.n) 1990 Oxford University Press 801
Nucleic Acids Research, Vol. 18, No. 4
Expression of the type I DNA topoisomerase adenovirus-5 infected human cells
gene
in
Helmut Romig and Arndt Richter* Universitat Konstanz, Fakultat fOr Biologie, D-7750 Konstanz, FRG Received November 14, 1989; Revised and Accepted January 17, 1990
ABSTRACT The amount of topoisomerase I specific mRNA increases three- to fivefold during the early phase of infection of HeLa cells with adenovirus-5. The observed increase in specific mRNA is mainly due to an increased rate of transcription of the gene. In human 293 cells, which constitutively express the viral El A and El B genes, we determined an elevated level of topoisomerase I mRNA, comparable to the amount of mRNA present in HeLa cells early after infection with adenovirus. In contrats, in HeLa cells infected with adenovirus d1312, a mutant were the ElA region had been deleted, the amount of topoisomerase I mRNA remained constant, unless the cells were superinfected with wild type virus. Our experiments indicate that the topoisomerase I gene is transactivated by an early adenovirus protein product coded by the ElA region. In contrast to the increase in mRNA synthesis, the amount of topoisomerase I protein and the topoismerase I activity remain constant up to 24 hours after infection. INTRODUCTION Type I DNA topoisomerases are enzymes present in high amounts in all eukaryotic cells. The enzymes relax superhelical DNA and catalyze changes of DNA topology. The reaction mechanism and the biochemical properties of this class of enzymes have been extensively studied and are summarized in several reviews (1,2). In vivo type I DNA topoisomerase (together with topoisomerase II) is involved in the fundamental biological processes of DNA replication (3 and references therein) and transcription (4,5), controlling the superhelical status of the chromosomal DNA. As the DNA recognition sequences of the enzyme are often found at the boundaries of recombined DNA, it has been proposed, that the enzyme is also involved in recombination processes (6). Challberg and Kelly (7) have developed an in vitro system for adenovirus replication. They could show that, besides other viral and host coded functions, a nuclear protein from uninfected cells is essential for adenovirus DNA replication in vitro. They have purified this protein and named it nuclear factor II (NF II). The protein had topoisomerase activity and could be replaced in vitro by purified eukaryotic topoisomerase I (8). Their experiments *
To whom
correspondence
should be addressed
strongly suggest a role of eukaryotic type I DNA topoisomerase in adenovirus DNA replication. The adenovirus early region IA (EIA) codes for proteins, which are necessary for the transactivation of viral and some cellular genes (9; for review see 10). However, the expression of the majority of the cellular genes is not affected or even repressed during the early phase of the infection process. Known exceptions are the cellular genes for dihydrofolate reductase (11), f-tubulin (12) and the heat shock protein 70 (13) which are activated by the adenovirus early proteins, or the growth factor inducible JE gene which is repressed by ElA products (14). It is not known, whether these proteins play a role in adenovirus infection. Therefore it would be interesting to find out whether the gene of a cellular protein which is probably involved in viral multiplication would be activated by adenovirus. For this reason we have investigated the expression of the human topoisomerase I gene in adenovirus infected and transformed cells using a recently isolated topoisomerase I specific cDNA (15). We report below an elevated level of topoisomerase I specific mRNA in transformed cells and in HeLa cells after infection with adenovirus-5. In HeLa cells, the increase of specific mRNA is due to an increased gene activity. However, in contrast to results reported by Chow and Pearson (16) we find, that the amount of protein, as well as the enzyme activity, remain constant throughout the infection cycle. MATERIALS AND METHODS Cell culture and infection with adenovirus 5 Cell line 293, a human embryonic kidney cell line transformed by the pre-early regions of adenovirus-5 (17) and HeLa S3 cells were maintained in DME and MEM medium, respectively, supplemented with 5 percent of fetal calf serum (Gibco) at 37°C in a 95 percent air, 5 percent CO2 atmosphere on plastic dishes. HeLa cells were infected at half confluency with adenovirus type 5 or d1312 (18) at a multiplicity of 100 plaque forming units per cell.
The titer of adenovirus d1312 was determined with 293
cells). For DNA synthesis 2 x 107 cells were collected and incubated in lml DME medium containing 10,Ci 3H-thymidine for 15 min at 370C. The cells were pelleted and the trichloroacetic acid precipitable radioactivity was determined in Hirt supematants (19).
