growing WI-38 fibroblasts were labeled at an early passage (P25) with 3H-uridine (left and center panel) or 3Hmethyl-methionine (right panel) and 14C-TdR for 24 hr. The cells were then grown in medium containing 5 mM uridine and 2.5 mM cytosine (left and center panels) or 15 pg/ml methionine over the indicated chase period. Cell growth (right panel) was halted by addition of medium depleted of growth factors. Cell growth curves in medium containing 10% (A- - -A) or 20% FCS (A---A) are represented on the lower panels. Remaining 3Hactivity in total acid-precipitable RNA (0- - -O), phenol-extracted RNA (V- - -V), and 28s and 18s rRNA (as determined by gel electrophoresis) was corrected for cell loss by dividing each value by the corresponding cpm remaining in 14C-DNA. In the center panel, the top decay curve is for cells grown in 10% serum and the bottom is in 20% serum.
doubling every 39 hr (left) or 30 hr (center). Decay was seen both in total acid-precipitable material, in phenol-extracted RNA, and in separated 28s and 18s rRNA, with a half-life of approximately 70 hr over a 5 day period. Thus in contrast to bacteria (Davern and Meselson, 1960) and certain cells in tissue culture (Rake and Graham, 1962; Abelson et al., 1974; Kolodny, 1975), these primary human cells actively degrade mature 28s and 18s rRNA during exponential growth. To determine whether rRNA stability would change with growth rate, the normal feeding medium overlying growing fibroblasts was replaced with medium depleted of serum growth factors (Figure 1, right). Growth continued for 2 days and then markedly slowed down. The decay rate of the total RNA in serum-depleted cells was the same as in growing cells (68-72 hr). The same half-life of RNA was observed in cells rendered nondividing
I
by 3H-irradiation (Ehmann et al., 1975; Marz et al., 1976; Liebhaber and Schlessinger, 1978) or by passage into the nondividing “crisis” state (Wolf, 1977; further data to be published). The half-life of RNA prelabeled with 3H-methylmethionine thus showed no change during the transition from rapid growth to a resting state. Furthermore, during the period of exponential growth (days l-5), decay was similar whether RNA was labeled with 3H-methionine or 3H-uridine (Figure 1, left), confirming the equivalence of the two pulse-chase techniques. Previous work with 3T3 cells (Abelson et al., 1974; Kolodny, 1975) and chick fibroblasts (Weber, 1972) has shown that the stability of rRNA in those cells was dependent upon cell density. During exponential growth, the rRNA was stable, but as cell growth slowed consequent to cell crowding, rRNA degradation was initiated. To be certain that the instability of rRNA reported here was not due to such an effect of cell density, cells in the experiment of Figure 1 were plated at 10% (left) or 4% (center) of confluent density and showed the same half-life, although the cell doubling time decreased from 39 to 30 hr in cells plated at lower initial densities. Similarly, increasing the concentration of fetal calf serum from lo-20% did not affect RNA halflife (Figure 1, middle panel). rRNA Is Stable in Growing SV40-Transformed Fibroblasts To determine whether viral transformation of the WI-38 primary fibroblasts was associated with an increase in stability of the cytoplasmic rRNA, VA13, an established line of SV40-transformed WI-38 cells (positive for T antigen) was tested. Decay rates of RNA were determined using 3H-methylmethionine and 3H-uridine in separate trials (Figure 2, left). The same results were obtained with both pulse-chase methods: there was minimal decay of label in these exponentially growing cells, which were doubling every 23 hr in one trial and every 30 hr in a second (Figure 2, left). The slight increase in decay rate of the labeled RNA seen once the cells reached maximal density on the plates may be analogous to the initiation of degradation found in 3T3 cells (Abelson et al., 1974; Kolodny, 1975) and chick fibroblasts (Weber, 1972) after density inhibition of growth. Several additional clones of SV40-transformed WI-38 cells were made by Dr. S. Gotoh in our laboratory by infecting early passage WI-38 cells with SV40 (Girardi, Jensen and Koprowski, 1966) and subculturing the resultant foci of transformed cells. The clone used here (SV-C52) was T antigenpositive and morphologically similar to the established clone VA-13. Different clones of SV40-transformed WI-38 cells had different doubling times.
Regulation 123
of rRNA
2 Figure
4
2. Stability
Levels
6
in Fibroblasts
8 10 2 DAYS OF CHASE
of 3H-RNA
in Transformed
A
2
II 4
6
Cells
Decay curves of 3H-RNA in 3H-uridine (0) and 3H-methyl-methionine (V) labeled cells growing in media with unlabeled uridine and cytosine or methionine, respectively, are plotted (after correction for slight ‘%-DNA loss). Growth curves of the 3H-U (a) and JH-methyl-methionine (A) labeled cells are shown in the lower curves.
