Nuclear RNA polymerase activities and poly(A)-containing mRNA accumulation in cultured AKR mouse embryo cells stimulated to proliferate

Printed in Sweden Copyright ~ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved ISSN 0014-4827

Experimental Cell Research 108 (1977) 157-165

NUCLEAR RNA POLYMERASE ACTIVITIES AND POLY(A)CONTAINING mRNA ACCUMULATION MOUSE EMBRYO CELLS STIMULATED

IN C U L T U R E D

AKR

TO P R O L I F E R A T E

E. W. BENZ, Jr, 1 M. J. GETZ, D. J. WELLS and H. L. M O S E S

Department of Pathology and Anatomy, Mayo Clinic and Foundation, Rochester, MN 55901, U S A

SUMMARY

When non-growing AKR-2B mouse embryo cells are stimulated to proliferate by changing from serum-deficient to fresh media containing 10% serum, there is a consistent lag of 12 h before the onset of DNA synthesis. Endogenous DNA-dependent RNA polymerase activities, binding and initiation sites on isolated chromatin for exogenous RNA polymerase, and the rate of accumulation of poly(A)-containing polysomal RNA has been examined in AKR-2B cells with emphasis on the interval between stimulation and the onset of DNA synthesis. RNA polymerase type II activity, which is responsible for transcription of heterogeneous nuclear RNA (hnRNA), was increased by 1 h after stimulation and at 6 h reached peak levels which were 60-100 % greater than the activity in resting cells. A rifampicin challenge method to assay for E. coli RNA polymerase binding and initiation sites on isolated chromatin was used to assay for changes in the amount of DNA available as a template for transcription. The results of this assay showed a slight decrease in the number of binding and initiation sites at 4 h followed by a slight increase at 6 h. The rate of accumulation of poly(A)-containing mRNA in polysomes showed a pattern and magnitude of increase following stimulation which was considerably different from that of RNA polymerase type II activity. There was a 4.5-fold increase over the resting levels by 2 h following stimulation. This enhanced level was maintained at all time points examined prior to the onset of DNA synthesis. These data suggest that both transcriptional and post-transcriptional mechanisms are responsible for the marked increase in polysomal poly(A)-containing mRNA observed after resting cells are stimulated to proliferate.

When non-growing cells in culture are stimulated to proliferate, they accumulate RNA and protein prior to the onset of DNA synthesis [1, 2]. All of the major classes of RNA accumulate long before DNA synthesis begins [3]. The coordinate increase in ribosomal RNA (rRNA) and transfer RNA (tRNA) is brought about by increased rates of synthesis and decreased rates of breakdown [4-6]. Cytoplasmic poly(A)-contain1 Present address: Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, New York, NY 10461, USA.

ing messenger RNA (mRNA) increases earlier and to a greater extent than the other RNAs and the increase in the rate of accumulation of mRNA after stimulation to grow more closely parallels the increase in protein synthesis than do increases in rRNA or tRNA accumulation [3]. Furthermore, recent studies using 5-fluoro-uridine (FUdR), an inhibitor of rRNA synthesis, have shown that both poly(A)-containing mRNA accumulation and the rate of protein synthesis increases during the first 8 h after the stimulus to proliferate, although riboExp Cell Res 108 (1977)

158

Benz et al.

some synthesis is completely blocked and tRNA accumulation is prevented [7]. This indicates that following stimulation of resting cells to proliferate, the level of m R N A is a major rate-controlling factor in the synthesis of proteins which are presumably necessary for initiation and maintenance of D N A synthesis prior to mitosis. The level at which this m R N A accumulation is controlled is, therefore, of considerable importance in discerning the mechanism of stimulation of cell proliferation. Although the increase in r R N A and tRNA is due to increased transcription and decreased breakdown [4-6], the mechanism of increased m R N A accumulation is much less clear. The rate of degradation of m R N A has been reported to be the same in resting and growing mouse fibroblasts lines, indicating that the accumulation o f poly(A)containing m R N A during the transition from the resting to the growing state is not due to increased m R N A stability, but to increased mRNA formation [6]. Johnson et al. [8] demonstrated that growing cells transfer twice as much of the poly(A)-containing R N A from the nucleus to the cytoplasm as do resting cells. In addition, increased incorporation of newly synthesized poly(A)-containing cytoplasmic R N A into polysomes following the stimulation of resting cells to proliferate has been reported [9, 10]. H o w e v e r , Mauck & Green [4] detected no increase in transcription of hnRNA, the apparent precursor of cytoplasmic m R N A [11, 12], during the transition from the resting to the growing state coinciding with the increased accumulation of cytoplasmic m R N A [3]. Detergent disrupted monolayers of 3T6 cells were utilized in this study, and no increase in the rate of h n R N A synthesis was detected until after the onset of D N A synthesis [4]. In the present study we have observed a Exp Cell Res I08 (1977)

