88,82-91
VIROLOGY
(1978)
Stimulation
SANDRA Department
of
of RNA Synthesis in Isolated Nucleoli by Preparations Simian Virus 40 T Antigen WHELLY,’
TOSHINORI
IDE,’
AND
RENATO
of
BASERGA3
Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Accepted March 26, 1978
Partially purified and highly purified preparations of simian virus 40 (SV40) T antigen from SV80 cells stimulate the incorporation of [3H]UTP into RNA in isolated rat liver nucleoli. The increased incorporation is dependent on an endogenous o-amanitin-resistant RNA polymerase, and ribosomal RNA sequences are present in the product, T antigen was also partially purified from Chinese hamster cells transformed by a tsA mutant of SV40. The complement-fixing activity of tsT was rapidly inactivated by heating at 50”, and this inactivation was paralleled by a marked decrease in its ability to stimulate nucleolar RNA synthesis in vitro. Under these same conditions, T preparations from wild-type transformed cells were more resistant, in terms of both complement-firing activity and ability to stimulate nucleolar RNA synthesis The stimulation of nucleolar RNA synthesis with wildtype T preparations of different degrees of purity correlated with the ability of the preparations to fix complement, even with highly purified preparations. Finally, cellular RNA synthesis was also stimulated when quiescent 3T3 cells were infected with wild-type SV40. The results do not prove, but strongly suggest, that one of the sites of action of SV40 T antigen may be at the level of transcription of nucleolar DNA.
(1) preparations of SV40 T antigen can directly stimulate RNA synthesis in isolated nucleoli from rat liver; (2) the nucleolar product has, at least partially, the characteristics of preribosomal RNA; (3) stimulation of nucleolar RNA synthesis can also be achieved with highly purified preparations of SV40 T antigen; and (4) an increased incorporation of [3H]uridine into RNA can also be demonstrated in vivo in SV40-infected 3T3 cells.
INTRODUCTION
In a previous communication (Ide et al. 1977), we reported that partially purified preparations of simian virus 40 (SV40) T antigen stimulated RNA synthesis in nuclei isolated from either rat liver or quiescent hamster cells in culture. The increase in nuclear RNA synthesis was abolished by pretreatment with anti-T antiserum. Although several lines of evidence suggested that the T antigen in these partially purified preparations was responsible for the stimulation of nuclear RNA synthesis, the preparations used were contaminated by other nuclear proteins. The present communication reports that:
MATERIALS
82 Inc. reserved.
METHODS
Cells. SV80 cells (a gift from D. M. Livingston, Sidney Farber Cancer Center) were grown in Dulbecco’s medium, as described by Jesse1et al. (1975). CHL cells were of two types. CHL wt 15 cells transformed by wild-type SV40, and CHL A239 Ll cells transformed by a tsA mutant of SV40. Both of these cell lines were obtained from Dr. Robert G. Martin and have been previously described (Martin and Chou, 1975; Tenen et al. 1977b). For in vivo experiments, 3T3 cells (kindly
’ Present address: University of Nebraska Medical Center, Obstetrics and Gynecology Research Laboratory, Conkling Hall, 42nd and Dewey Avenue, Omaha, Nebraska 68105. ‘Present address: Department of Virology, Institute of Medical Science, Tokyo University, Minatoku, Tokyo, Japan. 3 To whom requests for reprints should be sent.
0042-sS22/78/~1-0082$02.00/0 Copyright 0 1978 by Academic Press, All rights of reproduction in any form
AND
T ANTIGEN
AND
given to us by Daniel Nathans) were plated in 16-mm microwells and grown in Dulbecco’s medium plus 10% fetal calf serum until quiescent (7 days after plating). Isolation of nuclei and assay for nuclear RNA synthesis. The isolation of nuclei from
rat liver was carried out by the method of Marzluff et al. (1973) with slight modifications which have been described in detail in the previous paper (Ide et al. 1977). Similarly, the method of Marzluff et al. (1973) was followed for the determination of RNA synthesis in vitro. The dependence of the assay on the presence of the four ribonucleotide triphosphates, the inhibition by actinomycin D, etc., have already been discussed by Marzluff et al. (1973) and in papers from our laboratory (Kane et al. 1976; Baserga et al. 1978). The final volume of the assays was 125 ~1 and each assay contained approximately 5 pg of DNA as nuclei. All assays were performed at 37” and were terminated after 15 min of incubation, unless otherwise specified. The control values were always at least three to four times the background counts per minute. Isolation of nucleoli cleolar RNA synthesis.
and assay for nu-
Nucleoli were isolated by the method of Muramatsu et al. (1974) with slight modifications, as described by Huang and Baserga (1976). Although the definition of what constitutes a nucleolus may vary from one laboratory to another, the procedure we used is the one most commonly adopted for the preparation of isolated nucleoli (Yu and Feigelson, 1971; Grumm t 1975; S&mid and Sekeris, 1975; Matsui et al., 1976; Rothblum et al., 1977). These nucleoli are highly pure, as can be judged by electron microscopy. The criteria used for the purity of nucleoli have already been described in a previous paper from this laboratory (Huang and Baserga, 1976). Although a smah degree of contamination by perinucleolar chromatin cannot be ruled out, RNA synthesis in such nucleoli is 100% resistant to even high concentrations of a-amanitin (Coupar and Chesterton, 1975) and reasonably reproduces in uiuo synthesis (Ballal et al., 1977). The assay for nucleolar RNA synthesis was carried out according to the technique of Mar-
RNA
SYNTHESIS
83
zluff et al. (1973) for determination of RNA synthesis in isolated nuclei. The cofactor’s requirements for our nucleolar preparations were essentiahy the same as described by us and other investigators (Villalobos et al., 1964; Lindell, 1975; Ferencz and Seifart, 1975). The assay simply measures incorporation of r3H]UTP into RNA by an endogenous RNA polymerase resistant to aamanitin. For convenience and following the example of other investigators (see below), we shall briefly refer to this measurement as a measurement of nucleolar RNA synthesis. We prefer the term nucleolar RNA synthesis (rather than preribosomal RNA synthesis) because it is possible that the nucleolus may also synthesize nonribosomal RNA (Beebee and Butterworth, 1977). Preparation of T protein. Most of the experiments, except those described in Fig. 7, were carried out with partially purified preparations of SV40 T antigen, prepared by the method of Jesse1 et al. (1975), as previously described (Ide et al., 1977). Highly purified T antigen was prepared by a modification of the method of Tenen et al. (1977a) as follows. Active fractions of T antigen from a DE 52 column were concentrated by addition of ammonium sulfate (final SO%), dialyzed against 106 vol of buffer A [50 mu Tris-HCl, pH 7.4, 3 mu dithiothreitol (DTT), and 20% glycerol) for 5 hr with two changes of buffer, and applied on a heparin-Sepharose column (15~ml bed volume). The column was washed with 3 column vol of buffer A and eluted with a 1Zcolumn-vol linear gradient between 0 and 0.6 M NaCl in buffer A. T antigen-containing fractions (0.39-0.4 M NaCl fractions) as assayed by CF were pooled, adjusted to pH 6.0, and dialyzed against 100 vol of buffer B (50 mM Tris, pH 6.0,3 mM DTT, and 20% glycerol) for 4 hr with two changes of buffer. The dialyzed T preparations were applied to a DNA-cellulose column (4-m.l bed volume) very slowly. The column was washed with 3 column vol of buffer B and eluted with a 20-column-vol linear gradient between 0 and 0.8 M NaCl in buffer C (100 m&f Tris-HCl, pH 8.0, 3 mM DTT, and 20% glycerol). T antigen fractions were pooled,
a4
WHELLY,
IDE,
concentrated by addition of Sephadex G200 outside the dialysis bag, and dialyzed against 200 vol of TGMED (50 n&f Tris-HCl, 25% glycerol, Mg acetate 5 mM, DTT 1.5 mM, and EDTA 0.1 mM) for 4 hr with two changes of buffer. Complement fixation. Complement-fixation tests for T antigen were carried out by the microtiter technique of Sever (1961). RNA/DNA hybridization. Competitive hybridization of RNA to DNA on nitrocellulose filters was performed according to the procedure of Gillespie (1968). Purified rat liver nucleolar DNA was heat denatured at 100” in %o SSC (0.015 M NaCZ; 0.0015 M Na-citrate, pH 7.0). Heat denatured DNA was annealed to nitrocellulose filters in 4x SSC. The percentage of DNA binding to each filter was monitored by optical density. After drying, the DNA-nitrocelhilose filters were baked at 90” for 1 hr. Varying concentrations of unlabeled cytoplasmic ribosomal RNA (purified from rat liver) or unlabeled transfer RNA (Sigma) were hybridized to DNA-nitrocellulose filters at 66” for 30 hr in 4~ SSC. The filters were mildly treated with RNase (pretreated at 100’) and washed thoroughly. The RNA-DNA nitrocellulose filters were then incubated with the [3H]RNA product isolated from the nucleolar template assay made in the presence of T antigen. The competitive hybridization was carried out at 55” for 30 hr following digestion with RNase and washing of the filters. The amount of radioactivity still hybridized to nucleolar DNA was determined in a Packard scintillation counter. Controls were carried out in the absence of cold RNA. Values reported were corrected for nonspecific binding of [3H]RNA to the filters. SV40 infection of 3T3 cells. This was done essentially by the method of Scott et al. (1976). Quiescent 3T3 cells were infected at a m.o.i. of 1000 with a suspension of KBrpurified SV40 in phosphate-buffered saline. After adsorption, the virus suspension was diluted with fresh medium (no serum) and conditioned medium in a ratio of 9:l. For DNA synthesis, the cells were labeled for 1 hr with [3H]thymidine (10 fCi/welI), and for RNA synthesis, with [ Hluridine (20 &i/well) also for 1 hr. The amount of radioactivity incorporated into acid-precip-
AND
BASERGA
itable material was determined scintillation counting.