Clone SV-C.52, one of the fastest growing, had a doubling time similar to VA-13 (about 30 hr). Cells of SV-C52 were labeled at generations 44 and 53 with 3H-uridine and/or 3H-methyl-methionine in separate experiments. The labeled RNA in the acidprecipitated and phenol-extracted fractions of growing cells was as stable as in VA-13 cells (for example, Figure 1, middle panel). We emphasize that the shutdown of turnover in the SV40-transformed cells is not a simple correlate of a more rapid growth rate, because as indicated above, early passage cultures of human fibroblasts can divide as fast as many cultures of cloned transformed cells (for example, compare Figure 1, left, with Figure 2). When HeLa cells, an established epithelial cell line, were similarly labeled with 3H-uridine, they also exhibited stable rRNA. In this case, no turnover was seen even at maximum cell density (Figure 2, right panel). These results with two separately transformed and one established cell type suggest a characteristic of these cells that may be general: cytoplasmic rRNA is stable during growth, in contrast to the exponential degradation of rRNA in the growing primary cell cultures. Similar Nuclear rRNA Metabolism in Primary and Transformed Fibroblasts We next compared rates of transcription, processing and wastage of pre-rRNA as further potential sites of differential rRNA metabolism. An earlier paper (Wolf and Schlessinger, 1977) describes in detail the experimental design used here and justifies the assumptions of stochastic prOCeSSing
made in the quantitative analysis of the data. Briefly, steady state labeling kinetics of each of the pre-rRNA and rRNA species were determined for human fibroblasts in various growth states using 3H-methyl-methionine as the precursor. Results quantitated by this method and analyzed with the aid of a computer program have previously been shown to determine accurately the pre-t-RNA processing pathway and the processing half-lives in HeLa cells, and have also demonstrated that no wastage occurs in HeLa cells during rapid growth (Wolf and Schlessinger, 1977). The accumulation of 3H-methyl label in the individual pre-rRNA and rRNA species was determined from polyacrylamide gel profiles of purified total RNA extracted from VA-13 or WI-38 cells during continuous labeling. Subsequently, the kinetics of accumulation of 3Hlabeled rRNA were quantitated and analyzed using an IBM 360 computer with a simulation, analysis and modeling program (SAAM 23) (Berman, 1966). The program simultaneously obtains a least-squares fit to the accumulation data for all the RNA species, thereby determining rate constants for synthesis of the primary 45s transcript, processing of each of the pre-rRNA intermediates and any appreciable degradation of the pre-rRNA intermediates. The program also calculates the standard deviation of each rate constant and prints a table of the calculated accumulation values which correspond to the experimentally measured points (see Experimental Procedures). The intermediates of processing, as identified in polyacrylamide gels, were the same for both VA-13 and early passage WI-38 cells (Figure 3). For example, after 5 min (figure 3, top panels), the 45s precursor is clearly visible, and after 75 min, the 45s precursor, 32s intermediate, and 28s and 18s rRNA products can be seen in all the gel profiles (Figure 3, bottom panels). Computer analysis of the accumulation data produced good fits to the data with the standard prerRNA processing pathway. The calculated accumulation curves shown in Figure 4, for example, were obtained as a best fit using the well accepted processing of 45s to 32s and 2OS, and thence to 28s and 18s rRNA, in HeLa cells (for review, see Hadjiolov and Nikolaev, 1976). It can be seen in Table 1 that, in addition to following the same processing pathway, the intermediates in each of the cultures studied were processed with the same half-lives. The relative pool sizes also remained the same. Thus processing is indistinguishable in primary and transformed cells. The computer-assisted analysis of 3H-methyl-labeled RNA accumulation data was used to test for nuclear wastage of pre-rRNA species. Wastage of 45S, 32s and 20s pre-rRNAs was looked for by
Cell 124
Growing WI
ASS
38
17
325 285
I,
50
100
150
50
100
150
50
100
150
MINUTES
Figure 4. 3H-Methyl-Labeled Curves
GEL SLICE Figure
3. Polyacrylamide
Gel Profiles
of 3H-Methyl-Labeled
RNA
Cultures were labeled with 3H-methyl-methionine, and the RNA was extracted and run on polyacrylamide gels for 8 hr (as in Experimental Procedures). The top panels show profiles of RNA extracted after labeling for 5 min, and the lower panels after labeling for 75 min. 14C marker 23s and 16s E. coli rRNA was coelectrophoresed with the RNA from growing WI-38, and 14Cmarker 28s and 18s HeLa rRNA was co-electrophoresed with the RNA from VA-13.
including a pathway for wastage of each of the pre-rRNAs in the model used for the data analysis (Wolf and Schlessinger, 1977). In both cases, the pathways for wastage of 45S, 32s and 205 prerRNA were eliminated by the program as it reached the best fit to the data. For example, the solid lines in Figure 4 show the best computer fit for the measured accumulation data. The dashed lines represent predicted accumulation curves of 28s and 185 rRNA if 20% wastage is imposed on 32s and 20s pre-rRNA. It is clear that within a range of lo%, all the rRNA sequences which started in 45s pre-rRNA eventually appeared in 28s and 18s rRNA. Since the 45s pre-rRNA accumulation curves were comparable for both cellular growth states (Table 1) and no nuclear wastage was detected, the relative 455 synthesis rates could be estimated by labeling cellular RNA with a short pulse of 3Hmethyl-methionine and quantitating the label content of 45s pre-rRNA from polyacrylamide gel profiles. To eliminate the possibility of artifacts due to differences in the methionine-specific activity, the cells were labeled with a single lot of radioactive medium, and the methionine pool size in the cul-
Pre-rRNA
and
rRNA
Accumulation
The accumulation of label into pre-rRNA and rRNA species was quantitated and analyzed as described in the text and Experimental Procedures. The points represent the actual data, and the solid lines represent the calculated accumulation curves for the best computer fit to the data. The dashed lines illustrate theoretical accumulation curves assuming the same rates of synthesis and processing arrived in the “best fit,” but including 20% wastage of the synthesized 325 and 20s pre-rRNA. Relative moles were arrived at by dividing the accumulated counts in a species by the number of methyl groups per RNA chain of that species.
tures was measured directly (see Experimental Procedures). The methionine pool size was found to be the same in all cultures. Unexpectedly, the rate of rRNA synthesis in human diploid fibroblasts was found to vary in such a way that confluent cells contained about twice as much rRNA as rapidly growing cells. When confluent cells were replated, they doubled every 36 hr with a constant half-life of rRNA (as in Figures 1 and 2), but for 2-3 days, rRNA doubled only half as fast (Table 2). RNA then began to double as fast as the cells, and synthesis continued in confluent cultures at a rate sufficient to increase the content of rRNA 2 fold again. These findings will be detailed elsewhere, but one feature is relevant here: the net formation of rRNA could always be accounted for by the rates of rRNA synthesis and cytoplasmic turnover. For example (Table 2), when cells were increasing their rRNA content only 40% as fast as transformed cells, their rate of rRNA synthesis was 67% that of the transformed cells; the “extra” synthesis was just sufficient to balance rRNA turnover (see Discussion). Discussion These are the first data on the stability of rRNA and the maintenance of rRNA levels in diploid human fibroblasts. The half-life of rRNA was consistently about 70 hr in WI-38 cells (for example,
Regulatron 125
Table
of rRNA
1. Pre-rRNA
Levels
in Frbroblasts
Half-Lives
and Cellular
Pool Sizes
Table 2. rRNA Levels
Growing WI-38 T112 (min)
Growing
45s
16 + 5%
18 AZ 7%
pg RNA
325
37 2 8%
38 * 9%
cpm
VA-13 Cells per Culture
Pool
20s Pool Sizes
8 t 17% (Relative
Synthetic
7 * 14%
to 32s)
in 45s
ws RNA rRNA
Half-Life
rRNA
Doubling
45s
0.4 2 5%
0.5 k 7%
32s
1 .o ? 8%
1 .o * 9%
Measured
20s
0.2 + 17%
0.2 2 18%
Predicted
The half-lives were computed from the calculated rate constants as described in Experimental Procedures. The percentage errors are those arrived at by the SAAM program. Relative pool sizes were calculated by solving the differential equation for the model at the steady state limit. At that time, dqldt = 0 for each species q, and the equations reduce to simple algebraic relationships (see Wolf and Schlessinger, 1977). The level of 32s pre-rRNA was arbitrarily set equal to 1 .O and the other pool srzes calculated relative to the 32s soecies.