previously unreported early increase in h n R N A synthesis following the stimulation of resting mouse fibroblasts to proliferate. This study has utilized measurement of endogenous D N A dependent RNA polymerase type II activity in isolated nuclei from A K R mouse e m b r y o cells (AKR-2B). Rifampicin challenge assays for E. coli R N A polymerase binding and initiation sites on isolated chromatin [13-15] indicated approximately equal numbers of sites in chromatin from either resting or stimulated cells. This is consistent with our recent demonstration that the great majority of the increased m R N A content in growing AKR-2B cells consists of m R N A species which are c o m m o n to both growing and resting cells [16]. In the present study the maximum increase in R N A polymerase type II activity was found to occur later and be of considerably lower magnitude than the increase in the rate of accumulation of polysomal poly(A)-containing mRNA.

MATERIALS AND METHODS

Cell culture AKR-2B cloned mouse embryo cells [17] were grown

in McCoy's 5a medium supplemented with 10% heatinactivated fetal bovine serum, 100 U]ml penicillin G and 100 t~g/ml streptomycin under a 5 % CO2, 95 % air environment at 36°C. The cells were passaged weekl7 by trypsinization and subsequent seeding at 3× 104 cells/cm~ in 75 cm2 flasks (Falcon) or 490 cmz plastic roller bottles (Coming). Medium was changed on day 3 or 4, and the cells were confluent by day 7. Non-growingcultures were produced by shifting the serum concentration of confluent cultures down to 0.5% for 48 h. These non-growing cells were stimulated to begin a new round of DNA synthesis and cell division by replacing the serum-deficient medium with fresh medium containing 10% serum.

Assay for DNA synthesis Duplicate cultures in roller bottles were exposed to

complete media containing 0.5 /xCi/ml [methyl-3H]thymidine (6.7 Ci//zmole) for 60 min. The incorporation of ['~H]thymidine into acid-precipitable material and the quantity of DNA was determined as de-

R N A polymerase and poly(A) m R N A in cell proliferation scribed previously [16]. For autoradiography cells from the same cultures were placed on glass slides and allowed to air-dry. ,After fixation in cold methanol-glacial acetic acid (3: 1), the slides were rinsed in cold absolute methanol, 10% trichloroacetic acid (TCA) and 70% ethanol. The air-dried slides were then coated with Ilford L-4 nuclear emulsion (diluted 1:1 with water). After exposure in a vacuum dessicator at 4°C, the slides were developed with Kodak D-19 developer and stained with Giemsa. At least 500 cells were counted per time point.