by liquid
Materials Isotopes. [5-3H]UTP (22.0 Ci/mmol), [methyl-3H]thymidine (6.7 Ci/mmol), and [5-3H]uridine (26.7 Ci/mmol) were purchased from the New England Nuclear Corp. Heparin-Sepharose column. CNBr-Activated Sepharose 4B (Pharmacia Fine Chemicals) was coupled with heparin (Inolex Corporation, Chicago, Ill) in coupling buffer (0.1 M NaHC03, pH 8.3,0.5 M NaCl) for 2 hr at room temperature. The coupled Sepharose was washed exhaustively with coupling buffer, 1 M ethanolamine solution (pH 8.0), coupling buffer, acetate buffer (0.1 M, pH 4.0, 0.5 M NaCl), and coupling buffer. Then, the resin was equilibrated with buffer (50 r&f Tris-HCl, pH 7.4, 3 m&f DTT, and 20% glycerol) and poured into a column. DNA-cellulose column. DNA-Cellulose was prepared by the method of Alberts and Herrick (1971) by using calf thymus DNA (Type 1, Sigma) and cellulose powder (Cellex 410, Bio-Rad Laboratories). Amount of bound DNA was 1.37 mg/ml of packed we1 volume of cellulose. RESULTS
Effect of T-Antigen Preparations on RNA Synthesis in Isolated Nucleoli Figure 1 shows the effect of partially purified T-antigen preparations on RNA synthesis in isolated nuclei and nucleoli from rat liver. As reported in the previous paper (Ide et al., 1977) RNA synthesis in isolated nuclei is stimulated by the addition of T-antigen preparations, at least twofold. B in Fig. 1 shows that good stimulation of RNA synthesis by T-antigen preparations can also be obtained with isolated nucleoli. In this paper we use the term “RNA synthesis” for its convenient brevity to express the incorporation of [3H]UTP into acid-insoluble material (Marxhiff et al., 1973). Other investigators used, for the same assay, the expression “endogenous RNA polymerase activity” (Lindell, 1975; Weinmann et al., 1976; Hardin et al., 1976). As already reported in this, as well as other laboratories (Marxluff et al., 1973; Kane et
T ANTIGEN
100
AND
CY 0
5
10
15 TIME
20 0 1 MIN
4, 4,000
5
IO
15
20
i
FIG. 1. Time course of C3H]UTP incorporation into RNA in isolated rat liver nuclei and nucleoli, with or without the addition of SV40 T-antigen preparations, The abscissa is the time of incubation, The ordinate represents the counts per minute incorporated into RNA per microgram of DNA. The nuclei or nucleoli were preincubated with T-antigen preparations at 25” for 30 or 15 min, respectively. At the end of the preincubation period the ribonucleotide triphosphates and the necessary ions were added, and the reaction was stopped at the times indicated on the abscissa. (A) Nuclei; (B) nucleoli. (O---O) No T antigen added; (A---A) addition of T-antigen preparations, 8 ag of protein/assay.
RNA
85
SYNTHESIS
assay was terminated after a further 15 min. Table 1 shows that the stimulation of RNA synthesis in isolated rat liver nuclei by T-antigen preparations is largely aamanitin resistant. Nucleolar RNA synthesis is, as expected, 100% a-amanitin resistant, in the presence or absence of T. Although this does not exclude that extra nucleolar genes may also be activated, it seems to indicate that at least most of the increase in RNA synthesis can be attributed to an increased activity of the nucleolus. The nature of the product synthesized by the isolated nucleoli after T-antigen stimulation was further studied by RNA/DNA hybridization. Using the RNA/DNA hybridization technique of Gillespie (1968), we found that the product of nucleolar RNA synthesis did not appreciably hybridize to total cellular DNA, but it did hybridize to nucleolar DNA. In addition, Fig. 4 showed that hybridization of the nucleolar product to nucleolar DNA is largely competed out by cold ribosomal RNA. The addition of transfer RNA to the hybridization mixture
al., 1976; Chiu and Baserga, 1975), the assay
is dependent upon the presence of ribonucleotide triphosphates, the product is digestible with RNase, and the incorporation of [3H]UTP is totally inhibited by actinomytin D. This is true of nuclei as well as nucleoli (Villalobos et a& 1964; Grummt, 1975; Baserga et al, 1978). The extent of stimulation of RNA synthesis in isolated nucleoli is dependent on the amount of protein present in the Tantigen preparation. This is shown in Fig. 2 where addition of 16 pg of protein/assay causes a threefold increase in RNA synthesis in isolated nucleoli. A similar amount of proteins extracted from untransformed T antigen-negative CHL cells (“mock” T) had no effect on the incorporation of r3H]UTP into RNA (Ide et al., 1977). Preincubation with T antigen for at least 20 min was found to be necessary for stimulation of RNA synthesis in isolated nuclei (Ide et al., 1977). Figure 3 shows that the nucleoli have to be preincubated for only 5 min to obtain maximal stimulation of RNA synthesis. In all subsequent experiments preincubation with T preparations was 15 min long (unless otherwise stated) and the
0
4 pg
8
12
16
PROTEIN /ASSAY
FIG. 2. Stimulation of RNA synthesis in isolated rat liver nucleoli by increasing amounts of proteins from partially purified preparations of SV40 T antigen. Isolated rat liver nucleoli were assayed for RNA synthesis as described in Fig. 1 and under Materials and Methods. The abscissa gives the amount of protein present in the added T-antigen preparation, the ordinate, the amount of [3H]UTP incorporated into RNA per microgram of DNA. The isolated nucleoli were preincubated at 25” for 15 min in the absence of the ribonucleotide triphosphates. After addition of the triphosphates the assay mixture was incubated at 37” for another 15 min and the reaction was then terminated. Background counts per minute have been subtracted from all values.
WHELLY,
86
IDE, AND BASERGA
Preincubation of T-antigen preparations from wild-type transformed cells at 50” for as long as 2 hr did not decrease the stimulation of nucleolar RNA synthesis, whereas TABLE 1 EFFECT OF a-AMANITIN ON RNA SYNTHESIS IN ISOLATED NUCLEI AND NUCLEOLI” T antigen a-Amanitin DNA Nuclei 0
5 TIME
10
15
1 MIN
FIG. 3. Effect of time of preincubation with T-antigen preparations on RNA synthesis in isolated nucleoli. Rat liver nucleoli were isolated and preincubated for various times (shown on the abscissa) at 25’ with partially purified preparations of SV40 T antigen. After preincubation, the ribonucleotide triphosphates were added and incubation was carried out at 37” for another 15 min. In these experiments the T-antigen preparation had 8 pg of protein/assay and was prepared from SV80 cells. (O---O) Nucleoli; (A---A) nucleoli plus T-antigen preparations.
has no effect on the extent of RNA/DNA hybridization between the nucleolar product and nucleolar DNA. On the basis of these results it seems reasonable to conclude that the product of nucleolar RNA synthesis includes at least some preribosomal RNA sequences (see also Discussion).
Stimulation of Nucleolar RNA with T-Antigen Preparations Mutants
+ + +
+ Nucleoli
+ +
+ +
241 501 150 451 2600 6ooo 3600 6100
DRat liver nuclei or nucleoli were preincubated in the presence or absence of T-antigen preparations from wild-type SV40 transformed cells (SV80) for 30 or 15 min, respectively. The nuclei or nucleoli were then assayed for RNA synthesis in the presence or absence of a-amanitin, 5 pg/ml. The values reported represent incorporation of r3H]UTP into RNA per microgram of DNA. The assays were carried out for 15 min at 37’.
: 3500w
Synthesis from tsA
T antigen was partially purified from CHL A239 Ll cells, a cell line developed by Martin and Chou (1975) by transformation of CHL cells with a tsA mutant of SV40. The T antigen in these cells is temperature sensitive (Tenen et al., 1975). As reported previously (Ide et al., 1977), preincubation at 25” for 2 hr in TGMED of T antigen preparations from both CHL A239 Ll cells, and CHL wt 15 cells (CHL wt 15 cells are CHL cells transformed by wild-type SV40 virus), has no effect on complement-fixing activity. On the other hand preincubation at 50’ inactivates the tsT antigen much more rapidly and more profoundly than the T antigen from wild-type transformed cells. Figure 5 shows the effect of these T-antigen preparations on nucleolar RNA synthesis.