Figure 1 and Table 2). In more extensive studies (Wolf, 1977), the half-life of cytoplasmic rRNA was measured as diploid fibroblasts were passaged into the “crisis” phase after 50 -+ 2 doublings; the half-life remained 70 ? 7 hr at all growth rates-that is, throughout the range of growth rates expected for normal fibroblasts in vivo. The same half-life was also observed in other experiments with primary cultures of C3H mouse fibroblasts (unpublished results). These results are to be contrasted with the stability of rRNA in various growing transformed and established cell lines, including human VA-13 (Figure 2) and mouse 3T6 and 3T3 cells (Abelson et al., 1974; our confirmatory experiments). The near-constancy of rRNA half-life over a wide range of diploid fibroblast growth rates makes the curtailment of turnover in growing transformed cells seem very abrupt. No such qualitative difference between WI-38 and VA-13 cells was seen in nuclear rRNA metabolism. In both types of cell, the processing rates were very similar and no wastage was observed. Since they are the only control points at which changes are observed, the rates of rRNA synthesis and cytoplasmic turnover should fix the net rate of rRNA formation. Table 2 includes values consistent with this possibility. Using the relation in the legend to the table, the relevant rates predict a doubling time of 25 hr for RNA in VA-13 cells, compared to the observed 29 hr + 15%. In the same experiments, in a comparable analysis of nondividing cells, the corresponding synthesis and turnover rates predicted a doubling time of 180 hr, com-
x lo-’
and Turnover
Rates
and Steady
State
WI-36
VA-l 3
7.2 & 6%
a .2
12,11,13~10%
14,14,13+10%
340,312,303
545, 494,412
72hr*ll%
700 hr ? 10%
72 hr i 7%
29hrc15%
+ 6%
Time
25 hr
The number of cells per culture was determined by a hemocytometer count of a sample of cells from a petri dish treated in parallel with the labeled dishes. The net increase in extractable RNA was measured on a series of cell samples by ultraviolet absorption and by the orcinol reaction. Labeling, RNA extraction and gel electrophoresis were performed as described in Experimental Procedures. The data related to synthesis and pg RNA are from parallel samples in one experiment to ensure that the specific activity of the precursor in the medium was the same for all cultures. Note that in this experiment, the primary fibroblasts had been freshly plated and exhibited a doubling time of 72 hr for their RNA content for several days, even though the cell doubling time was 36 hr (see text). Half-lives of cytoplasmic turnover were determined from experiments like those in Figures 1 and 2. Predicted rRNA doubling times were calculated based on the following assumptions: the rate of rRNA production is proportional to the amount of existing rRNA, and the rate of rRNA decay is proportional to the amount of existing rRNA. (This is only a first-order approximation.) With those assumptions, dN/ dt = N-(In 2/Tl,z)N and hence, T,, = In 2/(a-In 2/T,,%). N is the amount of rRNA in the culture; T,,, is the cytoplasmic half-life of rRNA; TD is the rRNA doubling time; and 01 is the 45s rRNA synthesis rate constant. (Y is proportional to the incorporation rate per pg RNA listed in the Table. The proportionality constant was determined and then used to predict TD for other cultures (see text).
pared to the observed doubling time of 215 hr ? 10% (Wolf, 1977; data to be published). The adjustment of rRNA levels therefore occurs in very different ways in untransformed and transformed fibroblasts. In normal fibroblasts, changes of rRNA doubling time are associated with a relative balance of synthetic and degradative rates. This mode of regulation is different from that proposed by some speculative models, and in particular, it is not analogous to the regulation of rRNA levels in bacteria. In bacteria, regulation occurs almost exclusively by changes in the rate of synthesis during growth, and even in nutrient-limited bacteria, in which rRNA turnover begins, it proceeds at a rate only about 1% that of the maximal rate of synthesis (Koch, 1971). The established lines and viral transformed cells examined thus far resemble the bacterial paradigm more, since net rRNA accumulation is directly linked to the rate of synthesis. Another example of the selective curtailment of turnover has been re-
Cell 1’26
ported in kidney cells during renal hypertrophy (Melvin, Kumar and Malt, 1976), although net rRNA formation is transient in that case. The stabilization of rRNA in growing transformed fibroblasts does not seem to be a specific corollary of the faster growth of many transformed cells since it is observed in transformed cell cultures but not in normal cell cultures doubling at very similar rates (see Results, Figures 1 and 2). Comparable observations over a wider range of growth rates of transformed cells would strengthen this inference. We are attempting to test the possibility that the shutoff of t-RNA turnover is one of the pleiotropic effects of viral transformation, and that it may give the transformed cells a selective advantage. Experimental
Procedures
Cells Primary human fetal lung fibroblasts (WI-38), generation 18, and VA-13, an established cell line from a clone of SV40-transformed WI-38, were obtained from ATCC and grown at least eight generations before use. SV-C52 clone cells were obtained as a rapidly growing focus of cells after transformation at generation 20 by SV40 (the virus was obtained from Dr. D. Nathans, Johns Hopkins University). The cells were grown at 37°C in MEM-a with nonvolatile buffers fortified with 10% fetal calf serum (Kansas City Biologicals). Resting cells in Figure 1, right panel, were prepared by replacing normal growth media with MEM-a plus 2% FCS depleted of growth factors (by contact with a confluent monolayer of WI-38 cells for 72 hr.) Determination of 3H-Labeled RNA Decay On the indicated days, cells from two plates were trypsinized into 3 ml of 0.25 M sucrose and counted on a hemocytometer to obtain a growth curve. 