Assays for endogenous D N A dependent R N A polymerase activity For each time point cells from three roller bottles were harvested by trypsinization, collected in cold complete medium with serum and pooled for further processing. Cells were pelleted at 600 g at 4°C for 5 min and the pellets resuspended in 20 ml of 1.0 M sucrose in TKM buffer (0.01 M Tris-HC1, pH 7.5, 0.01 M KC1, 0.0015 M MgC12). The cells were disrupted by shearing with a Virtis homogenizer for 30 sec at 10000 rpm on ice. The nuclei were pelleted by centrifugation over a 4 cm cushion of 1.0 M sucrose in TKM for 15 rain at 1500 g in a swinging bucket rotor at 5°C. The nuclear pellet was immediately resuspended in 25 % glycerol, 0.0015 M MgC12, 0.01 M Tris-HCl, pH 7.4 (storage buffer) a t a concentration of 1-2 mg DNA as nuclei/ml. The reactions for the assay of endogenous RNA polymerase activities have been described elsewhere [18, 19]. Reactions were started by the addition of nuclei, and the mixtures (250/zl) were incubated at 15°C to minimize ribonuclease activity [18]. Assays for RNA polymerase type I activity (nucleolar; synthesizing preribosomal RNA) [20] were carried out for 20 min under low salt [0.05 M (NH4)zSO4] conditions in the presence of 0.8 /zg/ml of c~-amanitin. Assays for RNA polymerase II activity (nucleoplasmic; synthesizing DNA-like RNA) [20] were carried out for 10 rain under high salt [0.25 M (NH4)2SO4] conditions containing no c~-amanitin. The (NH4)2SO4 concentration needed for optimum polymerase type I! activity was the same for resting and growing cells and following the stimulus to proliferate. In some instances c~-amanitin at concentrations of 100 /zg/ml was used in reactions with both high and low saltconditions in order to determine the contribution of RNA polymerase III to the reaction [21]. The reactions were terminated by addition of 1.0 ml of cold 10% TCA, centrifuging, washing the pellet with 2.0 ml of cold 5% TCA containing 1% (w/w) Na4P2OT, and centrifuging again. The pellets, resuspended in the TCA-Na4P207 solution, were collected on Millipore filters, which were rinsed, dried and counted in a liquid scintillation spectrometer. The filters were then removed from the vials, dried and the DNA content]filter was determined by the diphenylamine reaction [22]. To confirm that the RNA product of the RNA polymerase II reaction was DNA-like and that for polymerase I was ribosomal RNA-like, the ratios of UMP to GMP incorporated were determined as described by Pogo et al. [23]. UMP to GMP ratios were 11-771801

159

determined using the endogenous polymerase assays in nuclei from cells 6 h after stimulation.

Rifampicin challenge assay Nuclei were isolated as described above for the RNA polymerase assay and further purified by resuspension, using a Teflon-glass homogenizer, in 0.5 M sucrose in TKM buffer containing 0.1% (v/v) Triton X-100. The homogenate was filtered through organza cloth (100 mesh) and centrifuged for 10 min at 10000 g. The pellets were subjected to a series of hypoosmotic buffers to obtain chromatin as described elsewhere [24]. Isolated chromatin (2-5 mg DNA/reaction) was used in rifampicin challenge assays as described elsewhere [13-15] in the presence of varying amounts of E. coli RNA polymerase partially purified according to the method of Burgess [25] through the agarose A-5 M chromatography step.

Labelling and extraction o f polysomal poly(A)-containing R N A For determination of the rate of accumulation of polysomal RNA during a serum stimulation experiment, duplicate cultures were labeled for 60 rain with [5-3H]uridine at a concentration of 1 p,Ci/ml prior to harvest. Unlabeled uridine was added to give a final concentration of 5.4× 10-6 M. This was in excess of that required to maintain linear incorporation in whole cell RNA for at least 3 h in non-confluent cells and for 2.0-2.5 h in confluent cultures. When the uridine concentration was lowered to 5.4× 10-7 M, incorporation was linear for only 1 h (fig. 1A); thus, radioactivity accumulating at the higher concentration represents mostly the accumulation of RNA rather than a change in the specific activity of internal precursor pools [26]. Since uridine concentrations in excess of 5.4× 10-6 M (constant specific activity) produced only very small increases in the specific activity of polysomal RNA obtained following a 3 h labelling period (fig. 1B), we conclude that this concentration is sufficient to virtually saturate internal precursor pools and to maintain these pools at constant specific activity for at least 120-180 min. Cells were harvested by a brief treatment with trypsin-EDTA and collected in fresh cold medium. Cells were disrupted with 0.5% NP-40 [27]. All buffers were sterilized with 0.01% diethylpyrocarbonate (DEP). The DEP was removed by heating at 70°C for 3 h before use. Crude nuclei were removed by low speed centrifugation and polysomes were prepared by adjusting the post-nuclear supernatant to 0.25 M sucrose and centrifuging for 10 min at 10000 rpm in the Beckman JS-13 rotor. The supernatant from this spin was made 1% in Triton X-100 and layered over 5 ml pads of 2 M sucrose in 35 ml polycarbonate tubes. Purified polysomal pellets were obtained by centrifuging 5-00-00 rpm for 2.5 h in the Beckman 60 Ti rotor. Polysomal RNA was extracted and poly(A)-containing RNA prepared by two cycles on oligo(dT)-cellulose columns as previously described [16, 28].