~9
RNA
FIG. 4. Competition by cold ribosomal RNA of the RNA/DNA hybridization between nucleolar DNA and the RNA synthesized in vitro by T antigen-stimulated nucleoli. Rat liver nucleoli were incubated as described in Fig. 1 and under Materials and Methods. The radioactive product was isolated and hybridized to nucleolar DNA by the method of Gillespie (1968). The ordinate gives the amount of radioactivity that was hybridized to nucleolar DNA, the abscissa, the amount of nonradioactive ribosomal (O---O) or transfer (w) RNA added to the hybridization mixture. The input in these reactions was 10,000 cpm/loO pg of DNA. Nonspecific binding for the filter was 65 cpm. Other details are given in Materials and Methods.
T ANTIGEN
AND
HOURS
FIG. 5. Effect of preincubation at either 25 or 50” on the stimulation of nucleolar RNA synthesis by Tantigen preparations from cells transformed by either wild-type or tsA SV40. The T-antigen preparations were preincubated at 25 or 50” for the time indicated and then added to isolated nucleoli. Nucleoli alone or with T-antigen preparation were incubated at 25 or 40” for an additional 10 min in the absence of the ribonucleotide triphosphates. After addition of the triphosphates the assay was terminated at 15 min. The ordinate gives the counts per minute incorporated into RNA per microgram of nucleolar DNA. The abscissa gives the time of preincubation at either 25 or 50’ of the T-antigen preparations. (A) Wild-type T antigen; (B) tsA T antigen. (Cl) No T antigen added, 25’; (m) no T antigen added, 40’; (w) wildtype T antigen preincubated at 25’; (O---O) wild-type T antigen preincubated at 50’; (A-A) tsA T antigen preincubated at 25’; (A---A) tsA T antigen preincubated at 50”. In T-antigen preparations from the wild-type transformed cells, 9.3 pg of protein was used per assay. In T-antigen preparations from the tsA mutant transformed cells 15 pg of protein was used per assay.
the same treatment almost completely abolished the RNA stimulating activity of T-antigen preparations obtained from tsA transformed cells. The increased RNA stimulating activity observed following 50” preincubationof wild-type T-antigen preparations has been repeated numerous times, but the reason for the stimulation is unknown. Stimulation of Nucleolar RNA Synthesis by Further Purified Preparations of T Antigen Figure 6 shows the effect of highly purified preparations of T antigen on the stimulation of RNA synthesis in isolated nucleoli. Rat liver nucleoli were incubated with different concentrations of T-antigen preparations from three successive steps of
RNA
87
SYNTHESIS
purification, the DNA cellulose step, in our purification procedure, replacing the glycerol gradient in the original procedure by Tenen et al. (1977a). For a given unit of complement-fixing activity of the T-antigen preparations, as determined by the method of Sever (1961), the amount of protein decreased from 40 pg in crude preparations to 0.36 pg after DEAE-cellulose, to 0.17 pg after heparin-Sepharose. Because of the small amount of product obtained, we have not been able to ascertain the degree of purity of the T preparation obtained from DNA-cellulose. However, it should not be worse than the degree of purity obtained by Tenen et al, (1977a) with heparin-sepharose, which is the next to last step in their procedure. Figure 6 shows that the stimulation of nucleolar RNA synthesis by three different preparations of T antigen is proportional to the complement-fixing activity of the preparation. In the experiments of Fig. 6 the control values for the unstimulated nucleoli ran consistently between 890 and 915 cpm/pg of DNA above background values. Stimulation of Cellular RNA Synthesis In Vivo The stimulation of cellular RNA and DNA was also investigated in quiescent
I 3
I 6
12
24
4E
W
FIG. 6. Stimulation of RNA synthesis in isolated nucleoli by highly purified preparations of SV40 T antigen. The assay for nucleolar RNA synthesis was the same as in Fig. 1. The amount of T antigen is expressed in complement-fling units per assay, and nucleolar RNA synthesis, in percentage of control values (no T added). The T preparation had different degrees of purity as follows: (O--O) from DEAEcellulose step; (O--O) from heparin-Sepharose step; (A-A) from the DNA-cellulose column.
WHELLY,
-sEr---
IDE,
co 80 hrs p.i.
FIG. 7. Stimulation of RNA and DNA synthesis by SV40 infection of quiescent 3T3 cells. Infection with SV40 and determinations of RNA and DNA synthesis were carried out as described under Materials and Methods. (M) Incorporation of C3H]uridine; (w) incorporation of [3H]thymidine; (X---X) mock infection. hbscissaz Time after infection.
monolayers of 3T3 cells. The cells were infected with SV40, as described in Materials and Methods. Figure 7 shows that both RNA and DNA synthesis are stimulated by SV40 infection and that the increase in RNA synthesis precedes the increase in DNA synthesis by several hours. DISCUSSION
There is substantial evidence that the A gene product of the SV40 genome is required for the initiation of SV40 DNA replication (Tegtmeyer, 1972)) the transcription of late SV40 genes (Cowan et a&, 1973), the establishment and maintenance of transformation (Brugge and Butel, 1975), and the stimulation of cellular DNA synthesis that occurs after SV40 infection (Scott et aZ., 1976; Postel and Levine, 1976). There is also good evidence that one of the two products of the early region is identifiable with the traditional T antigen (Tegtmeyer, 1975; see review by Levine, 1976). In a previous communication (Ide et al., 1977), we reported that partially purified preparations of SV40 T antigen stimulated RNA synthesis in isolated nuclei from either rat liver or hamster cells in culture. This was an interesting finding because it suggested that T antigen may operate at the level of transcription. Our present results indicate that T-antigen preparations can stimulate RNA synthesis not only in isolated rat liver nuclei, but also in isolated rat liver nucleoli. A threefold stimulation can be achieved by
AND
BASERGA
the addition of an appropriate amount of T-antigen preparations. The product of the nucleoli stimulated by T-antigen preparations, at least partially, preribosomal RNA, as can be judged by RNA/DNA hybridization methods. Some investigators believe that preribosomal RNA is the only product synthesized by the nucleolus, either in viuo or in vitro (Grummt, 1975; Matsui et al., 1976; Ballal et al., 1977). Furthermore, from the nucleolus one can isolate only RNA polymerase I (Coupar and Chester-ton, 1975; Matsui et al., 1976), which specifically transcribes rRNA genes (Chambon, 1975). However, Beebee and Butterworth (1977) have raised the possibility that the nucleolus may also synthesize nonribosomal RNA transcripts. For this reason, we can only say at this point that T-antigen preparations stimulate the incorporation of precursors into RNA by a nucleolar, endogenous, (Yamanitin-resistant RNA polymerase. Our results also show that the stimulation of nucleolar RNA synthesis is thermosensitive when T-antigen preparations are used, prepared from celIs that have been transformed by a tsA mutant of SV40. FinalIy, the stimulation of both nuclear or nucleolar RNA synthesis can be achieved by highly purified preparations of T antigen from SV40 transformed cells. True, in our experiments we have not demonstrated that T antigen has been completely purified. However, the following evidence seems to point to T antigen as the protein responsible for the stimulation of nucleolar RNA synthesis: (1) the effect of anti-T antiserum (Ide et aZ., 1977); (2) the thermosensitivity of T-antigen preparations from cells transformed by tsA mutants, in terms of both complement-fixing activity and ability to stimulate nucleolar RNA synthesis, (3) the stimulatory activity of nucleolar RNA synthesis of T preparations of different degrees of purity; and finally (4) the in viuo experiments mentioned below, especially those of SaIomon et al. (1977). This is the same type of evidence usually accepted as indicating a biological effect of T antigen (see for instance a recent paper by PersicoDiLauro et al., 1977). In another paper (Baserga et al., 1978) we have given the evidence excluding the role of RNA polymerase or DNase activity