0.5 ml of the suspended cells were precipitated with ice-cold 5% CCl&OOH. collected and washed on a glass fiber filter, and counted in toluene-based scintillation fluid with channels adjusted for separate counting of 3H and j4C. Decay rates of total 3H-labeled RNA were corrected for cell loss (usually i 1% per day) by plotting all values as 3H/‘4C. To measure the decay rates of individual rRNA species, the remaining 2.5 ml of cell suspension were brought to 100 mM Trisacetate (pH 5.4), 10 mM EDTA, 0.5% sodium dodecylsulfate, and RNA was extracted once at 55°C for 5 min with buffer-saturated phenol and a second time at 0°C with l/2 vol of phenol-chloroform-isoamyl alcohol (l:l:O.Ol). The final aqueous phases were precipitated with 2.5 vol of 100% ethanol and stored at -20°C until all samples were collected. Labeling and RNA Extraction to Determine Nuclear Metabolism of rRNA A detailed description of the protocol for 3H-methyl-methionine labeling and extracting rRNA is given in Wolf and Schlessinger (1977). To summarize, the washed cells were allowed to preincubate in fresh low methionine medium (1 .O pg/ml methionine) for 1 hr. Medium containing 3H-methyl-methionine was then added. For the kinetic analysis of pre-rRNA processing, the levels of methionine were as in Wolf and Schlessinger (1977), with about 100 &i/ml. At the indicated times, sample monolayers were rinsed with buffer and lysed in extraction buffer (0.1 M Trizma Base, 0.05 M EDTA, 0.5% sodium dodecylsulfate, adjusted with acetic acid to pH 5.4). The RNA was extracted several times with warm phenol and stored in 70% ethanol at -20°C. To determine the rates of synthesis of 45s pre-rRNA in the
cultures, the cells were preincubated in low methionine medium as described above and then labeled for 15 min in methioninefree medium containing 3H-methyl-methionine (11 Ci/mmole) at a concentration of 0.1 mCi/ml. In preparation for polyacrylamide gel electrophoresis, the samples were resuspended, and 0.1% bromophenol blue and marker YZ-labeled HeLa or E. coli ribosomal RNA were added. The samples were warmed at 55°C for 5 min, and a measured volume was then applied to the polyacrylamide gels. Polyacrylamide Disc Gel Electrophoresis and Quantitation The technique used for acrylamide gel electrophoresis was essentially that of Weinberg and Penman (1968). with the modifications described by Wolf and Schlessinger (1977). After electrophoresis, the gels were frozen and sliced. The slices were dissolved in vials containing 5% protosol (New England Nuclear) in toluene scintillator. The cpm were then summed up for each RNA species and analyzed as described below. Computer Modeling and Kinetic Analysis Based on the assumption of stochastic processing, the SAAM 23 program obtains a least-squares fit to the data for each pathway in a submitted model (Berman, 1966; Wolf and Schlessinger, 1977). A processing model is submitted to the SAAM 23 program by specifying an initial estimate of the rate constants for proposed steps, initial conditions, the accumulation data for the experiment and error estimates for the data. When desired, wastage pathways are included by submitting a rate constant for a pathway with no normal product. The program then iteratively adjusts the rate constants to minimize the sum of squares of the difference between the calculated and observed values (for further details, see Wolf and Schlessinger, 1977). Determination of Methionine Pool Sizes Cultures of cells in the growth states under study were incubated in low methionine medium (1.0 pg/ml methionine). After 1 hr, the medium was aspirated off, and macromolecules in the remaining monolayer were precipitated with 0.2 N perchloric acid at 0°C. The perchloric acid was pipetted off, and the monolayer was washed with fresh perchloric acid. The two perchloric acid washes were combined and neutralized with potassium hydroxide, and the potassium perchlorate was removed by centrifugation. The supernatant was removed, lyophilized, and then resuspended in a small volume and analyzed on a Beckman Model 1208 amino acid analyzer under standard conditions. The amount of methionine in each culture was quantitated from the elution profiles. Acknowledgments These investigations were aided by a grant from the NIH and a fellowship to S.A.L. Access to the IBM 360 and SAAM 23 program was through the Washington University Biomedical Computer Lab. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received
June
13,1977;
revised
November3,
1977
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13, 121-127,
January
1978, Copyright
0 1978 by MIT
Differences in rRNA Metabolism of Primary SV40-Transformed Human Fibroblasts
Stephen A. Liebhaber, Stanley Wolf and David Schlessinger Department of Microbiology and immunology Washington University School of Medicine St. Louis, Missouri 63110
Summary rRNA metabolism was compared in primary human diploid fibroblasts (WI-38) and in SV40-transformed cultures derived from WI-38 (VA-13 and SV-C52). Both in actively growing and nondividing diploid fibroblasts, mature rRNA, labeled with 3Huridine or 3H-methyl-methionine, decayed with a half-life of 72 f 4 hr. In contrast, in growing SV40-transformed cells, as in HeLa and other established lines, rRNA was stable, and only began to show a half-life shorter than 700 hr as the cells reached maximum density. To determine the relative importance of degradation and synthesis in maintaining the cellular content of rRNA, the net synthesis of rRNA was evaluated by measurements of the transcription, processing and wastage of pre-rRNA. Accumulation of 3H-methyl-methionine into precursors and mature rRNA was quantitated by an established computer program (Wolf and Schlessinger, 1977). The relative synthetic rates of 45s pre-rRNA increased by 50% or less in the transformed cells. In all cases, the rates of processing were identical and little if any nuclear wastage was observed: all of the 45s pre-rRNA gave rise to mature 28s and 18s rRNA. In growing normal cells, the net level of RNA could therefore be accounted for by the balance of the transcription of 45s pre-rRNA and cytoplasmic decay of 18s and 28s rRNA. In growing transformed cells, 18s and 28s rRNA were stable, and hence the net formation of rRNA was determined only by the rate of synthesis. This qualitative difference could give transformed cells a selective advantage. Introduction The rate of cell proliferation is dependent upon coordinated synthesis of ribosomes and protein (Brachet, 1941; Caspersson, 1941; Koch, 1971). Thus changes in growth state-for example, from nondividing to exponentially growing cells-necessitate parallel adjustments in the net accumulation of ribosomes (Daskal and Sinclair, 1975). This close relationship between cell growth and ribosome production suggests that changes in RNA metabolism are important in the regulation of cell growth and might accompany the transition of normal primary diploid cells to longer-lived viral transformed cells.