Exp CellRes 108 (1977)

Benz et al.

160 18 t6 t4

t2 tC

o

0.5

70

1.0

2.0

2.5

3.0

315

B

60 5O 40 50 20 t0

0

"1.5

~.--------,r-

018

'1'6

i

2,4

312

410

i

4,8

5[6

Fig. 1. Abscissa: (A) time (hours); (B) uridine M ×106; ordinate: (A) dpm xl0-S/mg DNA; (B) dpm × 10-4/mg DNA. ©, 5.4× 10-6 M uridine, subconfluent; m, 5.4x 10-7 M uridine, subconfluent; Q, 5.4x 10-6 M uridine, confluent; A, 5.4x 10-7 M uridine, confluent. (A) Rate of incorporation of pH]uridine into whole cell RNA as a function of external uridine conc. Duplicate roller bottles of confluent or sub-confluent AKR2B cells were adjusted to a final external uridine conc. of either 5.4× 10-6 or 5.4× 10-~ M unlabeled uridine containing 1 t~Ci/ml [5-3H]uridine. Cells were allowed to incorporate for the indicated time periods, chilled, harvested, and the TCA-precipitable material collected on filters. TCA-precipitable pH]uridine was determined by liquid scintillation counting and the DNA content of the filters determined by the diphenylamine reaction. The data are plotted as dpm/mg culture DNA to correct for small differences in the number of cells/ culture; (B) spec. act. of polysomal RNA as a function of external uridine conc. Duplicate roller bottles of rapidly growing AKR-2B cells were incubated with the indicated external uridine conc. (spec. act. held constant) for 3 h. Aliquots were taken for the measurement of DNA content and the radioactivity in polyribosomes determined as described in Materials and Methods.

that less than 0.5% of the cells are synthesizing DNA under these conditions. After shifting these non-growing cells to fresh medium containing 10% serum (0 time), there is a consistent lag period of 12 h before the onset of DNA synthesis as determined by [2H]thymidine incorporation [16] or by autoradiography (fig. 2). The data indicates that a minimum of 55 % of the cells undergo DNA synthesis between 12 and 26 h after stimulation (fig. 2). This compares favorably with other cell culture models for the study of cell proliferation [2]. A wave of mitotic figures follows the DNA synthesis by about 6-7 h beginning 19 h after stimulation (data not shown).

Endogenous nuclear R N A polymerase activities The procedures utilized for assay of endogenous nuclear RNA polymerase activities gave reproducible results with linear reactions for 25 rain of incubation with the low salt assay and 15 min with the high salt assay. The incorporation was shown to be DNA and nucleotide dependent, and the 5O

30

20

10

RESULTS

Stimulation of proliferation in AKR-2B cells Previous studies have shown that the culture of confluent or near-confluent AKR-2B cells in medium containing 0.5 % serum for 48 h results in a decline in the rate of DNA synthesis as determined by pH]thymidine incorporation to levels which are barely detectable [16]. Autoradiography indicates Exp Cell Res 108 (1977)

0

6

12

16 20

24

28

42

Fig. 2. Abscissa: time after stimulation (hours); ordinate: % labelled nuclei [3H]TMP. Autoradiography of [3H]thymidine incorporation into AKR-2B cells following the stimulation to proliferate. The cells were allowed to grow to confluence and were shifted to medium containing 0.5 % serum for 48 h. They were then stimulated to grow by changing to fresh medium containing 10% serum at zero time. Cells were exposed to [3H]thymidine for 60 rain prior to harvest at the time points indicated and autoradiography was performed as indicated in Materials and M e t h o d s .

RNA polymerase and poly(A) mRNA in cell proliferation Table 1. U[G ratio of polymerases I and H RNA product UMP ~

GMP b

U/G (Range)

Polymerase I 12.8+1.40 15.7_+0.75 0.82 (0.71~).96) Polymerase II 35.5-+1.14 22.8-+1.10 1.56 (1.33-1.72) a pmol [3H]UMP incorporated mg DNA -1. pmol [3H]GMP incorporated mg DNA -1.