T ANTIGEN
89
AND RNA SYNTHESIS
in stimulating RNA synthesis in isolated nuclei treated with T-antigen preparations. Accordingly, all the preparations of T antigen used in the present experiments were devoid of any RNA polymerase or DNase activities. It should also be noted that addition of large quantities of RNA polymerase I to isolated nucleoli has very little effect on the synthesis of RNA (personal communication from H. Busch; unpublished results from our own laboratory). In fact, Daubert et al. (1977) have reported that addition of RNA polymerase I to isolated nucleoli actually causes a decrease in RNA synthesis. It should be clearly stated that the term “stimulation” is used here in an operational sense to indicate an increased incorporation of precursors into nucleolar RNA. The data presented thus far cannot discriminate between an increased initiation of RNA chains or a decreased processing. Work is in progress to elucidate which of these two alternatives may be the correct one. Recently, Graessmann and Graessmann (1976) induced DNA synthesis in primary mouse kidney cells microinjected with mRNA transcribed from the A gene of the SV40 genome. They concluded that SV40 T antigen (the microinjected cells became T-antigen positive) provided the necessary information for the stimulation of cellular DNA synthesis. On the other hand there is considerable evidence that stimulation of cellular DNA synthesis is preceded, by several hours, by a marked increase in the synthesis of rRNA (Tsukada and Lieberman, 1964a,b; Mauck and Green, 1973; Nicolette and Babler, 1974; Schmid and Sekeris, 1975). Thus it is tempting to correlate the ability of T antigen to stimulate cellular DNA synthesis in viuo with its ability to stimulate in vitro nucleolar RNA synthesis. Indeed, an increase in the amount and synthesis of ribosomal RNA in resting cells infected with either SV40 or polyoma virus has been reported by Benjamin (1966), Weil et al. (1975), and May et al. (1976). We have confirmed these in uiuo findings and extended them to show that an increase in RNA synthesis clearly precedes the entry of cells into S phase. The stimulation of cellular RNA synthesis also occurs when the expression of late genes is
inhibited by FUdr, provided the T antigen is expressed (Salomon et al., 1977). Stimulation of RNA synthesis has also been reported in BHK cells infected by adenovirus 12 (Raska et al., 1971). The stimulation of RNA synthesis is modest compared to the extent of DNA synthesis that can be obtained when resting cells are stimulated to proliferate. But, as is clear from the literature, RNA synthesis in mammalian cells rarely varies by more than a factor of 2 (see review by Baserga, 1976). Our results seem to indicate that highly purified preparations of SV40 T antigen can stimulate RNA synthesis in isolated nuclei and nucleoli. It should be clearly pointed out, however, that this does not rule out that the T antigen may also act on extra nucleolar genes or even directly on DNA synthesis. The results simply point out that T antigen can stimulate the synthesis of nucleolar RNA. We would like to suggest, therefore, that one of the key mechanisms for control of cell proliferation is the synthesis of nucleolar RNA and that the T antigen from SV40 may be involved in the regulation of its synthesis. ACKNOWLEDGMENTS This work was supported by USPHS Research Grant CA-12923 and Wistar Contract HD-66323 from the National Institutes of Health. REFERENCES ALBERTS, B., and HERRICK, G. (1971). DNA-Cellulose chromatography. In “Methods in Enzymology, Vol. 21” (S. P. Colowick and N. 0. Kaplan, eds.), pp. 196-217. Academic Press, New York. BALLAL, N. R., CHOI, Y. C., MOUCHE, R., and BUSCH, H. (1977). Fidelity of synthesis of preribosomal RNA in isolated nucleoli and nucleolar chromatin. Biochemistry 74,2446-2450. BASERGA, R. (1976). “Multiplication and Division in Mammalian Cells.” Marcel Dekker, New York. BA~ERGA, R., IDE, T., and WHELLY, S. (1978). S&ulation of ribosomal RNA synthesis in isolated nuclei and nucleoli by partially purified preparations of SV40 T antigen. CoM Spring Harbor Symp. Quant. Bid., in press. BEEBEE, T. J. C., and BUTTERWORTH, P. H. W. (1977). Transcription fidelity and structural integrity of isolated nucleoli. Eur. J. Biochem. 17, W-348. BENJAMIN, T. L. (1966). Virus-specific RNA in cells productively infected or transformed by polyoma virus. J. Mol. Biol. l&359-373. BRUGGE, J. S., and BUTEL, J. S. (1975). The role of
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WHELLY,
IDE,
simian virus 40 gene A function in maintenance of transformation. J. Viral. 16, 619-635. CHAMBON, P. (1975). Eukaryotic nuclear RNA polymerases. Annu. Rev. Biochem. 44,613~638. CHIU, N., and BASERGA, R. (1975). Changes in template activity and structure of nuclei from WI-38 cells in the prereplicative phase. Biochemistry 14, 3126-3132. COUPAR, B. E. H., and CHESTERTON, C. J. (1975). Purification of form AI and AI1 DNA-dependent RNA polymerases from rat liver nucleoli using lowionic strength extraction conditions. Eur. J. Biothem. 69,25-34. COWAN, K., TEGTMEYER, P., and ANTHONY, D. D. (1973). Relationship of replication and transcription of simian virus 40 DNA. Proc. Nat. Acad. Sci. USA 70, 1927-1930. DAUBERT, S., PETERS, D., and DAHMUS, M. E. (1977). Selected transcription of ribosomal sequences in vitro by RNA polymerase I. Arch. B&hem. Biophys. 178,381-386. FERENCZ, A., and SEIFART, K. H. (1975). Comparative effect of heparin on RNA synthesis of isolated rat liver nucleoli and purified RNA polymerase A. Eur. J. Biochem. 53,605-613. GILLESPIE, D. (1968). The formation and detection of DNA/RNA hybrids. In “Methods in Enzymology Vol. 12” (L. Grossman and K. Moldave, eds.), pp. 641-668. Academic Press, New York. GRAESSMANN, M., and GUESSMANN, A. (1976). Early simian virus 40 specific RNA contains information for tumor antigen formation and chromatin replication. Proc. Nat. Acad. Sci. USA 73, 366-370. GRUMMT, I. (1975). Synthesis of RNA molecules larger than 45s by isolated rat liver nucleoli. Eur. J. Biothem. 57, 159-167. HARDIN, J. W., CLARK, J. H., GLASSER, S. R., and PECK, E. J., JR. (1976). RNA polymerase activity in uterine growth. Differential stimulation by estradiol, estriol and Nafoxidine. Biochemistry 15,1370-1374. HUANG, C. H., and BASERGA, R. (1976). Circular dichroic studies of DNA and RNA of nucleoli. Biochemistry 15,2829-2836. IDE, T., WHELLY, S., and BASERGA, R. (1977). Stimulation of RNA synthesis in isolated nuclei by partially purified preparations of simian virus 40 T antigen. Proc. Nat. Acad. Sci. USA 74, 3189-3192. JESSEL, D., HUDSON, J., LANDAU, T., TENEN, D., and LIVINGSTON, D. M. (1975). Interaction of partially purified simian virus 40 T antigen, with circular viral DNA molecules. Proc. Nat. Acad. Sci. USA 72, 1960-1964. KANE, A., BASILICO, C., and BASERGA, R. (1976). Transcriptional activity and chromatin structural changes in a temperature-sensitive mutant of BHK cells blocked in early G1. Exp. Cell Res. 99,165-173. LEVINE, A. J. (1976). SV40 and adenovirus early functions involved in DNA replication and transformation. Biochim. Biophys. Acta 458,213-241.
AND BASERGA LINDELL, T. J. (1975). Inhibition of eukaryotic DNA dependent RNA polymerase release from isolated nuclei and nucleoli by phenylmethylsulfonllfluoride. Arch. Biochem. Biophys. 171,268-275. MARTIN, R. G., and CHOU, J. Y. (1975). Simian virus 40 functions required for the establishment and maintenance of malignant transformation. J. Viral. 16, 599-612. MAFULUFF, W. F., JR., MURPHY, E. C., JR., and HUANG, R. C. (1973). Transcription of ribonucleic acid in isolated mouse myeloma nuclei. Biochemistry12,3440-3446.
MATSUI, T., ONISHI, T., and MURAMATSU, M. (1976). Nucleolar DNA-dependent RNA polymerase from rat liver. Eur. J. Biochem. 71,351-360. MAUCK, J. C., and GREEN, H. (1973). Regulation of RNA synthesis in fibroblasta during transition from resting to growing state. Proc. Nat. Acad. Sci. USA 70,2819-2822.