802 Nucleic Acids Research RNA preparation and analysis Total cellular RNA was prepared from 2 x 107 cells by the guanidinium isothiocyanate method described by Chirgwin et al. (20). 30 jig of total cellular RNA per time point were denatured for 10 minutes at 65°C in 50 percent formamide, 6.5 percent formaldehyde, 0.5x3-(N-morpholino) propanosulfonic acid buffer (MOPS) and separated on a 0.8 percent agarose gel containing 0.66 M formaldehyde. The RNA was transferred to Hybond N membranes (Amersham Buchler) and immobilized for 2 hours at 80°C in vacuo. A 2.1 kb 3-actin cDNA in pUC-8, kindly provided by Dr.H.Arnold, University of Hamburg, a 1975 bp DNA fragment coding for 18 S rRNA cloned in pBR 322 (21) and a 2.2 kb fragment of human topoisomerase I cDNA cloned in pUC-8 (22) were used in this study. All DNA probes were nick-translated with 32P-dATP to a specific activity of 108 cpm / Zg DNA. Hybridization was performed in 5 x SSC, 50 percent formamide, 0.5 percent sodium dodecyl sulfate at 42°C. After washing in 0.2 x SSC at 600C, the filters were blotted dry with 3MM paper (Whatman) and exposed to X-ray film with intensifying screen. Filters were dehybridized in 5 mM Tris-HCl pH 8.0, 2 mM EDTA for one hour at 65°C and were re-used with different radioactive probes. The amount of specific mRNA was measured by scanning autoradiograms with a LKB laser densitometer. The amount of polyA-mRNA was determined according to Harley (23). Nuclear run on transcription Nuclear run on transcription was performed as described (24) with some modifications. Briefly, nuclei were isolated from infected or mock-infected HeLa cells and frozen in a solution containing 50 mM Tris-HCl pH 8.3, 5 mM magnesium chloride, 0.1 mM EDTA and 40 percent glycerol at a concentration of x 108 nuclei per ml. In vitro transcription reactions were carried out at 30°C for 20 minutes in a solution containing one volume of isolated nuclei (corresponding to 107 cells) and one volume of a twofold concentrated reaction buffer with 10 mM Tris-HCl pH 8.0, 5 mM magnesium chloride, 300 mM potassiun chloride, 1 mM each of ATP, CTP,GTP and 100 4Ci of 32plabeled UTP. After the in vitro reaction, RNA was prepared from the nuclei by the guanidinium isothiocyanate method as described above. 1-2 x 106 cpm of labeled RNA was then hybridized to 2 4g of isolated topoisomerase I cDNA immobilized on Hybond N membranes.
DNA blot hybridization Adenovirus DNA was purified from Hirt supernatants, digested with HindIll and separated on 1 percent agarose gels. After transfer to nitrocellulose the filters were hybridized with radioactively labeled pAdS-XhoC DNA (25). pAd5-XhoC contains the adenovirus Xho-C fragment and was kindly provided by T.Shenk.
Preparation and analysis of nuclear extracts For this purpose we adapted a quantitative extraction procedure developed for the purification of topoisomerases from eukaryotic cells (26). Cells from one plate (2x107) were harvested and lysed in 0.25 percent Triton-X-100, 10 mM EDTA, 10 mM mercaptoethanol, 10 mM Tris-HCl pH 7.5. Nuclei were pelleted by a 5 minute centrifugation at 500xg and washed two times in the same buffer. The washed nuclei were then extracted into 500 /A of lysis buffer containing 650 mM sodium chloride. After
30 minutes on ice, nuclei were pelleted for 30 min at 20000 x g and 0°C (Sorvall centrifuge, SS34 rotor) and the supernatant was recovered. For Western analysis, nuclear extracts were prepared as described above. Sodium dodecylsulfate polyacrylamide gel electrophoresis (27) and immunoblotting analysis with antibodies against topoisomerase I was performed essentially as described by Towbin et al. (28) and Oddou et al. (22). The signals were quantitated by scanning of the stained filters with a LKB laser densitometer.
Determination of the topoisomerase I activity Nuclear extracts were prepared as described above. Nuclear extracts were serially diluted into 20 mM Tris-HCl pH 7.5, 10 mM 3-mercaptoethanol, 50 mM sodium chloride, 0.1 mM EDTA and 5 percent glycerol. Topoisomerase I activity was then determined by incubation of superhelical SV40 DNA with the diluted extracts at 37°C as described (22).
RESULTS Adenovirus infection increases the amount of topisomerase I mRNA Human type I DNA topoisomerase specific cDNA hybridized to one single mRNA species in agreement with the data published by D'Arpa et al. (29). This mRNA migrated under denaturing conditions in agarose gels as an RNA species of 4.2 kb relative to 28 S and 18 S ribosomal RNA (Fig. lA). Using the same cDNA probe, we examined the effect of adenovirus-5 infection on the amount of the topoisomerase I mRNA throughout the infection cycle up to sixteen hours after viral infection. For these experiments, total cellular RNA was isolated at various times after infection, fractionated on denaturing agarose gels, transferred to membranes and hybridized to a set of different radioactively labeled DNA probes. As shown in Figure lB, infection of HeLa cells with adenovirus-5 resulted in an increase in mRNA coding for topoisomerase I. The amount of the mRNA increased progressively starting already two hours after infection. A maximum value was reached four hours after adenovirus infection. At that time, the level of topoisomerase I mRNA was three- to fivefold higher compared to uninfected HeLa cells or to HeLa cells four hours after a 'mock' infection (not shown). Thereafter, the amount of topoisomerase I mRNA continuously decreased, reaching the basal level at 12 hours after infection. After sixteen hours, less than 30 percent of topoisomerase I mRNA was present compared to uninfected cells. As a control we determined the relative amount of 3-actin mRNA (Fig. IC), ribosomal RNA (Fig. ID) and of total poly (A)RNA (Fig. lE) at various times after adenovirus infection. Our results are summarized in Figure 2, demonstrating that the level of total poly(A)-RNA as well as the level of 3-actin mRNA remained constant during the early phase of the infection but declined later, reaching a value corresponding to 50 percent and 20 percent of the amount detectable in uninfected cells 16 hours after infection, respectively. The amount of ribosomal RNA remained constant throughout the entire infection cycle.