product was shown to be RNA. The conditions for incubation (15°C) resulted in little or no detectable RNase activity. The reaction under low salt conditions in the presence of o~-amanitin (0.8 /xg/ml) would reflect both RNA polymerase type I and type III activities [21]; however, high concentrations of o~-amanitin (100 /xg/ml) gave little or no inhibition of the activity indicating that polymerase type III contributes very little to the reaction. The low salt assay was shown to yield an RNA with a ratio of uridine to guanosine similar to that of rRNA (table 1). The high salt reaction would reflect polymerase type II activity almost exclusively since 95% of the reactivity is abolished when 0.2/xg of o~-amanitin is included in the reaction mixture. The high salt assay gives an RNA with a uridine to guanosine ratio of 1.56 indicating that it represents DNA-like RNA (table 1). Table 2 shows the response of RNA polymerase type I to stimulation of cell proliferation. There is a significant increase over resting levels (0 time) at the earliest time point tested (1 h), and all time points tested following stimulation had polymerase type I levels significantly greater than resting with a maximum increase of 400 % obtained at 24 h. Fig. 3 illustrates the typical response of polymerase type II as the cells approach the resting stage at 0 time and following the stimulation to proliferate. The results of several repeat experiments on resting and

161

stimulated cells are presented in table 3. There is a significant increase in polymerase type II activity at 1 h, and the peak of activity was obtained at 6 h where the levels are 60-100% greater than those obtained with resting (0 time) cells. This increase was observed consistently and the differences from resting levels were statistically significant for the 1, 2, 4, 6 and 8 h time points (table 3). Fig. 3 shows the results of a simultaneous analysis of RNA polymerase II activity and poly(A)-containing mRNA accumulation as determined on the same batch of cells during the approach to the resting state, at the resting state, and following the stimulus to proliferate. The RNA data has been published previously [16] but is shown here for comparison. The RNA polymerase II activity exhibits a pattern very similar to that described in table 3 while the rate of accumulation of poly(A)-containing mRNA exhibits a considerably different pattern. The rate decreased very markedly as the cells ceased proliferation, and there was a marked (4.5-fold) increase in the rate of accumulation at 2 h following stimulation. Table 2. The effect of stimulation on polymerase I Time (hours)

Activity a after stimulation

Ratio stimulated: resting

pb

0 (3) 1 (3) 2 (4) 4 (4) 6 (3) 8 (3) 12 (3) 24 (3)

17.60_+ 2.15 25.63_+ 1.34 28.03_+ 1.14 33.10+ 3.39 39.65_+ 0.85 33.37_+ 2.57 28.77_+ 2.95 88.82_+ 14.02

1.00 1.46 1.59 1.88 2.25 1.90 1.64 5.05

<0.05 <0,05 <0,05 <0.05 <0.05 <0.05 <0,05

The numbers in parentheses are the numbers of independent experiments. Each determination was performed in triplicate for each experiment. a pmol [3H]UMP incorporated mg DNA -1 20 rain 1. b p values determined from the r statistic for means of two samples. Exp Cell Res 108 (1977)

162

Benz et al.

but reflects in large measure, increased RNA accumulation (fig. 1 and Materials and Methods). Exogenous R N A polymerase binding and initiation on isolated chromatin

Under the assay conditions used, the endogenous RNA polymerase type II activity has two potential rate limiting factors: the amount of active enzyme present and the amount of DNA available as template for transcription [19]. In order to investigate -72 -48 -24 0"+2 112 '18 24 Fig. 3. Abscissa: time relative to serum stimulation the latter parameter, we utilized a rifampi(hours); ordinate: ratio of activity to resting levels cin challenge assay described by Tsai et al. (zero time). Endogenous RNA polymerase type II activity (I-q) [13, 15] for E. coli RNA polymerase bindand rate of accumulation of poly(A)-containing mRNA ing and initiation sites on isolated chromainto polysomes (11) as AKR-2B cells approach the resting state (minus 72 h to zero time) and following the tin. After preincubation of chromatin with stimulation to proliferate at zero time. The cells were varying amounts of E. coli RNA polymerin media containing 0.5% serum from minus 48 h to zero time when they were changed to fresh medium ase in the absence of nucleotides to allow containing 10 % serum. Cells for determination of the binding of polymerase to chromatin, nurate of poly(A)-containing RNA accumulation were pulsed with [3H]uridine for 1 h prior to harvest at the cleotides are added to permit elongation time indicated in the abscissa. Poly(A)-containing along with rifampicin and heparin which polysomal RNA was isolated and the activity of incorporated tritium was determined. Replicate bottles prevents reinitiation [13]. The point at of cells were harvested at the same times for deter- which the saturation curve underwent a mination of endogenous RNA polymerase type II activity. The results are expressed as the ratio of the change in slope was determined and the activity to that of resting cells (zero time). values of [14CLUMP incorporated and This same rate of accumulation was maintained at the 4, 6 and 8 h time points. Even greater rates of accumulation were observed at the 18 and 24 h time points following stimulation. Thus in the same cells, RNA polymerase type II activity and polysomal poly(A)-containing mRNA accumulation exhibit a non-coordinated response to a stimulus to proliferate. It is important to note that a 4-5-fold increased poly(A)-containing mRNA content of growing cells as compared with resting cells was demonstrated in a previous study [16] on the basis of the absorbance at 260 nm of purified poly(A)-containing RNA. The increase shown in fig. 3 is, therefore, not due simply to changing precursor pool concentrations Exp Cell Res 108 (1977)