MAY, P., MAY, E., and BORDB, J. (1976). Stimulation of cellular RNA synthesis in mouse kidney cell cultures infected with SV40 virus. Exp. Cell Res. 100,433-436. MURAMATSU, M., HAYASHI, Y., ONISHI, T., SAKAI, M., TAKAI, K., and KASHIYAMA, T. (1974). Rapid isolation of nucleoli from detergent purified nuclei of various tumor and tissue culture cells. Exp. Cell Res. 88,345-351. NICOLETTE, A. A., and BABLER, M. (1974). The selective inhibitor effect of NH&l on estrogen stimulated rat uterine in vitro RNA synthesis. Arch. Biochem. Biophys. 163,656-665. PERSICO-DILAURO, M., MARTIN, R. G., and LIVINGSTON, D. M. (1977). Interaction of simian virus 40 chromatin with simian virus 40 T-antigen. J. Viral. 24,451-460. POSTEL, E. H., and LEVINE,
A. J. (1976). The requirement of simian virus 40 gene A product for the stimulation of cellular thymidine kinase activity after viral infection. Virology 73,206-215. RASKA, K., JR., STROHL, W. A., HOLOWCZAK, J. A., and ZIMMERMAN, J. (1971). The response of BHK 21 cells to infection with type 12 adenovirus. Virology44,296-306. ROTHBLUM, L. I., MAMRACK, P. M., KUNKLE, H. M., OLSON, M. 0. J., and BUSCH, H. (1977). Fractiona-
tion of nucleoli. Enzymatic and two-dimensional polyacrylamide gel electrophoretic analysis. Biochemistry 16,4716-4721. SALOMON, C., TWIRLER, H., and WEIL, R. (1977). Polyoma-induced stimulation of cellular RNA synthesis is paralleled by changed expression of the viral genome. Nucleic Acids Res. 4, 1463-1503. SCHMID, W., and SEKERIS, C. E. (1975). Nucleolar RNA synthesis in the liver of partially hepatectomized and cortisol-treated rats. Biochim. Biophys. Acta 402, 244-252. SCOTT, W. A., BROCKMAN, W. W., and NATHANS, D. (1976). Biological activities of deletion mutants of
T ANTIGEN
AND RNA SYNTHESIS
simian virus 40. Virology X,319-334. SEVER, J. L. (1961). Application of microtechnique to viral serological investigations. J. Zmmunol. 88, 320-329. TEGTMEYER, P. (1972). Simian virus 40 deoxyribonucleic acid synthesis: The viral replicon. J. Viral. 10, 591-598. TEGTMEYER, P. (1975). Function of simian virus 40 gene A in transforming infection. J. Virol. 16, 613-618. TENEN, D. C., BAYGELL, P., and LIVINGSTON, D. M. (1975). Thermolabile T (tumor) antigen from celIs transformed by a temperature-sensitive mutant of simian virus 40. Proc. Nat. Acad. Sci. USA 72, 4351-4355. TENEN, D. G., GAREWAL, H., HAINES, L. L., HUDSON, J., WOODARD, V., LIGHT, S., and LIVINGSTON, D. M. (1977a). Purification of simian virus 40 tumor antigen from a line of simian virus 40-transformed ceils. Proc. Nat. Acad. Sci. USA 74,3745-3749. TENEN, D. G., MARTIN, R. G., ANDERSON, J., and LIVINGSTON, D. M. (1977b). Biological and biochemical studies of cells transformed by simian virus 40 temperature-sensitive gene A mutants and A mu-
91
tant revertants. J. Viral. 22,210-218. TSUKADA, K., and LIEBERMAN, I. (1964a). Metabolism of nucleolar ribonucleic acid after partial hepatectomy. J. Biol. Chem. 239,1564-1568. TSUKADA, K., and LIEBERMAN, 1. (1964b). Synthesis of ribonucleic acid by liver nuclear and nucleolar preparations after partial hepatectomy. J. Biol. Chem. 239,2952-2956. VILLALOBOS, J. G., JR., STELE, J., and BUSCH, H. (1964). Effects of thiocetamide on labeling of ribonucleic acid of isolated nucleoli in vitro. Biochim. Biophys. Acta g&233-238. WEIL, R., SALOMON, C., MAY, E., and MAY, P. (1975). A simplifying concept in tumor virology: Virua-specik “pleiotropic effector.” Cold Spring Harbor Symp. Quant. Biol. 39, 381-395. WEINMANN, R., JAEHNING, J. A., RASKAS, H. J., and ROEDER, R. G. (1976). Viral RNA synthesis and levels of DNA dependent RNA polymerases during replication of adenovirus II. J. Virol. 17, 114-126. Yu, F., and FEIGELSON, P. (1971). Cortisone stimulation of nucleolar RNA polymerase activity. Proc. Nat. Acad. Sci. USA 68.2177-2180.
VIROLOGY
(1978)
Stimulation
SANDRA Department
of
of RNA Synthesis in Isolated Nucleoli by Preparations Simian Virus 40 T Antigen WHELLY,’
TOSHINORI
IDE,’
AND
RENATO
of
BASERGA3
Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Accepted March 26, 1978
Partially purified and highly purified preparations of simian virus 40 (SV40) T antigen from SV80 cells stimulate the incorporation of [3H]UTP into RNA in isolated rat liver nucleoli. The increased incorporation is dependent on an endogenous o-amanitin-resistant RNA polymerase, and ribosomal RNA sequences are present in the product, T antigen was also partially purified from Chinese hamster cells transformed by a tsA mutant of SV40. The complement-fixing activity of tsT was rapidly inactivated by heating at 50”, and this inactivation was paralleled by a marked decrease in its ability to stimulate nucleolar RNA synthesis in vitro. Under these same conditions, T preparations from wild-type transformed cells were more resistant, in terms of both complement-firing activity and ability to stimulate nucleolar RNA synthesis The stimulation of nucleolar RNA synthesis with wildtype T preparations of different degrees of purity correlated with the ability of the preparations to fix complement, even with highly purified preparations. Finally, cellular RNA synthesis was also stimulated when quiescent 3T3 cells were infected with wild-type SV40. The results do not prove, but strongly suggest, that one of the sites of action of SV40 T antigen may be at the level of transcription of nucleolar DNA.
(1) preparations of SV40 T antigen can directly stimulate RNA synthesis in isolated nucleoli from rat liver; (2) the nucleolar product has, at least partially, the characteristics of preribosomal RNA; (3) stimulation of nucleolar RNA synthesis can also be achieved with highly purified preparations of SV40 T antigen; and (4) an increased incorporation of [3H]uridine into RNA can also be demonstrated in vivo in SV40-infected 3T3 cells.
INTRODUCTION
In a previous communication (Ide et al. 1977), we reported that partially purified preparations of simian virus 40 (SV40) T antigen stimulated RNA synthesis in nuclei isolated from either rat liver or quiescent hamster cells in culture. The increase in nuclear RNA synthesis was abolished by pretreatment with anti-T antiserum. Although several lines of evidence suggested that the T antigen in these partially purified preparations was responsible for the stimulation of nuclear RNA synthesis, the preparations used were contaminated by other nuclear proteins. The present communication reports that:
MATERIALS
82 Inc. reserved.
METHODS
Cells. SV80 cells (a gift from D. M. Livingston, Sidney Farber Cancer Center) were grown in Dulbecco’s medium, as described by Jesse1et al. (1975). CHL cells were of two types. CHL wt 15 cells transformed by wild-type SV40, and CHL A239 Ll cells transformed by a tsA mutant of SV40. Both of these cell lines were obtained from Dr. Robert G. Martin and have been previously described (Martin and Chou, 1975; Tenen et al. 1977b). For in vivo experiments, 3T3 cells (kindly
’ Present address: University of Nebraska Medical Center, Obstetrics and Gynecology Research Laboratory, Conkling Hall, 42nd and Dewey Avenue, Omaha, Nebraska 68105. ‘Present address: Department of Virology, Institute of Medical Science, Tokyo University, Minatoku, Tokyo, Japan. 3 To whom requests for reprints should be sent.
0042-sS22/78/~1-0082$02.00/0 Copyright 0 1978 by Academic Press, All rights of reproduction in any form
AND
T ANTIGEN
AND
given to us by Daniel Nathans) were plated in 16-mm microwells and grown in Dulbecco’s medium plus 10% fetal calf serum until quiescent (7 days after plating). Isolation of nuclei and assay for nuclear RNA synthesis. The isolation of nuclei from
rat liver was carried out by the method of Marzluff et al. (1973) with slight modifications which have been described in detail in the previous paper (Ide et al. 1977). Similarly, the method of Marzluff et al. (1973) was followed for the determination of RNA synthesis in vitro. The dependence of the assay on the presence of the four ribonucleotide triphosphates, the inhibition by actinomycin D, etc., have already been discussed by Marzluff et al. (1973) and in papers from our laboratory (Kane et al. 1976; Baserga et al. 1978). The final volume of the assays was 125 ~1 and each assay contained approximately 5 pg of DNA as nuclei. All assays were performed at 37” and were terminated after 15 min of incubation, unless otherwise specified. The control values were always at least three to four times the background counts per minute. Isolation of nucleoli cleolar RNA synthesis.