and
Possible mechanisms for the regulation of eucaryotic cell ribosome content include the following: the rate of rRNA transcription (Maden et al., 1969; Emerson, 1971) could vary as is seen in procaryotes (Kjeldgaard and Gausing, 1974); the time required to process rRNA precursors could vary widely with growth rate [as is observed in bacteria (Schlessinger, 1974)]; a variable fraction of newly formed i-RNA could be discarded (“wastage”) before leaving the nucleus in completed ribosomes (Cooper, 1973); and the rate of breakdown of mature cytoplasmic ribosomes could vary (Hirsch and Hiatt, 1966; Weber, 1972; Abelson et al., 1974). Thus to determine how rRNA levels are regulated, measurements of synthesis, nuclear wastage and cytoplasmic turnover are required. Studies with cultured cells have examined one or two of these parameters. Here we assess and compare quantitatively all of these possible control points of net rRNA accumulation in exponentially growing primary human diploid fibroblasts and in these fibroblasts transformed by simian virus 40 (SV40). The critical control points are inferred to be the rates of synthesis and cytoplasmic turnover. One major difference was found: the normal cells showed cytoplasmic rRNA turnover, whereas transformed cells did not. Results How important are rates of cytoplasmic turnover compared to nuclear events in maintaining the rRNA content of fibroblasts? Independent measures of the rates of decay and synthesis are required to answer this question. rRNA Decays in Growing Primary Fibroblasts The turnover rate of rRNA in diploid fibroblasts was measured by labeling RNA with 3H-uridine or 3H-methyl-methionine and monitoring decay over a chase period of approximately 1 week. Chases of both uridine and methyl label are highly efficient (Weber, 1972; Murphy and Attardi, 1973). The initial labeling period (18-24 hr) was prolonged to ensure the labeling of all cells. A low level of 14C-thymidine was added during the labeling period to permit corrections of the decay rate for cell loss during manipulations (in general these were ~1% per day). Once labeled, cells were dispersed into 60 mm petri dishes, and medium was changed every 48 hr. RNA was extracted from plates on subsequent days and its specific radioactivity was measured. For some experiments, the radioactivity in individual RNA species was estimated after they were separated by polyacrylamide gel electrophoresis. Figure 1 displays the resultant decay curves of labeled RNA in exponentially growing fibroblasts
Cell
122
2
t
It .2 IO 51
2
I
L
2
11
4
6
2
4
DAYS Figure
1. Decay
of %H-Labeled
6 OF
a 10
I
1
I
,
2
4
6
a
CHASE
RNA from
Growing
WI-38 Cells
Exponentially growing WI-38 fibroblasts were labeled at an early passage (P25) with 3H-uridine (left and center panel) or 3Hmethyl-methionine (right panel) and 14C-TdR for 24 hr. The cells were then grown in medium containing 5 mM uridine and 2.5 mM cytosine (left and center panels) or 15 pg/ml methionine over the indicated chase period. Cell growth (right panel) was halted by addition of medium depleted of growth factors. Cell growth curves in medium containing 10% (A- - -A) or 20% FCS (A---A) are represented on the lower panels. Remaining 3Hactivity in total acid-precipitable RNA (0- - -O), phenol-extracted RNA (V- - -V), and 28s and 18s rRNA (as determined by gel electrophoresis) was corrected for cell loss by dividing each value by the corresponding cpm remaining in 14C-DNA. In the center panel, the top decay curve is for cells grown in 10% serum and the bottom is in 20% serum.
doubling every 39 hr (left) or 30 hr (center). Decay was seen both in total acid-precipitable material, in phenol-extracted RNA, and in separated 28s and 18s rRNA, with a half-life of approximately 70 hr over a 5 day period. Thus in contrast to bacteria (Davern and Meselson, 1960) and certain cells in tissue culture (Rake and Graham, 1962; Abelson et al., 1974; Kolodny, 1975), these primary human cells actively degrade mature 28s and 18s rRNA during exponential growth. To determine whether rRNA stability would change with growth rate, the normal feeding medium overlying growing fibroblasts was replaced with medium depleted of serum growth factors (Figure 1, right). Growth continued for 2 days and then markedly slowed down. The decay rate of the total RNA in serum-depleted cells was the same as in growing cells (68-72 hr). The same half-life of RNA was observed in cells rendered nondividing
I
by 3H-irradiation (Ehmann et al., 1975; Marz et al., 1976; Liebhaber and Schlessinger, 1978) or by passage into the nondividing “crisis” state (Wolf, 1977; further data to be published). The half-life of RNA prelabeled with 3H-methylmethionine thus showed no change during the transition from rapid growth to a resting state. Furthermore, during the period of exponential growth (days l-5), decay was similar whether RNA was labeled with 3H-methionine or 3H-uridine (Figure 1, left), confirming the equivalence of the two pulse-chase techniques. Previous work with 3T3 cells (Abelson et al., 1974; Kolodny, 1975) and chick fibroblasts (Weber, 1972) has shown that the stability of rRNA in those cells was dependent upon cell density. During exponential growth, the rRNA was stable, but as cell growth slowed consequent to cell crowding, rRNA degradation was initiated. To be certain that the instability of rRNA reported here was not due to such an effect of cell density, cells in the experiment of Figure 1 were plated at 10% (left) or 4% (center) of confluent density and showed the same half-life, although the cell doubling time decreased from 39 to 30 hr in cells plated at lower initial densities. Similarly, increasing the concentration of fetal calf serum from lo-20% did not affect RNA halflife (Figure 1, middle panel). rRNA Is Stable in Growing SV40-Transformed Fibroblasts To determine whether viral transformation of the WI-38 primary fibroblasts was associated with an increase in stability of the cytoplasmic rRNA, VA13, an established line of SV40-transformed WI-38 cells (positive for T antigen) was tested. Decay rates of RNA were determined using 3H-methylmethionine and 3H-uridine in separate trials (Figure 2, left). The same results were obtained with both pulse-chase methods: there was minimal decay of label in these exponentially growing cells, which were doubling every 23 hr in one trial and every 30 hr in a second (Figure 2, left). The slight increase in decay rate of the labeled RNA seen once the cells reached maximal density on the plates may be analogous to the initiation of degradation found in 3T3 cells (Abelson et al., 1974; Kolodny, 1975) and chick fibroblasts (Weber, 1972) after density inhibition of growth. Several additional clones of SV40-transformed WI-38 cells were made by Dr. S. Gotoh in our laboratory by infecting early passage WI-38 cells with SV40 (Girardi, Jensen and Koprowski, 1966) and subculturing the resultant foci of transformed cells. The clone used here (SV-C52) was T antigenpositive and morphologically similar to the established clone VA-13. Different clones of SV40-transformed WI-38 cells had different doubling times.