Adenovirus infection increases the rate of transcription of the topoisomerase I gene The adenovirus induced increase of topoisomerase I specific mRNA may be due to an altered stability of the topoisomerase I mRNA or to an increased rate of transcription of the
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Fig. 1: Effect of adenovirus infection on the expression of cellular genes. (A) Northern analysis of total cellular RNA prepared from uninfected HeLa cells with a topoisomerase I cDNA probe (3.2.4). Linearized pUC3.2.4 DNA, containing the topoisomerase I cDNA (4.9 kb) or isolated 3.2.4 cDNA (2.2 kb) were run in a parallel lane and served as size markers (M). 28 S and 18 S shows the migration of 28 S and 18 S rRNA. kb = kilobases. (B-D) HeLa S3 cells were infected with adenovirus 5. At the times indicated total cellular RNA was prepared and 30 jg were separated on agarose gels. After transfer of RNA the Nylon membranes were hybridized with topoisomerase I cDNA (B), (3-actin cDNA (C) and a cloned rRNA gene fragment (D) successively. For analysis of poly(A)-RNA lig of total cellular RNA was spotted onto nitrocellulose filters and hybridized to end-labeled oligo-dTI8 (E).
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hours post Infection Fig. 2: Increase of the steady state level of topoisomerase I mRNA during adenovirus infection. Three independent experiments performed as described in the legend of Fig. 1 are summarized. After hybridization the radioactive bands were excised from the filters and quantitated by liquid scintillation counting. One arbitrary unit corresponds to an average of 229 counts per minute (topo I), 3400 counts per minute (,B-actin) and 590 counts per minute (poly(A)-RNA).
topoisomerase I gene. To distinguish between these possibilities, we performed run on transcription experiments with nuclei isolated from HeLa cells at different times after infection. For these experiments, identical amounts of in vitro labeled radioactive total nuclear RNA prepared from 107 nuclei were hybridized with 2 ug of the isolated topoisomerase I cDNA fragment immobilized on nitrocellulose filters. The same amount of pUC-8 DNA was used as a control. As shown in Figure 3, already two hours after adenovirus-5 infection we detected a fourfold higher amount of hybridized RNA compared to the signal obtained with RNA from uninfected cells. This value increased to approximately 15-fold four hours after infection. At later times, the amount of hybridized RNA decreased drastically. Eight hours after infection the radioactive signal was as low as in uninfected
cells. 12 hours after infection, when still considerable amounts of topoisomerase I mRNA are present in the cells, the signal further declined below the level of detection under our experimental conditions. The results shown in Figure 3 suggest, that the increase in the total amount of topoisomerase I mRNA observed early after adenovirus infection, is mainly due to newly synthesized mRNA and not to an increase in the stability of the topoisomerase I mRNA already present in the cells. This also means, that the topoisomerase I gene is specifically induced after adenovirus infection, a phenomenon previously observed for the cellular 3tubulin gene (12) or the heat shock protein 70 gene (13). Furthermore, there are striking similarities between the activation pattern observed for the expression of the topoisomerase I gene
804 Nucleic Acids Research
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Fig. 3: Transcriptional activation of the topoisomerase I gene. (A) Nuclear run on transcription experiments were performed as described in Material and Methods with nuclei isolated at the times indicated after adenovirus infection of HeLa cells. 1 -2 x 106 counts per minute were hybridized to 2 /kg of isolated topoisomerase I cDNA fragment. 2 j4g of pUC-8 DNA was immobilized on the filter and used as a control (lane C). The radioactive signals were quantitated by scanning the autoradiogram depicted in (A) with a laser densitometer (B).
early in infection and the activation of the early viral genes. This may be due to an effect of the viral ElA products. If this assumption is correct, we would expect to find a higher topoisomerase I mRNA level in human 293 cells which express the adenovirus EIA and E1B products constitutively. Indeed, when identical amounts of total RNA isolated from uninfected HeLa cells or from adenotransformed 293 cells were analysed by hybridization, we found an fourfold higher amount of mRNA specific for topoisomerase I in the adenotransformed 293 cells compared to HeLa cells (Fig.4). The amount of topoisomerase I mRNA present in 293 cells corresponded approximately to the amount of mRNA present in HeLa cells four hours after infection
with adenovirus-5.