and assay for nu-
Nucleoli were isolated by the method of Muramatsu et al. (1974) with slight modifications, as described by Huang and Baserga (1976). Although the definition of what constitutes a nucleolus may vary from one laboratory to another, the procedure we used is the one most commonly adopted for the preparation of isolated nucleoli (Yu and Feigelson, 1971; Grumm t 1975; S&mid and Sekeris, 1975; Matsui et al., 1976; Rothblum et al., 1977). These nucleoli are highly pure, as can be judged by electron microscopy. The criteria used for the purity of nucleoli have already been described in a previous paper from this laboratory (Huang and Baserga, 1976). Although a smah degree of contamination by perinucleolar chromatin cannot be ruled out, RNA synthesis in such nucleoli is 100% resistant to even high concentrations of a-amanitin (Coupar and Chesterton, 1975) and reasonably reproduces in uiuo synthesis (Ballal et al., 1977). The assay for nucleolar RNA synthesis was carried out according to the technique of Mar-
RNA
SYNTHESIS
83
zluff et al. (1973) for determination of RNA synthesis in isolated nuclei. The cofactor’s requirements for our nucleolar preparations were essentiahy the same as described by us and other investigators (Villalobos et al., 1964; Lindell, 1975; Ferencz and Seifart, 1975). The assay simply measures incorporation of r3H]UTP into RNA by an endogenous RNA polymerase resistant to aamanitin. For convenience and following the example of other investigators (see below), we shall briefly refer to this measurement as a measurement of nucleolar RNA synthesis. We prefer the term nucleolar RNA synthesis (rather than preribosomal RNA synthesis) because it is possible that the nucleolus may also synthesize nonribosomal RNA (Beebee and Butterworth, 1977). Preparation of T protein. Most of the experiments, except those described in Fig. 7, were carried out with partially purified preparations of SV40 T antigen, prepared by the method of Jesse1 et al. (1975), as previously described (Ide et al., 1977). Highly purified T antigen was prepared by a modification of the method of Tenen et al. (1977a) as follows. Active fractions of T antigen from a DE 52 column were concentrated by addition of ammonium sulfate (final SO%), dialyzed against 106 vol of buffer A [50 mu Tris-HCl, pH 7.4, 3 mu dithiothreitol (DTT), and 20% glycerol) for 5 hr with two changes of buffer, and applied on a heparin-Sepharose column (15~ml bed volume). The column was washed with 3 column vol of buffer A and eluted with a 1Zcolumn-vol linear gradient between 0 and 0.6 M NaCl in buffer A. T antigen-containing fractions (0.39-0.4 M NaCl fractions) as assayed by CF were pooled, adjusted to pH 6.0, and dialyzed against 100 vol of buffer B (50 mM Tris, pH 6.0,3 mM DTT, and 20% glycerol) for 4 hr with two changes of buffer. The dialyzed T preparations were applied to a DNA-cellulose column (4-m.l bed volume) very slowly. The column was washed with 3 column vol of buffer B and eluted with a 20-column-vol linear gradient between 0 and 0.8 M NaCl in buffer C (100 m&f Tris-HCl, pH 8.0, 3 mM DTT, and 20% glycerol). T antigen fractions were pooled,
a4
WHELLY,
IDE,
concentrated by addition of Sephadex G200 outside the dialysis bag, and dialyzed against 200 vol of TGMED (50 n&f Tris-HCl, 25% glycerol, Mg acetate 5 mM, DTT 1.5 mM, and EDTA 0.1 mM) for 4 hr with two changes of buffer. Complement fixation. Complement-fixation tests for T antigen were carried out by the microtiter technique of Sever (1961). RNA/DNA hybridization. Competitive hybridization of RNA to DNA on nitrocellulose filters was performed according to the procedure of Gillespie (1968). Purified rat liver nucleolar DNA was heat denatured at 100” in %o SSC (0.015 M NaCZ; 0.0015 M Na-citrate, pH 7.0). Heat denatured DNA was annealed to nitrocellulose filters in 4x SSC. The percentage of DNA binding to each filter was monitored by optical density. After drying, the DNA-nitrocelhilose filters were baked at 90” for 1 hr. Varying concentrations of unlabeled cytoplasmic ribosomal RNA (purified from rat liver) or unlabeled transfer RNA (Sigma) were hybridized to DNA-nitrocellulose filters at 66” for 30 hr in 4~ SSC. The filters were mildly treated with RNase (pretreated at 100’) and washed thoroughly. The RNA-DNA nitrocellulose filters were then incubated with the [3H]RNA product isolated from the nucleolar template assay made in the presence of T antigen. The competitive hybridization was carried out at 55” for 30 hr following digestion with RNase and washing of the filters. The amount of radioactivity still hybridized to nucleolar DNA was determined in a Packard scintillation counter. Controls were carried out in the absence of cold RNA. Values reported were corrected for nonspecific binding of [3H]RNA to the filters. SV40 infection of 3T3 cells. This was done essentially by the method of Scott et al. (1976). Quiescent 3T3 cells were infected at a m.o.i. of 1000 with a suspension of KBrpurified SV40 in phosphate-buffered saline. After adsorption, the virus suspension was diluted with fresh medium (no serum) and conditioned medium in a ratio of 9:l. For DNA synthesis, the cells were labeled for 1 hr with [3H]thymidine (10 fCi/welI), and for RNA synthesis, with [ Hluridine (20 &i/well) also for 1 hr. The amount of radioactivity incorporated into acid-precip-
AND
BASERGA
itable material was determined scintillation counting.
by liquid
Materials Isotopes. [5-3H]UTP (22.0 Ci/mmol), [methyl-3H]thymidine (6.7 Ci/mmol), and [5-3H]uridine (26.7 Ci/mmol) were purchased from the New England Nuclear Corp. Heparin-Sepharose column. CNBr-Activated Sepharose 4B (Pharmacia Fine Chemicals) was coupled with heparin (Inolex Corporation, Chicago, Ill) in coupling buffer (0.1 M NaHC03, pH 8.3,0.5 M NaCl) for 2 hr at room temperature. The coupled Sepharose was washed exhaustively with coupling buffer, 1 M ethanolamine solution (pH 8.0), coupling buffer, acetate buffer (0.1 M, pH 4.0, 0.5 M NaCl), and coupling buffer. Then, the resin was equilibrated with buffer (50 r&f Tris-HCl, pH 7.4, 3 m&f DTT, and 20% glycerol) and poured into a column. DNA-cellulose column. DNA-Cellulose was prepared by the method of Alberts and Herrick (1971) by using calf thymus DNA (Type 1, Sigma) and cellulose powder (Cellex 410, Bio-Rad Laboratories). Amount of bound DNA was 1.37 mg/ml of packed we1 volume of cellulose. RESULTS
Effect of T-Antigen Preparations on RNA Synthesis in Isolated Nucleoli Figure 1 shows the effect of partially purified T-antigen preparations on RNA synthesis in isolated nuclei and nucleoli from rat liver. As reported in the previous paper (Ide et al., 1977) RNA synthesis in isolated nuclei is stimulated by the addition of T-antigen preparations, at least twofold. B in Fig. 1 shows that good stimulation of RNA synthesis by T-antigen preparations can also be obtained with isolated nucleoli. In this paper we use the term “RNA synthesis” for its convenient brevity to express the incorporation of [3H]UTP into acid-insoluble material (Marxhiff et al., 1973). Other investigators used, for the same assay, the expression “endogenous RNA polymerase activity” (Lindell, 1975; Weinmann et al., 1976; Hardin et al., 1976). As already reported in this, as well as other laboratories (Marxluff et al., 1973; Kane et
T ANTIGEN
100
AND
CY 0
5
10
15 TIME
20 0 1 MIN
4, 4,000
5
IO
15
20
i
FIG. 1. Time course of C3H]UTP incorporation into RNA in isolated rat liver nuclei and nucleoli, with or without the addition of SV40 T-antigen preparations, The abscissa is the time of incubation, The ordinate represents the counts per minute incorporated into RNA per microgram of DNA. The nuclei or nucleoli were preincubated with T-antigen preparations at 25” for 30 or 15 min, respectively. At the end of the preincubation period the ribonucleotide triphosphates and the necessary ions were added, and the reaction was stopped at the times indicated on the abscissa. (A) Nuclei; (B) nucleoli. (O---O) No T antigen added; (A---A) addition of T-antigen preparations, 8 ag of protein/assay.
RNA
85
SYNTHESIS
assay was terminated after a further 15 min. Table 1 shows that the stimulation of RNA synthesis in isolated rat liver nuclei by T-antigen preparations is largely aamanitin resistant. Nucleolar RNA synthesis is, as expected, 100% a-amanitin resistant, in the presence or absence of T. Although this does not exclude that extra nucleolar genes may also be activated, it seems to indicate that at least most of the increase in RNA synthesis can be attributed to an increased activity of the nucleolus. The nature of the product synthesized by the isolated nucleoli after T-antigen stimulation was further studied by RNA/DNA hybridization. Using the RNA/DNA hybridization technique of Gillespie (1968), we found that the product of nucleolar RNA synthesis did not appreciably hybridize to total cellular DNA, but it did hybridize to nucleolar DNA. In addition, Fig. 4 showed that hybridization of the nucleolar product to nucleolar DNA is largely competed out by cold ribosomal RNA. The addition of transfer RNA to the hybridization mixture
al., 1976; Chiu and Baserga, 1975), the assay
is dependent upon the presence of ribonucleotide triphosphates, the product is digestible with RNase, and the incorporation of [3H]UTP is totally inhibited by actinomytin D. This is true of nuclei as well as nucleoli (Villalobos et a& 1964; Grummt, 1975; Baserga et al, 1978). The extent of stimulation of RNA synthesis in isolated nucleoli is dependent on the amount of protein present in the Tantigen preparation. This is shown in Fig. 2 where addition of 16 pg of protein/assay causes a threefold increase in RNA synthesis in isolated nucleoli. A similar amount of proteins extracted from untransformed T antigen-negative CHL cells (“mock” T) had no effect on the incorporation of r3H]UTP into RNA (Ide et al., 1977). Preincubation with T antigen for at least 20 min was found to be necessary for stimulation of RNA synthesis in isolated nuclei (Ide et al., 1977). Figure 3 shows that the nucleoli have to be preincubated for only 5 min to obtain maximal stimulation of RNA synthesis. In all subsequent experiments preincubation with T preparations was 15 min long (unless otherwise stated) and the
0
4 pg
8
12
16
PROTEIN /ASSAY
FIG. 2. Stimulation of RNA synthesis in isolated rat liver nucleoli by increasing amounts of proteins from partially purified preparations of SV40 T antigen. Isolated rat liver nucleoli were assayed for RNA synthesis as described in Fig. 1 and under Materials and Methods. The abscissa gives the amount of protein present in the added T-antigen preparation, the ordinate, the amount of [3H]UTP incorporated into RNA per microgram of DNA. The isolated nucleoli were preincubated at 25” for 15 min in the absence of the ribonucleotide triphosphates. After addition of the triphosphates the assay mixture was incubated at 37” for another 15 min and the reaction was then terminated. Background counts per minute have been subtracted from all values.