Regulation 123
of rRNA
2 Figure
4
2. Stability
Levels
6
in Fibroblasts
8 10 2 DAYS OF CHASE
of 3H-RNA
in Transformed
A
2
II 4
6
Cells
Decay curves of 3H-RNA in 3H-uridine (0) and 3H-methyl-methionine (V) labeled cells growing in media with unlabeled uridine and cytosine or methionine, respectively, are plotted (after correction for slight ‘%-DNA loss). Growth curves of the 3H-U (a) and JH-methyl-methionine (A) labeled cells are shown in the lower curves.
Clone SV-C.52, one of the fastest growing, had a doubling time similar to VA-13 (about 30 hr). Cells of SV-C52 were labeled at generations 44 and 53 with 3H-uridine and/or 3H-methyl-methionine in separate experiments. The labeled RNA in the acidprecipitated and phenol-extracted fractions of growing cells was as stable as in VA-13 cells (for example, Figure 1, middle panel). We emphasize that the shutdown of turnover in the SV40-transformed cells is not a simple correlate of a more rapid growth rate, because as indicated above, early passage cultures of human fibroblasts can divide as fast as many cultures of cloned transformed cells (for example, compare Figure 1, left, with Figure 2). When HeLa cells, an established epithelial cell line, were similarly labeled with 3H-uridine, they also exhibited stable rRNA. In this case, no turnover was seen even at maximum cell density (Figure 2, right panel). These results with two separately transformed and one established cell type suggest a characteristic of these cells that may be general: cytoplasmic rRNA is stable during growth, in contrast to the exponential degradation of rRNA in the growing primary cell cultures. Similar Nuclear rRNA Metabolism in Primary and Transformed Fibroblasts We next compared rates of transcription, processing and wastage of pre-rRNA as further potential sites of differential rRNA metabolism. An earlier paper (Wolf and Schlessinger, 1977) describes in detail the experimental design used here and justifies the assumptions of stochastic prOCeSSing
made in the quantitative analysis of the data. Briefly, steady state labeling kinetics of each of the pre-rRNA and rRNA species were determined for human fibroblasts in various growth states using 3H-methyl-methionine as the precursor. Results quantitated by this method and analyzed with the aid of a computer program have previously been shown to determine accurately the pre-t-RNA processing pathway and the processing half-lives in HeLa cells, and have also demonstrated that no wastage occurs in HeLa cells during rapid growth (Wolf and Schlessinger, 1977). The accumulation of 3H-methyl label in the individual pre-rRNA and rRNA species was determined from polyacrylamide gel profiles of purified total RNA extracted from VA-13 or WI-38 cells during continuous labeling. Subsequently, the kinetics of accumulation of 3Hlabeled rRNA were quantitated and analyzed using an IBM 360 computer with a simulation, analysis and modeling program (SAAM 23) (Berman, 1966). The program simultaneously obtains a least-squares fit to the accumulation data for all the RNA species, thereby determining rate constants for synthesis of the primary 45s transcript, processing of each of the pre-rRNA intermediates and any appreciable degradation of the pre-rRNA intermediates. The program also calculates the standard deviation of each rate constant and prints a table of the calculated accumulation values which correspond to the experimentally measured points (see Experimental Procedures). The intermediates of processing, as identified in polyacrylamide gels, were the same for both VA-13 and early passage WI-38 cells (Figure 3). For example, after 5 min (figure 3, top panels), the 45s precursor is clearly visible, and after 75 min, the 45s precursor, 32s intermediate, and 28s and 18s rRNA products can be seen in all the gel profiles (Figure 3, bottom panels). Computer analysis of the accumulation data produced good fits to the data with the standard prerRNA processing pathway. The calculated accumulation curves shown in Figure 4, for example, were obtained as a best fit using the well accepted processing of 45s to 32s and 2OS, and thence to 28s and 18s rRNA, in HeLa cells (for review, see Hadjiolov and Nikolaev, 1976). It can be seen in Table 1 that, in addition to following the same processing pathway, the intermediates in each of the cultures studied were processed with the same half-lives. The relative pool sizes also remained the same. Thus processing is indistinguishable in primary and transformed cells. The computer-assisted analysis of 3H-methyl-labeled RNA accumulation data was used to test for nuclear wastage of pre-rRNA species. Wastage of 45S, 32s and 20s pre-rRNAs was looked for by
Cell 124
Growing WI
ASS
38
17
325 285
I,
50
100
150
50
100
150
50
100
150
MINUTES
Figure 4. 3H-Methyl-Labeled Curves
GEL SLICE Figure
3. Polyacrylamide
Gel Profiles
of 3H-Methyl-Labeled
RNA
Cultures were labeled with 3H-methyl-methionine, and the RNA was extracted and run on polyacrylamide gels for 8 hr (as in Experimental Procedures). The top panels show profiles of RNA extracted after labeling for 5 min, and the lower panels after labeling for 75 min. 14C marker 23s and 16s E. coli rRNA was coelectrophoresed with the RNA from growing WI-38, and 14Cmarker 28s and 18s HeLa rRNA was co-electrophoresed with the RNA from VA-13.
including a pathway for wastage of each of the pre-rRNAs in the model used for the data analysis (Wolf and Schlessinger, 1977). In both cases, the pathways for wastage of 45S, 32s and 205 prerRNA were eliminated by the program as it reached the best fit to the data. For example, the solid lines in Figure 4 show the best computer fit for the measured accumulation data. The dashed lines represent predicted accumulation curves of 28s and 185 rRNA if 20% wastage is imposed on 32s and 20s pre-rRNA. It is clear that within a range of lo%, all the rRNA sequences which started in 45s pre-rRNA eventually appeared in 28s and 18s rRNA. Since the 45s pre-rRNA accumulation curves were comparable for both cellular growth states (Table 1) and no nuclear wastage was detected, the relative 455 synthesis rates could be estimated by labeling cellular RNA with a short pulse of 3Hmethyl-methionine and quantitating the label content of 45s pre-rRNA from polyacrylamide gel profiles. To eliminate the possibility of artifacts due to differences in the methionine-specific activity, the cells were labeled with a single lot of radioactive medium, and the methionine pool size in the cul-
Pre-rRNA
and
rRNA
Accumulation
The accumulation of label into pre-rRNA and rRNA species was quantitated and analyzed as described in the text and Experimental Procedures. The points represent the actual data, and the solid lines represent the calculated accumulation curves for the best computer fit to the data. The dashed lines illustrate theoretical accumulation curves assuming the same rates of synthesis and processing arrived in the “best fit,” but including 20% wastage of the synthesized 325 and 20s pre-rRNA. Relative moles were arrived at by dividing the accumulated counts in a species by the number of methyl groups per RNA chain of that species.