Infection of HeLa cells with adenovirus wild type but not with the mutant virus dL312 increases the amount of topoisomerase I mRNA To further confirm this conclusion we infected HeLa cells with Ad d1312, an adenovirus mutant were the region coding for ElA/E1B has been deleted (18). Four hours after infection with the mutant virus we then superinfected the cells with wild type virus. The amount of topoisomerase I mRNA was determined after northern blotting of total cellular RNA. Figure 5A shows the result of such an experiment. After infection of HeLa cells with the deletion mutant d1312, the amount topoisomerase I mRNA remains approximately constant (lane 1 and 3: 1- vs 1.3-fold; in two other experiments this value for d1312 was 1). However, in cells superinfected with the adenovirus wild type the amount of topoisomerase I mRNA increased 3.8-fold four
Fig. 4: Comparision of the amount of topoisomerase I mRNA in HeLa and 293 cells. Total cellular RNA was prepared from HeLa and 293 cells. 30 mg each was subjected to agarose gel electrophoresis, blotted to nylon membranes and visualized after hybridization against topoisomerase I cDNA.
hours later (lane 4). This is a topoisomerase I mRNA level comparable to the level reached four hours after infection of HeLa cells with adenovirus wild type alone (lane 2; 3.4-fold). The same results were obtained when the cells were superinfected with wild type eight hours instead of four hours after infection with the mutant virus (data not shown). Similar to the situation after infection with wild type virus alone, eight hours after superinfection the amount of topoisomerase I mRNA again declined. Thus, the kinetics of topoisomerase I mRNA expression are the same in cells solely infected with the wild type virus, or infected with d1312 and wild type. As a control, we determined the amounts of viral DNA present in the infected cells. Adenovirus DNA was isolated from Hirt supernatants, purified and digested with restriction enzyme Hind mI. After separation of the DNA on agarose gels and transfer to nitrocellulose membranes, the filters were hybridized with the Xho-C fragment of pAdXhoC. Due to the deleted EIA region in the mutant DNA we expect one hybridizing fragment 1.8 kbp in length in samples containing d1312 DNA and, after superinfection a second fragment, 2.8 kbp long, derived from the adenovirus wild type. Fig. 5B clearly shows, that comparable amounts of DNA derived from mutant and wild type virus were present in the cells. Obviously, infection with the mutant virus d1312, were the E1A region is deleted, did not affect topoisomerase I expression.
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Fig. 5: The level of topoisomerase I mRNA in HeLa cells infected with the ad- d1312. (A) HeLa cells were either mock infected (lane 1), infected with wild type virus (lane 2), the mutant d1312 (lane 3 and 4). Total cellular RNA was prepared from the cells four hours later (lane 1, 2, 3). At that time, cells infected with d1312 were superinfected with wild type virus and after four hours total cellular RNA was prepared from these cells (lane 4). Topoisomerase I mRNA was then visulalized by northern blotting experiments. The relative amounts of topoisomerase I mRNA were determined by scintillation counting of the excised bands and were 1 (lane 1; 158 counts per minute), 3.4 (lane 2), 1.3 (lane 3) and 3.8 (lane 4) arbitrary units. (B) In a parallel experiment adenovirus DNA was prepared from the cells, digested with Hind III and separated on agarose gels. The adenovirus DNA was identified by hybridization to the nick-translated adenovirus Xho-C fragment DNA.
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Fig. 6: Analysis of the amount of topoisomerase I present in adenovirus infected cells. (A) HeLa cells were fractionated into cytosol (C), nuclear extract (NE) and residual pellet (P) as described in the text. An equal amount of total protein was then separated on polyacrylamide gels, transferred to filters and stained with topoisomerase I specific antibodies. (B) High-salt nuclear extracts were prepared at different times after adenovirus infection. 150 ug (lane 1) and 30 jig (lane 2) of extract were separated on denaturing polyacrylamide gels, transferred to filters and stained with topoisomerase I specific antibodies. As a control, 25 ng of purified topoisomerase I from calf thymus was applied onto the same gel (lane T). The solid triangles mark the position of the adenovirus coded 110 kDa protein. h = hours post infection. (-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa) ovalbumin (45 kDa) and carbonic anhydrase (29 kDa) were used as molecular weight markers.
The amount of topoisomerase I protein and the specific activity of the enzyme remain constant upon adenovirus infection We have further analysed, whether the elevated level of topoisomerase I mRNA in HeLa cells upon adenovirus infection is accompanied by an increase in the amount of topoisomerase I protein in infected cells. For this purpose nuclei were pelleted, washed several times in lysis buffer (see Material and Methods) and then extracted into the same buffer containing additionally 700 mM sodium chloride. After centrifugation, the supernatant
and the residual nuclear structures were collected. As shown in Figure 6A, this method resulted in an almost quantitative recovery of topoisomerase I in the salt extract when analysed in Western blots. Less than two percent of the topoisomerase I protein are found in the cytosol fraction or in the residual nuclear pellet. The same distribution of topoisomerase I is obtained when adenovirus infected HeLa cells are fractionated by this procedure (data not shown). This method therefore seems to be reliable to quantitatively assess changes of type I topoisomerase during infection.