WHELLY,
86
IDE, AND BASERGA
Preincubation of T-antigen preparations from wild-type transformed cells at 50” for as long as 2 hr did not decrease the stimulation of nucleolar RNA synthesis, whereas TABLE 1 EFFECT OF a-AMANITIN ON RNA SYNTHESIS IN ISOLATED NUCLEI AND NUCLEOLI” T antigen a-Amanitin DNA Nuclei 0
5 TIME
10
15
1 MIN
FIG. 3. Effect of time of preincubation with T-antigen preparations on RNA synthesis in isolated nucleoli. Rat liver nucleoli were isolated and preincubated for various times (shown on the abscissa) at 25’ with partially purified preparations of SV40 T antigen. After preincubation, the ribonucleotide triphosphates were added and incubation was carried out at 37” for another 15 min. In these experiments the T-antigen preparation had 8 pg of protein/assay and was prepared from SV80 cells. (O---O) Nucleoli; (A---A) nucleoli plus T-antigen preparations.
has no effect on the extent of RNA/DNA hybridization between the nucleolar product and nucleolar DNA. On the basis of these results it seems reasonable to conclude that the product of nucleolar RNA synthesis includes at least some preribosomal RNA sequences (see also Discussion).
Stimulation of Nucleolar RNA with T-Antigen Preparations Mutants
+ + +
+ Nucleoli
+ +
+ +
241 501 150 451 2600 6ooo 3600 6100
DRat liver nuclei or nucleoli were preincubated in the presence or absence of T-antigen preparations from wild-type SV40 transformed cells (SV80) for 30 or 15 min, respectively. The nuclei or nucleoli were then assayed for RNA synthesis in the presence or absence of a-amanitin, 5 pg/ml. The values reported represent incorporation of r3H]UTP into RNA per microgram of DNA. The assays were carried out for 15 min at 37’.
: 3500w
Synthesis from tsA
T antigen was partially purified from CHL A239 Ll cells, a cell line developed by Martin and Chou (1975) by transformation of CHL cells with a tsA mutant of SV40. The T antigen in these cells is temperature sensitive (Tenen et al., 1975). As reported previously (Ide et al., 1977), preincubation at 25” for 2 hr in TGMED of T antigen preparations from both CHL A239 Ll cells, and CHL wt 15 cells (CHL wt 15 cells are CHL cells transformed by wild-type SV40 virus), has no effect on complement-fixing activity. On the other hand preincubation at 50’ inactivates the tsT antigen much more rapidly and more profoundly than the T antigen from wild-type transformed cells. Figure 5 shows the effect of these T-antigen preparations on nucleolar RNA synthesis.
~9
RNA
FIG. 4. Competition by cold ribosomal RNA of the RNA/DNA hybridization between nucleolar DNA and the RNA synthesized in vitro by T antigen-stimulated nucleoli. Rat liver nucleoli were incubated as described in Fig. 1 and under Materials and Methods. The radioactive product was isolated and hybridized to nucleolar DNA by the method of Gillespie (1968). The ordinate gives the amount of radioactivity that was hybridized to nucleolar DNA, the abscissa, the amount of nonradioactive ribosomal (O---O) or transfer (w) RNA added to the hybridization mixture. The input in these reactions was 10,000 cpm/loO pg of DNA. Nonspecific binding for the filter was 65 cpm. Other details are given in Materials and Methods.
T ANTIGEN
AND
HOURS
FIG. 5. Effect of preincubation at either 25 or 50” on the stimulation of nucleolar RNA synthesis by Tantigen preparations from cells transformed by either wild-type or tsA SV40. The T-antigen preparations were preincubated at 25 or 50” for the time indicated and then added to isolated nucleoli. Nucleoli alone or with T-antigen preparation were incubated at 25 or 40” for an additional 10 min in the absence of the ribonucleotide triphosphates. After addition of the triphosphates the assay was terminated at 15 min. The ordinate gives the counts per minute incorporated into RNA per microgram of nucleolar DNA. The abscissa gives the time of preincubation at either 25 or 50’ of the T-antigen preparations. (A) Wild-type T antigen; (B) tsA T antigen. (Cl) No T antigen added, 25’; (m) no T antigen added, 40’; (w) wildtype T antigen preincubated at 25’; (O---O) wild-type T antigen preincubated at 50’; (A-A) tsA T antigen preincubated at 25’; (A---A) tsA T antigen preincubated at 50”. In T-antigen preparations from the wild-type transformed cells, 9.3 pg of protein was used per assay. In T-antigen preparations from the tsA mutant transformed cells 15 pg of protein was used per assay.
the same treatment almost completely abolished the RNA stimulating activity of T-antigen preparations obtained from tsA transformed cells. The increased RNA stimulating activity observed following 50” preincubationof wild-type T-antigen preparations has been repeated numerous times, but the reason for the stimulation is unknown. Stimulation of Nucleolar RNA Synthesis by Further Purified Preparations of T Antigen Figure 6 shows the effect of highly purified preparations of T antigen on the stimulation of RNA synthesis in isolated nucleoli. Rat liver nucleoli were incubated with different concentrations of T-antigen preparations from three successive steps of
RNA
87
SYNTHESIS
purification, the DNA cellulose step, in our purification procedure, replacing the glycerol gradient in the original procedure by Tenen et al. (1977a). For a given unit of complement-fixing activity of the T-antigen preparations, as determined by the method of Sever (1961), the amount of protein decreased from 40 pg in crude preparations to 0.36 pg after DEAE-cellulose, to 0.17 pg after heparin-Sepharose. Because of the small amount of product obtained, we have not been able to ascertain the degree of purity of the T preparation obtained from DNA-cellulose. However, it should not be worse than the degree of purity obtained by Tenen et al, (1977a) with heparin-sepharose, which is the next to last step in their procedure. Figure 6 shows that the stimulation of nucleolar RNA synthesis by three different preparations of T antigen is proportional to the complement-fixing activity of the preparation. In the experiments of Fig. 6 the control values for the unstimulated nucleoli ran consistently between 890 and 915 cpm/pg of DNA above background values. Stimulation of Cellular RNA Synthesis In Vivo The stimulation of cellular RNA and DNA was also investigated in quiescent
I 3
I 6
12
24
4E
W
FIG. 6. Stimulation of RNA synthesis in isolated nucleoli by highly purified preparations of SV40 T antigen. The assay for nucleolar RNA synthesis was the same as in Fig. 1. The amount of T antigen is expressed in complement-fling units per assay, and nucleolar RNA synthesis, in percentage of control values (no T added). The T preparation had different degrees of purity as follows: (O--O) from DEAEcellulose step; (O--O) from heparin-Sepharose step; (A-A) from the DNA-cellulose column.
WHELLY,
-sEr---
IDE,
co 80 hrs p.i.