Table 3. Effect of stimulation on polymerase H Time (hours)

Activity a after stimulation

0 (3) 1 (3) 2 (4) 4 (5) 6 (3) 8 (3) 12 (3) 24 (3)

32.30_+4.00 41.13_+2.80 51.80+7.28 51.40+5.30 59.90__+7.28 54.66+2.91 38.73-+4.61 50.20_+8.01

Ratio stimulated: resting 1.00 1.27 1.60 1.59 1.85 1.69 1.20 1.55

pb

<0.05 <0.05 <0.05 <0.05 <0.05 NS <0.05

The numbers in parentheses are the numbers of independent experiments. Each determination was performed in triplicate for each experiment. a pmol [aH]UMP incorporated mg DNA -1 10 min-L b p values determined from the ~" statistic for means of two samples. NS, not significant.

RNA polymerase and poly(A) mRNA in cell proliferation z.oV

P~

"L8

"12

'10

OB

4h 6h 6h tO%serum "~0%serum 0.5%serum

Fig. 4. Ordinate: ratio stimulated/resting. The relative activity of endogenous RNA polymerase type II (P II) in nuclei and the numbers of initiation sites (A) and binding sites (B) for E. coli RNA polymerase on isolated chromatin of AKR-2B cells. The cells were harvested 4 h (left bars) and 6 h (middle bars) after stimulation of resting cells to proliferate by changing to fresh medium containing 10% serum. A control group was changed to medium containing 0.5 % serum and harvested 6 h later (right bars). All of the data in this graph were obtained from the same population of cells and the results are expressed as the ratio of the activity to that of resting cells (zero time).

amount of polymerase present at this point were utilized to calculate estimated binding and initiation sites as described previously [13]. The 4 and 6 h time points were chosen for study in comparison to resting cells, and the results are shown in fig. 4 in comparison to polymerase type II activity obtained from assays on the Same batch of cells utilized for the rifampicin challenge assays. The data in fig. 4 are presented as ratios of stimulated to resting levels. The resting values obtained for initiation sites was 2.43 x 105/pg DNA and for binding sites was 4.68x10~/pg DNA. These values are not presented as an indication of the number of actual binding and initiation sites for endogenous RNA polymerase in the living cells, and the assay is utilized in this study only as an indicator for relative changes in chromatin template capacity under different experimental conditions. As shown in fig. 4, there was a decrease in both initiation (A)

163

and binding (B) sites at 4 h following stimulation in conjunction with a 55 % increase in polymerase type II activity. There was an 11% increase in initiation sites and a 21% increase in binding sites at 6 h in comparison to a 95 % increase in polymerase type II activity. As a further control, cells in which the media was changed at 0 time, but replaced with media containing only 0.5% serum instead of 10% serum, were assayed for polymerase type II activity and E. coli RNA polymerase binding and initiation sites. As shown in fig. 4, only slight changes relative to the values obtained for resting cells were observed.