806 Nucleic Acids Research The relative amounts of topoisomerase I protein were determined by Western analysis. For this purpose, nuclear extracts prepared from HeLa cells at different times after infection were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and transferred to nitrocellulose membranes. For our studies we used polyclonal rabbit antibodies, directed against a topoisomerase I cDNA fragment expressed in E.coli or the monoclonal antibody B7 described by Oddou et al. (22). The filters were stained and the signals quantitated by laser densitometry. Comparing the signals from different dilution steps of the nuclear extracts, we were able to determine the relative amounts of topoisomerase I protein (30).
A typical result is shown in Figure 6B. In this experiment nuclear
extracts were prepared 0,
6, 12, 18 and 24 hours after infection. 50 ng of purified topoisomerase I from calf thymus was separated on the gel as a control (Fig.6B, lane T). With both topoisomerase I specific antibodies we detected one major crossreacting band, corresponding to a protein migrating with 100 kDa, the molecular weight of undegraded topoisomerase I. One additional signal was present in extracts prepared 18 or 24 hours after infection, migrating with a slightly reduced mobility in the gel, compared to topoisomerase I. This signal is probably derived from the adenovirus 110 kDa protein. This viral protein constitutes the major Coomassie blue stainable band at late times after infection and we do not know, whether this weak crossreactivity is of any significance. In
a
series of experiments of this type, we never observe
changes in the amount of topoisomerase I up to 24 hours after infection. We have also analysed the amount of topoisomerase I present in nuclear extracts in two hours intervalls, starting from four hours after infection, when the topoisomerase I gene is maximally expressed, up to 14 hours after infection, when the
amount of mRNA is reduced and the topoisomerase I gene is already repressed, compared to uninfected cells. Again, we did not find any variation in the amount of topoisomerase I.
Fig. 7: Enzymatic activity of topoisomerase I throughout the infection cycle. Highsalt nuclear extracts were prepared at different times after adenovirus infection of HeLa cells. Nuclear extracts were diluted 1:40 and incubated for 30 minutes (lane 1) or 15 minutes (lane 2) with 0.5 pAg of supercoiled SV40 DNA. The DNA was purified from the samples and separated on 0.8% agarose gels in 40 mM Tris-acetate pH 8.0, 5 mM sodium acetate and 2 mM EDTA. F I and F IV marks the position of supercoiled and relaxed SV40 DNA respectively.
Finally, we have examined the enzymatic activity of topoisomerase I after adenovirus infection in extracts prepared as described above. First, the relaxation activity of topoisomerase I was determined in serial dilutions of these extracts prepared from uninfected cells. The dilution step, which resulted in the complete relaxation of 0.5 mg of supercoiled SV40 DNA in 30 minutes at 37°C, was then used to compare the topoisomerase I activities in extracts prepared at different times after infection. As shown in Figure 7 the relaxation activity was constant at all times after infection and not distinguishable from the enzyme activity present in uninfected cells. Essentially the same results were obtained, when the activity of the enzyme was monitored by a DNA binding assay (not shown) which measures the transfer
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hours post Infection Fig.8: Activation of the topoisomerase I gene during the early phase of adenovirus-5 infection. Fig.8 summarizes the data given in Fig. lB and Fig.3B in relation to the infection cycle. Adenovirus DNA synthesis was determined after in vivo incubation with tritiated thymidine in Hirt supematants prepared at different times
after infection.
Nucleic Acids Research 807 of a radioactively labeled oligonucleotide to topoisomerase I as described by Chow and Pearson (16). This indicates, that the relaxation activity as well as the DNA binding activity of topoisomerase I remained unaffected, throughout the infection cycle.
DISCUSSION The expression of most of the cellular genes remaines unaffected during the first ten hours after adenovirus infection (31, 32). As summarized in Figure 8 and in contrast to most of the known cellular genes, the transcriptional activity of the topoisomerase I gene was greatly stimulated upon infection. This stimulation resulted in a three- to fivefold increase in the steady state level of topoisomerase I mRNA compared to uninfected cells and is specific for the adenovirus system. Mock infection, or infection of african green monkey kidney cells with another DNA tumor virus, the Simian virus 40 (not shown), had no effect on the transcription of the topoisomerase I gene or the steady state level of the mRNA. The increase in gene activity as well as of the amount of topoisomerase I specific mRNA was restricted to a time period between two and four hours after infection. During this time, the transcription of early adenovirus genes is activated by the products of the ElA region. Thereafter, from 4 to 8 hours after infection, these early viral genes are repressed by an unknown mechanism and the transcription rate of these genes declines. This biphasic kinetics of transcription is typical for the regulation of early viral genes by the EIA products. The regulation of the cellular topoisomerase I gene followed the same kinetic parameters as that of the early adenoviral genes. In agreement with the data published by Berk (10) and Stein and Ziff (12), the amount of total cellular RNA, represented mainly by ribosomal RNA, and the amount of polyadenylated mRNA remained constant throughout this period of time. The same was also found for the expression of the 3-actin gene which may be representative for the majority of cellular genes. We have compared the expression of the topoisomerase I gene in HeLa cells with its expression in human adenotransformed embryonic kidney cells. These transformed cells, which constitutively express the EIA and E1B