FIG. 7. Stimulation of RNA and DNA synthesis by SV40 infection of quiescent 3T3 cells. Infection with SV40 and determinations of RNA and DNA synthesis were carried out as described under Materials and Methods. (M) Incorporation of C3H]uridine; (w) incorporation of [3H]thymidine; (X---X) mock infection. hbscissaz Time after infection.
monolayers of 3T3 cells. The cells were infected with SV40, as described in Materials and Methods. Figure 7 shows that both RNA and DNA synthesis are stimulated by SV40 infection and that the increase in RNA synthesis precedes the increase in DNA synthesis by several hours. DISCUSSION
There is substantial evidence that the A gene product of the SV40 genome is required for the initiation of SV40 DNA replication (Tegtmeyer, 1972)) the transcription of late SV40 genes (Cowan et a&, 1973), the establishment and maintenance of transformation (Brugge and Butel, 1975), and the stimulation of cellular DNA synthesis that occurs after SV40 infection (Scott et aZ., 1976; Postel and Levine, 1976). There is also good evidence that one of the two products of the early region is identifiable with the traditional T antigen (Tegtmeyer, 1975; see review by Levine, 1976). In a previous communication (Ide et al., 1977), we reported that partially purified preparations of SV40 T antigen stimulated RNA synthesis in isolated nuclei from either rat liver or hamster cells in culture. This was an interesting finding because it suggested that T antigen may operate at the level of transcription. Our present results indicate that T-antigen preparations can stimulate RNA synthesis not only in isolated rat liver nuclei, but also in isolated rat liver nucleoli. A threefold stimulation can be achieved by
AND
BASERGA
the addition of an appropriate amount of T-antigen preparations. The product of the nucleoli stimulated by T-antigen preparations, at least partially, preribosomal RNA, as can be judged by RNA/DNA hybridization methods. Some investigators believe that preribosomal RNA is the only product synthesized by the nucleolus, either in viuo or in vitro (Grummt, 1975; Matsui et al., 1976; Ballal et al., 1977). Furthermore, from the nucleolus one can isolate only RNA polymerase I (Coupar and Chester-ton, 1975; Matsui et al., 1976), which specifically transcribes rRNA genes (Chambon, 1975). However, Beebee and Butterworth (1977) have raised the possibility that the nucleolus may also synthesize nonribosomal RNA transcripts. For this reason, we can only say at this point that T-antigen preparations stimulate the incorporation of precursors into RNA by a nucleolar, endogenous, (Yamanitin-resistant RNA polymerase. Our results also show that the stimulation of nucleolar RNA synthesis is thermosensitive when T-antigen preparations are used, prepared from celIs that have been transformed by a tsA mutant of SV40. FinalIy, the stimulation of both nuclear or nucleolar RNA synthesis can be achieved by highly purified preparations of T antigen from SV40 transformed cells. True, in our experiments we have not demonstrated that T antigen has been completely purified. However, the following evidence seems to point to T antigen as the protein responsible for the stimulation of nucleolar RNA synthesis: (1) the effect of anti-T antiserum (Ide et aZ., 1977); (2) the thermosensitivity of T-antigen preparations from cells transformed by tsA mutants, in terms of both complement-fixing activity and ability to stimulate nucleolar RNA synthesis, (3) the stimulatory activity of nucleolar RNA synthesis of T preparations of different degrees of purity; and finally (4) the in viuo experiments mentioned below, especially those of SaIomon et al. (1977). This is the same type of evidence usually accepted as indicating a biological effect of T antigen (see for instance a recent paper by PersicoDiLauro et al., 1977). In another paper (Baserga et al., 1978) we have given the evidence excluding the role of RNA polymerase or DNase activity
T ANTIGEN
89
AND RNA SYNTHESIS
in stimulating RNA synthesis in isolated nuclei treated with T-antigen preparations. Accordingly, all the preparations of T antigen used in the present experiments were devoid of any RNA polymerase or DNase activities. It should also be noted that addition of large quantities of RNA polymerase I to isolated nucleoli has very little effect on the synthesis of RNA (personal communication from H. Busch; unpublished results from our own laboratory). In fact, Daubert et al. (1977) have reported that addition of RNA polymerase I to isolated nucleoli actually causes a decrease in RNA synthesis. It should be clearly stated that the term “stimulation” is used here in an operational sense to indicate an increased incorporation of precursors into nucleolar RNA. The data presented thus far cannot discriminate between an increased initiation of RNA chains or a decreased processing. Work is in progress to elucidate which of these two alternatives may be the correct one. Recently, Graessmann and Graessmann (1976) induced DNA synthesis in primary mouse kidney cells microinjected with mRNA transcribed from the A gene of the SV40 genome. They concluded that SV40 T antigen (the microinjected cells became T-antigen positive) provided the necessary information for the stimulation of cellular DNA synthesis. On the other hand there is considerable evidence that stimulation of cellular DNA synthesis is preceded, by several hours, by a marked increase in the synthesis of rRNA (Tsukada and Lieberman, 1964a,b; Mauck and Green, 1973; Nicolette and Babler, 1974; Schmid and Sekeris, 1975). Thus it is tempting to correlate the ability of T antigen to stimulate cellular DNA synthesis in viuo with its ability to stimulate in vitro nucleolar RNA synthesis. Indeed, an increase in the amount and synthesis of ribosomal RNA in resting cells infected with either SV40 or polyoma virus has been reported by Benjamin (1966), Weil et al. (1975), and May et al. (1976). We have confirmed these in uiuo findings and extended them to show that an increase in RNA synthesis clearly precedes the entry of cells into S phase. The stimulation of cellular RNA synthesis also occurs when the expression of late genes is
inhibited by FUdr, provided the T antigen is expressed (Salomon et al., 1977). Stimulation of RNA synthesis has also been reported in BHK cells infected by adenovirus 12 (Raska et al., 1971). The stimulation of RNA synthesis is modest compared to the extent of DNA synthesis that can be obtained when resting cells are stimulated to proliferate. But, as is clear from the literature, RNA synthesis in mammalian cells rarely varies by more than a factor of 2 (see review by Baserga, 1976). Our results seem to indicate that highly purified preparations of SV40 T antigen can stimulate RNA synthesis in isolated nuclei and nucleoli. It should be clearly pointed out, however, that this does not rule out that the T antigen may also act on extra nucleolar genes or even directly on DNA synthesis. The results simply point out that T antigen can stimulate the synthesis of nucleolar RNA. We would like to suggest, therefore, that one of the key mechanisms for control of cell proliferation is the synthesis of nucleolar RNA and that the T antigen from SV40 may be involved in the regulation of its synthesis. ACKNOWLEDGMENTS This work was supported by USPHS Research Grant CA-12923 and Wistar Contract HD-66323 from the National Institutes of Health. REFERENCES ALBERTS, B., and HERRICK, G. (1971). DNA-Cellulose chromatography. In “Methods in Enzymology, Vol. 21” (S. P. Colowick and N. 0. Kaplan, eds.), pp. 196-217. Academic Press, New York. BALLAL, N. R., CHOI, Y. C., MOUCHE, R., and BUSCH, H. (1977). Fidelity of synthesis of preribosomal RNA in isolated nucleoli and nucleolar chromatin. Biochemistry 74,2446-2450. BASERGA, R. (1976). “Multiplication and Division in Mammalian Cells.” Marcel Dekker, New York. BA~ERGA, R., IDE, T., and WHELLY, S. (1978). S&ulation of ribosomal RNA synthesis in isolated nuclei and nucleoli by partially purified preparations of SV40 T antigen. CoM Spring Harbor Symp. Quant. Bid., in press. BEEBEE, T. J. C., and BUTTERWORTH, P. H. W. (1977). Transcription fidelity and structural integrity of isolated nucleoli. Eur. J. Biochem. 17, W-348. BENJAMIN, T. L. (1966). Virus-specific RNA in cells productively infected or transformed by polyoma virus. J. Mol. Biol. l&359-373. BRUGGE, J. S., and BUTEL, J. S. (1975). The role of
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AND BASERGA LINDELL, T. J. (1975). Inhibition of eukaryotic DNA dependent RNA polymerase release from isolated nuclei and nucleoli by phenylmethylsulfonllfluoride. Arch. Biochem. Biophys. 171,268-275. MARTIN, R. G., and CHOU, J. Y. (1975). Simian virus 40 functions required for the establishment and maintenance of malignant transformation. J. Viral. 16, 599-612. MAFULUFF, W. F., JR., MURPHY, E. C., JR., and HUANG, R. C. (1973). Transcription of ribonucleic acid in isolated mouse myeloma nuclei. Biochemistry12,3440-3446.
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MAY, P., MAY, E., and BORDB, J. (1976). Stimulation of cellular RNA synthesis in mouse kidney cell cultures infected with SV40 virus. Exp. Cell Res. 100,433-436. MURAMATSU, M., HAYASHI, Y., ONISHI, T., SAKAI, M., TAKAI, K., and KASHIYAMA, T. (1974). Rapid isolation of nucleoli from detergent purified nuclei of various tumor and tissue culture cells. Exp. Cell Res. 88,345-351. NICOLETTE, A. A., and BABLER, M. (1974). The selective inhibitor effect of NH&l on estrogen stimulated rat uterine in vitro RNA synthesis. Arch. Biochem. Biophys. 163,656-665. PERSICO-DILAURO, M., MARTIN, R. G., and LIVINGSTON, D. M. (1977). Interaction of simian virus 40 chromatin with simian virus 40 T-antigen. J. Viral. 24,451-460. POSTEL, E. H., and LEVINE,
A. J. (1976). The requirement of simian virus 40 gene A product for the stimulation of cellular thymidine kinase activity after viral infection. Virology 73,206-215. RASKA, K., JR., STROHL, W. A., HOLOWCZAK, J. A., and ZIMMERMAN, J. (1971). The response of BHK 21 cells to infection with type 12 adenovirus. Virology44,296-306. ROTHBLUM, L. I., MAMRACK, P. M., KUNKLE, H. M., OLSON, M. 0. J., and BUSCH, H. (1977). Fractiona-
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