tures was measured directly (see Experimental Procedures). The methionine pool size was found to be the same in all cultures. Unexpectedly, the rate of rRNA synthesis in human diploid fibroblasts was found to vary in such a way that confluent cells contained about twice as much rRNA as rapidly growing cells. When confluent cells were replated, they doubled every 36 hr with a constant half-life of rRNA (as in Figures 1 and 2), but for 2-3 days, rRNA doubled only half as fast (Table 2). RNA then began to double as fast as the cells, and synthesis continued in confluent cultures at a rate sufficient to increase the content of rRNA 2 fold again. These findings will be detailed elsewhere, but one feature is relevant here: the net formation of rRNA could always be accounted for by the rates of rRNA synthesis and cytoplasmic turnover. For example (Table 2), when cells were increasing their rRNA content only 40% as fast as transformed cells, their rate of rRNA synthesis was 67% that of the transformed cells; the “extra” synthesis was just sufficient to balance rRNA turnover (see Discussion). Discussion These are the first data on the stability of rRNA and the maintenance of rRNA levels in diploid human fibroblasts. The half-life of rRNA was consistently about 70 hr in WI-38 cells (for example,
Regulatron 125
Table
of rRNA
1. Pre-rRNA
Levels
in Frbroblasts
Half-Lives
and Cellular
Pool Sizes
Table 2. rRNA Levels
Growing WI-38 T112 (min)
Growing
45s
16 + 5%
18 AZ 7%
pg RNA
325
37 2 8%
38 * 9%
cpm
VA-13 Cells per Culture
Pool
20s Pool Sizes
8 t 17% (Relative
Synthetic
7 * 14%
to 32s)
in 45s
ws RNA rRNA
Half-Life
rRNA
Doubling
45s
0.4 2 5%
0.5 k 7%
32s
1 .o ? 8%
1 .o * 9%
Measured
20s
0.2 + 17%
0.2 2 18%
Predicted
The half-lives were computed from the calculated rate constants as described in Experimental Procedures. The percentage errors are those arrived at by the SAAM program. Relative pool sizes were calculated by solving the differential equation for the model at the steady state limit. At that time, dqldt = 0 for each species q, and the equations reduce to simple algebraic relationships (see Wolf and Schlessinger, 1977). The level of 32s pre-rRNA was arbitrarily set equal to 1 .O and the other pool srzes calculated relative to the 32s soecies.
Figure 1 and Table 2). In more extensive studies (Wolf, 1977), the half-life of cytoplasmic rRNA was measured as diploid fibroblasts were passaged into the “crisis” phase after 50 -+ 2 doublings; the half-life remained 70 ? 7 hr at all growth rates-that is, throughout the range of growth rates expected for normal fibroblasts in vivo. The same half-life was also observed in other experiments with primary cultures of C3H mouse fibroblasts (unpublished results). These results are to be contrasted with the stability of rRNA in various growing transformed and established cell lines, including human VA-13 (Figure 2) and mouse 3T6 and 3T3 cells (Abelson et al., 1974; our confirmatory experiments). The near-constancy of rRNA half-life over a wide range of diploid fibroblast growth rates makes the curtailment of turnover in growing transformed cells seem very abrupt. No such qualitative difference between WI-38 and VA-13 cells was seen in nuclear rRNA metabolism. In both types of cell, the processing rates were very similar and no wastage was observed. Since they are the only control points at which changes are observed, the rates of rRNA synthesis and cytoplasmic turnover should fix the net rate of rRNA formation. Table 2 includes values consistent with this possibility. Using the relation in the legend to the table, the relevant rates predict a doubling time of 25 hr for RNA in VA-13 cells, compared to the observed 29 hr + 15%. In the same experiments, in a comparable analysis of nondividing cells, the corresponding synthesis and turnover rates predicted a doubling time of 180 hr, com-
x lo-’
and Turnover
Rates
and Steady
State
WI-36
VA-l 3
7.2 & 6%
a .2
12,11,13~10%
14,14,13+10%
340,312,303
545, 494,412
72hr*ll%
700 hr ? 10%
72 hr i 7%
29hrc15%
+ 6%
Time
25 hr
The number of cells per culture was determined by a hemocytometer count of a sample of cells from a petri dish treated in parallel with the labeled dishes. The net increase in extractable RNA was measured on a series of cell samples by ultraviolet absorption and by the orcinol reaction. Labeling, RNA extraction and gel electrophoresis were performed as described in Experimental Procedures. The data related to synthesis and pg RNA are from parallel samples in one experiment to ensure that the specific activity of the precursor in the medium was the same for all cultures. Note that in this experiment, the primary fibroblasts had been freshly plated and exhibited a doubling time of 72 hr for their RNA content for several days, even though the cell doubling time was 36 hr (see text). Half-lives of cytoplasmic turnover were determined from experiments like those in Figures 1 and 2. Predicted rRNA doubling times were calculated based on the following assumptions: the rate of rRNA production is proportional to the amount of existing rRNA, and the rate of rRNA decay is proportional to the amount of existing rRNA. (This is only a first-order approximation.) With those assumptions, dN/ dt = N-(In 2/Tl,z)N and hence, T,, = In 2/(a-In 2/T,,%). N is the amount of rRNA in the culture; T,,, is the cytoplasmic half-life of rRNA; TD is the rRNA doubling time; and 01 is the 45s rRNA synthesis rate constant. (Y is proportional to the incorporation rate per pg RNA listed in the Table. The proportionality constant was determined and then used to predict TD for other cultures (see text).