genes, showed a permanently elevated level of DNA topoisomerase I mRNA. The relative amounts of mRNA present in these cells corresponded exactly to the amount of mRNA found in HeLa cells at four hours after infection with adenovirus-5 (Fig. 4). Interestingly, the amount of topoisomerase I mRNA present in transformed HeLa cells is comparable to the amount of mRNA present in untransformed human fibroblast cells (Hel 299 cells; data not shown). This makes it rather unlikely that the transformed phenotype per se is responsible for the increase in topoisomerase I mRNA observed in the adenotransformed 293 cells. Furthermore, we could show that infection of HeLa cells with the adenovirus mutant d1312, in which the region coding for the EIA proteins had been deleted, did not result in an increase of the amount of topoisomerase I mRNA (Fig.5). Taken together, these results strongly suggest, that the increase of DNA topoisomerase I specific mRNA in infected HeLa cells and the elevated level in adenotransformed cells is a consequence of transactivation of the gene by the adenovirus ElA products. The increase in the steady state level of topoisomerase I mRNA was not accompanied by a comparable increase on the protein level. Instead we have found, that the amount of topoisomerase I remained constant after adenovirus infection of HeLa cells
(Fig.6). This was surprising, as Chow and Pearson have reported
increase in the amount of enzyme as well as of topoisomerase I activity upon adenovirus infection of HeLa cells (16). We could not detect an net increase in the enzyme activity in an relaxation assay (Fig. 7) or in an assay measuring the covalent complex formation (data not shown). The reason for this discrepancy between our results and the results published by Chow and Pearson may be due to the different preparation procedures used. With our procedure, topoisomerase I is extracted quantitatively in one high salt fraction from purified nuclei. In contrast, Chow and Pearson have used a buffer with moderately low salt concentrations (33). The observed increase of topoisomerase I in their extracts could therefore result from an altered distribution of the enzyme upon adenovirus infection between different nuclear compartments, rendering part of the enzyme extractable at low salt concentrations. Given an 3- to 5-fold increase of the amount of topoisomerase I mRNA over a period of several hours, one might expect to find a significant increase in the amount of topoisomerase protein as well. Obviously this was not the case. This finding may be, however, less surprising if we realize the consequence of the increase of topoisomerase I mRNA after serum stimulation of quiescent human fibroblast cells on the level of topoisomerase I protein. In this cells we observed an continuous increase in topoisomerase I mRNA over a period of 25 hours. After that time, serum stimulated cells contained the 6-fold amount of topoisomerase I mRNA compared to resting cells. During the same period of time the amount of topoisomerase I protein and the enzyme activity increased only twofold (H.Romig and A.Richter, submitted). This finding may indicate that topoisomerase I protein synthesis is regulated at the level of translation as well. Assuming a similar relationship between mRNA level and translation in adenovirus infected HeLa cells, one would not expect that the increase in the amount of topoisomerase I mRNA over a period of 3-4 hour in these cells would result in a drastic increase of topoisomerase I protein and enzyme activity. Infection of human cells with adenovirus result in the shut-off of host protein synthesis late in infection (for review see 34). This is caused by the blockade of the transport of mRNA from the nucleus to the cytoplasm by proteins encoded by the virus (35) and the inhibition of host mRNA translation. The availability of host proteins essential for viral metabolism therefore greatly depends on the half-life of these proteins and the amount of their mRNA synthesized early in infection. Heck et al. (36) have determined the half-live of topoisomerase I in transformed chicken hepatoma cells and primary chicken embryo fibroblast cells. The half-life of topoisomerase I was found to be 23 hours in these primary cells and 16 hours in the transformed cells. Providing the half-live of HeLa topoisomerase I is within the same range, a considerable amount of enzyme would have decayed late in infection after host shut-off. At this time maximal adenovirus DNA synthesis and transcription of late viral genes takes place. The constancy in the level of topoisomerase I protein observed late in the infection cycle may be a consequence of the transcriptional activation of the gene. The increased amount of topoisomerase I mRNA may compensate for a depressed synthesis of protein later in infection thus providing enough enzyme to substitute those topoisomerase I molecules which decay after host shut-off. The transcriptional activation of the topoisomerase I gene may be a useful reaction, if the virus needs an active topoisomerase I for its replication and/or transcription. The participation of an
808 Nucleic Acids Research topoisomerase I in transcription of cellular genes has been shown in several systems (4, 5, 38). Though a direct experimental evidence is not available, a function of the enzyme in transcription of adenovirus DNA cannot be excluded. More data have been presented which strongly argue in favour of a participation of topoisomerase I in adenovirus replication. Nagata et al. (8) have purified a cellular factor (nuclear factor II; NF II) essential for the replication of adenovirus DNA in vitro which is most probably topoisomerase I. Furthermore, in a recent publication, Schaack et al. (37) have shown that topoisomerase I (together with topoisomerase II) is involved in adenovirus replication in vivo as well. Thus, the biological function of the transcriptional activation of the topoisomerase I gene may be, to provide an excess of transcripts, needed for a constant supply of active topoisomerase I necessary for the propagation of the virus during late stages after viral infection.