DISCUSSION We have shown that endogenous nuclear RNA polymerase type II activity is higher in growing than in resting cells and that there is an early (1-2 h) increase following the stimulation of resting cells to proliferate. This enzyme is responsible for the synthesis of hnRNA, at least a fraction of which contains sequences destined to become cytoplasmic mRNA, and the demonstrated increases in activity may account for some of the increased mRNA present in growing cells and resting cells following growth stimulation. However, determinations of the rate of accumulation of polysomal poly(A)-containing mRNA revealed a different pattern and considerably greater magnitude of increase than the increases in polymerase type II. This suggests that transcription is not the rate-limiting step controlling the initial increased mRNA accumulation in stimulated cells, and that posttranscriptional mechanisms may play a role. The major uncertainty regarding this conclusion is our lack of knowledge concerning the exact fraction of polymerase II Exp Cell Res 108 (1977)

164

B e n z et al.

transcripts which correspond to proteinencoding sequences. Recent studies by Johnson et al. [29] indicate that the efficiency with which cytoplasmic mRNA is produced from hnRNA is low and suggest that not every hnRNA molecule gives rise to a cytoplasmic message. If this is true and if the increased polymerase type II activity is selective for those enzyme molecules transcribing sequences destined to become mRNA, transcription could be the rate limiting factor controlling the increased mRNA accumulation in cells following stimulation. Alternatively, the early increase in cytoplasmic levels of mRNA may be'accounted for by increased mRNA transport. Johnson et al. [8] have also reported an increased transport of poly(A)-containing RNA from the nucleus to the cytoplasm in growing cells as compared to resting cells under conditions where further polyadenylation was inhibited by cordycepin. Another likely mechanism which may explain the discrepancy between rates of mRNA synthesis and the accumulation in polysomes after growth stimulation is the rapid movement of poly(A)-containing RNA from cytoplasmic ribonucleoprotein particles into polysomes as reported by other investigators [9, 10]. Mauck & Green [4] did not observe an increase in hnRNA synthesis following growth stimulation in mouse 3T6 cells until the onset of DNA synthesis, and the increase was then proportional to the increase in DNA. One possible reason for the discrepancy of their results with ours is the different cell lines used, although both are continuous lines of fibroblasts derived from mouse embryos and have many common properties. An alternative possibility for this discrepancy is the different methods used for assay of RNA polymerase type II activity (hnRNA synthesis). Mauck & Exp Cell Res 108 (1977)

Green used detergent disrupted monolayers of cells for their assays [4]. We have previously shown that detergent treatment of chick oviduct nuclei significantly reduces the nuclear endogenous polymerase type II activity [30]. The mechanism of the early increase in polymerase II activity in fibroblasts after stimulation to grow is still open to question. There are several possible mechanisms for the increase in polymerase type II activity as measured in this assay. Among these are changes in genome restriction to provide additional DNA template for transcription. The results of our previous study [16] suggest that if this were the explanation for the increased polymerase type II activity, it is not reflected as an increase in the number of diverse mRNA species on the polysomes. Williams & Penman [31] have also demonstrated that cell proliferation does not detectably alter the diversity of mRNA in cultured mouse fibroblasts. Thus, unless the new polymerase type II transcripts are restricted to and degraded in the nucleus, the increase in polymerase type II activity associated with growth stimulation probably is not due to transcription of previously non-transcribed structural genes. Alternatively, the increased activity could reflect an increase in the transcription rate of sequences which are also transcribed in resting cells. This is consistent with the measurement of approximately equal numbers of E. coli RNA polymerase binding and initiation sites in isolated chromatin from resting and stimulated cells (fig. 3). Farber et al. [32] and Baserga et al. [33] reported early increases in chromatin template activity in WI38 cells following the stimulation of resting cells to proliferate. More recent studies from the same laboratory, however, have shown that this change

RNA polymerase and poly(A) mRNA in cell proliferation in template activity is related to changes in nucleolar structure and not alterations in templating for hnRNA synthesis [34]. The method of chromatin isolation utilized in our study, which involves exposure of chromatin to sodium chloride concentrations as high as 0.35 M [24], would probably prevent this nucleolar effect [35]. Although the mechanism of increased nuclear RNA polymerase activity has not been firmly established, the observation of an early increase in endogenous RNA polymerase type II activity following the stimulation of resting fibroblasts to proliferate is a factor which must be taken into account when considering the mechanism of stimulated cell proliferation. We thank Drs W. Rowe and N. Teich for the original seed stock of AKR-2B cells and Dr T. C. Spelsberg for helpful advice and critical review of the manuscript. Mary E. Volkenant provided skilled technical assistance. This investigation was supported by Grant no. CA 16816, awarded by the NCI, DHEW; Grant no. NP192, awarded by the ACS; and the Mayo Foundation.