pared to the observed doubling time of 215 hr ? 10% (Wolf, 1977; data to be published). The adjustment of rRNA levels therefore occurs in very different ways in untransformed and transformed fibroblasts. In normal fibroblasts, changes of rRNA doubling time are associated with a relative balance of synthetic and degradative rates. This mode of regulation is different from that proposed by some speculative models, and in particular, it is not analogous to the regulation of rRNA levels in bacteria. In bacteria, regulation occurs almost exclusively by changes in the rate of synthesis during growth, and even in nutrient-limited bacteria, in which rRNA turnover begins, it proceeds at a rate only about 1% that of the maximal rate of synthesis (Koch, 1971). The established lines and viral transformed cells examined thus far resemble the bacterial paradigm more, since net rRNA accumulation is directly linked to the rate of synthesis. Another example of the selective curtailment of turnover has been re-
Cell 1’26
ported in kidney cells during renal hypertrophy (Melvin, Kumar and Malt, 1976), although net rRNA formation is transient in that case. The stabilization of rRNA in growing transformed fibroblasts does not seem to be a specific corollary of the faster growth of many transformed cells since it is observed in transformed cell cultures but not in normal cell cultures doubling at very similar rates (see Results, Figures 1 and 2). Comparable observations over a wider range of growth rates of transformed cells would strengthen this inference. We are attempting to test the possibility that the shutoff of t-RNA turnover is one of the pleiotropic effects of viral transformation, and that it may give the transformed cells a selective advantage. Experimental
Procedures
Cells Primary human fetal lung fibroblasts (WI-38), generation 18, and VA-13, an established cell line from a clone of SV40-transformed WI-38, were obtained from ATCC and grown at least eight generations before use. SV-C52 clone cells were obtained as a rapidly growing focus of cells after transformation at generation 20 by SV40 (the virus was obtained from Dr. D. Nathans, Johns Hopkins University). The cells were grown at 37°C in MEM-a with nonvolatile buffers fortified with 10% fetal calf serum (Kansas City Biologicals). Resting cells in Figure 1, right panel, were prepared by replacing normal growth media with MEM-a plus 2% FCS depleted of growth factors (by contact with a confluent monolayer of WI-38 cells for 72 hr.) Determination of 3H-Labeled RNA Decay On the indicated days, cells from two plates were trypsinized into 3 ml of 0.25 M sucrose and counted on a hemocytometer to obtain a growth curve. 0.5 ml of the suspended cells were precipitated with ice-cold 5% CCl&OOH. collected and washed on a glass fiber filter, and counted in toluene-based scintillation fluid with channels adjusted for separate counting of 3H and j4C. Decay rates of total 3H-labeled RNA were corrected for cell loss (usually i 1% per day) by plotting all values as 3H/‘4C. To measure the decay rates of individual rRNA species, the remaining 2.5 ml of cell suspension were brought to 100 mM Trisacetate (pH 5.4), 10 mM EDTA, 0.5% sodium dodecylsulfate, and RNA was extracted once at 55°C for 5 min with buffer-saturated phenol and a second time at 0°C with l/2 vol of phenol-chloroform-isoamyl alcohol (l:l:O.Ol). The final aqueous phases were precipitated with 2.5 vol of 100% ethanol and stored at -20°C until all samples were collected. Labeling and RNA Extraction to Determine Nuclear Metabolism of rRNA A detailed description of the protocol for 3H-methyl-methionine labeling and extracting rRNA is given in Wolf and Schlessinger (1977). To summarize, the washed cells were allowed to preincubate in fresh low methionine medium (1 .O pg/ml methionine) for 1 hr. Medium containing 3H-methyl-methionine was then added. For the kinetic analysis of pre-rRNA processing, the levels of methionine were as in Wolf and Schlessinger (1977), with about 100 &i/ml. At the indicated times, sample monolayers were rinsed with buffer and lysed in extraction buffer (0.1 M Trizma Base, 0.05 M EDTA, 0.5% sodium dodecylsulfate, adjusted with acetic acid to pH 5.4). The RNA was extracted several times with warm phenol and stored in 70% ethanol at -20°C. To determine the rates of synthesis of 45s pre-rRNA in the
cultures, the cells were preincubated in low methionine medium as described above and then labeled for 15 min in methioninefree medium containing 3H-methyl-methionine (11 Ci/mmole) at a concentration of 0.1 mCi/ml. In preparation for polyacrylamide gel electrophoresis, the samples were resuspended, and 0.1% bromophenol blue and marker YZ-labeled HeLa or E. coli ribosomal RNA were added. The samples were warmed at 55°C for 5 min, and a measured volume was then applied to the polyacrylamide gels. Polyacrylamide Disc Gel Electrophoresis and Quantitation The technique used for acrylamide gel electrophoresis was essentially that of Weinberg and Penman (1968). with the modifications described by Wolf and Schlessinger (1977). After electrophoresis, the gels were frozen and sliced. The slices were dissolved in vials containing 5% protosol (New England Nuclear) in toluene scintillator. The cpm were then summed up for each RNA species and analyzed as described below. Computer Modeling and Kinetic Analysis Based on the assumption of stochastic processing, the SAAM 23 program obtains a least-squares fit to the data for each pathway in a submitted model (Berman, 1966; Wolf and Schlessinger, 1977). A processing model is submitted to the SAAM 23 program by specifying an initial estimate of the rate constants for proposed steps, initial conditions, the accumulation data for the experiment and error estimates for the data. When desired, wastage pathways are included by submitting a rate constant for a pathway with no normal product. The program then iteratively adjusts the rate constants to minimize the sum of squares of the difference between the calculated and observed values (for further details, see Wolf and Schlessinger, 1977). Determination of Methionine Pool Sizes Cultures of cells in the growth states under study were incubated in low methionine medium (1.0 pg/ml methionine). After 1 hr, the medium was aspirated off, and macromolecules in the remaining monolayer were precipitated with 0.2 N perchloric acid at 0°C. The perchloric acid was pipetted off, and the monolayer was washed with fresh perchloric acid. The two perchloric acid washes were combined and neutralized with potassium hydroxide, and the potassium perchlorate was removed by centrifugation. The supernatant was removed, lyophilized, and then resuspended in a small volume and analyzed on a Beckman Model 1208 amino acid analyzer under standard conditions. The amount of methionine in each culture was quantitated from the elution profiles. Acknowledgments These investigations were aided by a grant from the NIH and a fellowship to S.A.L. Access to the IBM 360 and SAAM 23 program was through the Washington University Biomedical Computer Lab. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received
June
13,1977;
revised
November3,
1977
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