ACKNOWLEDGEMENT The author wish to thank Drs. T.Shenk, J.L.Bos and E.Fanning for providing plasmids harbouring ElA/ElB and a stock lysate of the adenovirus mutant d1312. We further thank Dr. R.Knippers for helpful discussions and critical reading of the manuscript and M.Mollerbernd for preparation of plasmids and virus. The work was supported by the Deutsche Forschungsgemeinschaft through SFB 156.
REFERENCES 1. Vosberg, H-P. (1985). Curr.Top.Microbiol.Immunol.114. 19-102. 2. Wang, J.C. (1987). Biochim. Biophys.Acta 909. 1-9. 3. Avemnann,K., Knippers R., Koller T. and Sogo J.M. (1988). Mol.Cell.Biol. 8. 3026-3034. 4. Brill, J.S. and Sternglanz R. (1988). Cell 54. 403-411. 5. Zhang,H., Wang J.C. and Liu L.F. (1988). Proc.Natl.Acad. Sci.USA 85. 1060-1064. 6. Bullock,P., Champoux J.J. and Botchan M. (1985). Science 230. 954-958. 7. Challberg M.D. and Kelly T.J. (1979). Proc.Natl.Acad.Sci.USA 76.655-659. 8. Nagata, K., Guggenheimer R.A. and Hurwitz J. (1983b). Proc.Natl.Acad.Sci. USA 80. 4266-4270. 9. Lillie,W.L. and Green M.R. (1989). Nature 338. 39-44. 10. Berk, J.A. (1986). Ann.Rev.Genet.20. 45-79. 11. Yoder, S.S., Robberson B.L., Leys E.J., Hook A.G., Al-Ubaidi M., Yeung C-Y., Kellems R.E. and Berget S.M. (1983). Mol.Cell.Biol.3. 819-828. 12. Stein,R. and Ziff E.B. (1984). Mol.Cell.Biol.4. 2792-2801. 13. Kao, H-T. and Nevins J.R. (1983). Mol.Cell.Biol.3. 2058-2065. 14. Timmers, H.T., vanDam H., Pronk G.J., Bos J.L. and A.J. van der Eb. (1989). J.Virol. 63. 1470-1473. 15. Kunze, N., Yang, G.C., Jiang, Z.Y., Hameister, H., Adolph, S., Wiedom, K-H., Richter, A. and Knippers, R. (1989). Human genetics 84. 6-10. 16. Chow, K-C. and Pearson G.D. (1985). Proc.Natl.Acad.Sci.USA 82. 2247-2251. 17. Graham,F.L., Smiley J., Russell W.C. and Naim R. (1977). J.Gen.Virol. 36. 59- 72. 18. Jones, H. and Shenk, T. (1979). Proc.Natl.Acad.Sci. USA 76. 3665-3669. 19. Hirt, B. 1969. J.Mol.Biol.26 365-369. 20. Chirgwin, J.M., Przybyla A.E., MacDonald R.J. and Rutter W.J. (1979). Biochemistry 18. 5294-52 21. Michot,B., Bachellerie J-P. and Raynal F. (1983). Nucl.Acids Res. 11. 3375-3391. 22. Oddou, P., Schmidt U., Knippers R. and Richter A. (1988). Eur.J.Biochem.177. 523-529. 23. Harley,B.C. (1987). Gene Anal.Techn.4. 17-22. 24. Groudine, M., Peretz M. and Weintraub H. (1983). Mol.Cell.Biol.l. 281-288. 25. Logan,J., Pilder, S. and Shenk, T. (1984). Cancer Cells 2. 527.
26. Strausfeld, U. and Richter A. (1989).Prep.Biochem 19. 37-48. 27. Lammli, U.K. 1970. Nature 227. 680-685. 28. Towbin,H., Staehelin T. and Gordon J. (1979). Proc.Natl.Acad.Sci.USA 76. 4350- 4354. 29. D'Arpa,P., Machlin P.S., Ratrie H.R., Rothfield N.F., Cleveland D.W. and Earnshaw W.C. (1988). Proc.Natl.Acad.Sci.USA 85. 2543-2547. 30. Heck, M.M.S. and Earnshaw W.C. (1986). J.Cell.Biol.103. 2569-2581. 31. Babich,A., Feldman L.T., Nevins J.R, Damell J.E and Weinberger C. (1983). Mol.Cell.Biol.3. 1212-1221. 32. Gaynor, R.B. and Berk A.J. (1983). Cell 33. 683-693. 33. Pearson, G.D., Chow K-C., Enns R.E., Ahern K.G., Corden J.L. and Harpst J.A. (1983). Gene 23. 293-305. 34. Schneider, R.J. and Shenk T. (1987). Ann.Rev.Biochem. 56. 317-332. 35. Sarnow, P., Hearing, P.,Anderson, C.W., Halbert, D.N., Shenk, T. and Levine, A.J. (1984). J.Virol. 49. 692-700. 36. Heck, M.M.S., Hittelman W.N. and Earnshaw W.C. (1988). Proc.Natl.Acad.Sci. USA 85. 1089-1090. 37. Schaack, J., Schedl,P. and Shenk,T. (1990). J.Virol. 64. 78-85. 38. Stewart, A.F. and Schiitz, G. (1987).Cell 50: 1109-1117.