REFERENCES 1. Todaro, G J, Lazar, G K & Green, H, J cell comp physio166 (1963) 325. 2. Todaro, T, Matsuya, Y, Bloom, S, Robbins, A & Green, H, Wistar inst symp monograph (ed V Defendi & M Stoker) no. 7, pp. 87-101. Philadelphia, Pa (1967). 3. Johnson, L F, Abelson, H T, Green, H & Penman, S, Cell 1 (1974) 95. 4. Mauck, J C & Green, H, Proc natl acad sci US 70 (1973) 2819. 5. - - Cell 3 (1974) 171. 6. Abelson, H T, Johnson, L F, Penman, S & Green, H, Cell 1 (1974) 161. 7. Johnson, L F, Penman, S & Green, H, J cell physiol 87 (1976) 141.

165

8. Johnson, L F, Williams, J G, Abelson, H T, Green, H & Penman, S, Cell 4 (1975) 69. 9. Bandman, E & Gurney, T, Jr, Exp cell res 90 (1975) 159. 10. Rudland, P S, Proc natl acad sci US 71 (1974) 750. 11. Edmonds, M, Vaughan, M H, Jr & Nakazato, H, Proc natl acad sci US 68 (1971) 1336. 12. Lewin, B, Cell 4 (1975) 77. 13. Tsai, M-J, Schwartz, R J, Tsai, S Y & O'Malley, B W, J biol chem 250 (1975) 5165. 14. Schwartz, R J, Tsai, M-J, Tsai, S Y & O'Malley, B W, J biol chem 250 (1975) 5175. 15. Tsai, S Y, Tsai, M-J, Schwartz, R, Kalimi, M, Clark, J H & O'Malley, B, Proc natl acad sci US 72 (1975) 4228. 16. Getz, M J, Elder, P K, Benz, E W, Jr, Stephens, R E & Moses, H L, Cell 7 (1976) 255. 17. Teich, N, Lowy, D R, Hartley, J W & Rowe, W P, Virology 51 (1973) 163. 18. Glasser, S R, Chytil, F & Spelsberg, T C, Biochem j 130 (1972) 947. 19. Moses, H L, Spelsberg, T C, Korinek, J & Chytil, F, Mol pharmacol 12 (1976) 731. 20. Chambon, P, Gissinger, F, Mandel, J L, Jr, Kedinger, C, Gnizadowski, M & Meihlac, M, Cold Spring Harbor symp quant bio165 (1970) 693. 21. Weinman, R & Roeder, R G, Proc natl acad sci US 71 (1974) 1790. 22. Burton, K, Biochemj 62 (1956) 315. 23. Pogo, A O, Littau, V C, Allfrey, V G & Mirsky, A E, Biochemistry 57 (1967) 743. 24. Spelsberg, T C, Steggles, A W & O'Malley, B W, J biol chem 246 (1971) 4188. 25. Burgess, R R, J biol chem 244 (1969) 6160. 26. Perry, R P, Kelley, D E & LaTorre, J, J mol biol 82 (1974) 315. 27. Borun, TW, Scharff, M C & Robbins, E, Biochim biophys acta 149 (1967) 302. 28. Getz, M J, Birnie, G D, Young, B D, MacPhail, E & Paul, J, Cell 4 (1975) 121. 29. Johnson, L F, Levis, R, Abelson, H T, Green, H & Penman, S, J cell bio171 (1976) 933. 30. Knowler, J T, Moses, H L & Spelsberg, T C, J cell biol 59 (1973) 685. 31. Williams, J G & Penman, S, Cell 6 (1975) 197. 32. Farber, J, Robera, G & Baserga, R, Biochem j 122 (1971) 189. 33. Baserga, R, Costlow, M & Rovera, G, Fed proc 32 (1973) 2115. 34. Baserga, R, Huang, C-H, Rossini, M, Chang, H & Ming, P-M L, Cancer res 36 (1976) 4297. 35. Baserga, R & Nicolini, C, Biochim biophys acta 458 (1976) 109. Received January 28, 1977 Accepted February 7, 1977

Exp Cell Res 108 (1977)