Nucleic Acids Research, Vol. 18, No. 4
Expression of the type I DNA topoisomerase adenovirus-5 infected human cells
gene
in
Helmut Romig and Arndt Richter* Universitat Konstanz, Fakultat fOr Biologie, D-7750 Konstanz, FRG Received November 14, 1989; Revised and Accepted January 17, 1990
ABSTRACT The amount of topoisomerase I specific mRNA increases three- to fivefold during the early phase of infection of HeLa cells with adenovirus-5. The observed increase in specific mRNA is mainly due to an increased rate of transcription of the gene. In human 293 cells, which constitutively express the viral El A and El B genes, we determined an elevated level of topoisomerase I mRNA, comparable to the amount of mRNA present in HeLa cells early after infection with adenovirus. In contrats, in HeLa cells infected with adenovirus d1312, a mutant were the ElA region had been deleted, the amount of topoisomerase I mRNA remained constant, unless the cells were superinfected with wild type virus. Our experiments indicate that the topoisomerase I gene is transactivated by an early adenovirus protein product coded by the ElA region. In contrast to the increase in mRNA synthesis, the amount of topoisomerase I protein and the topoismerase I activity remain constant up to 24 hours after infection. INTRODUCTION Type I DNA topoisomerases are enzymes present in high amounts in all eukaryotic cells. The enzymes relax superhelical DNA and catalyze changes of DNA topology. The reaction mechanism and the biochemical properties of this class of enzymes have been extensively studied and are summarized in several reviews (1,2). In vivo type I DNA topoisomerase (together with topoisomerase II) is involved in the fundamental biological processes of DNA replication (3 and references therein) and transcription (4,5), controlling the superhelical status of the chromosomal DNA. As the DNA recognition sequences of the enzyme are often found at the boundaries of recombined DNA, it has been proposed, that the enzyme is also involved in recombination processes (6). Challberg and Kelly (7) have developed an in vitro system for adenovirus replication. They could show that, besides other viral and host coded functions, a nuclear protein from uninfected cells is essential for adenovirus DNA replication in vitro. They have purified this protein and named it nuclear factor II (NF II). The protein had topoisomerase activity and could be replaced in vitro by purified eukaryotic topoisomerase I (8). Their experiments *
To whom
correspondence
should be addressed
strongly suggest a role of eukaryotic type I DNA topoisomerase in adenovirus DNA replication. The adenovirus early region IA (EIA) codes for proteins, which are necessary for the transactivation of viral and some cellular genes (9; for review see 10). However, the expression of the majority of the cellular genes is not affected or even repressed during the early phase of the infection process. Known exceptions are the cellular genes for dihydrofolate reductase (11), f-tubulin (12) and the heat shock protein 70 (13) which are activated by the adenovirus early proteins, or the growth factor inducible JE gene which is repressed by ElA products (14). It is not known, whether these proteins play a role in adenovirus infection. Therefore it would be interesting to find out whether the gene of a cellular protein which is probably involved in viral multiplication would be activated by adenovirus. For this reason we have investigated the expression of the human topoisomerase I gene in adenovirus infected and transformed cells using a recently isolated topoisomerase I specific cDNA (15). We report below an elevated level of topoisomerase I specific mRNA in transformed cells and in HeLa cells after infection with adenovirus-5. In HeLa cells, the increase of specific mRNA is due to an increased gene activity. However, in contrast to results reported by Chow and Pearson (16) we find, that the amount of protein, as well as the enzyme activity, remain constant throughout the infection cycle. MATERIALS AND METHODS Cell culture and infection with adenovirus 5 Cell line 293, a human embryonic kidney cell line transformed by the pre-early regions of adenovirus-5 (17) and HeLa S3 cells were maintained in DME and MEM medium, respectively, supplemented with 5 percent of fetal calf serum (Gibco) at 37°C in a 95 percent air, 5 percent CO2 atmosphere on plastic dishes. HeLa cells were infected at half confluency with adenovirus type 5 or d1312 (18) at a multiplicity of 100 plaque forming units per cell.
The titer of adenovirus d1312 was determined with 293
cells). For DNA synthesis 2 x 107 cells were collected and incubated in lml DME medium containing 10,Ci 3H-thymidine for 15 min at 370C. The cells were pelleted and the trichloroacetic acid precipitable radioactivity was determined in Hirt supematants (19).
802 Nucleic Acids Research RNA preparation and analysis Total cellular RNA was prepared from 2 x 107 cells by the guanidinium isothiocyanate method described by Chirgwin et al. (20). 30 jig of total cellular RNA per time point were denatured for 10 minutes at 65°C in 50 percent formamide, 6.5 percent formaldehyde, 0.5x3-(N-morpholino) propanosulfonic acid buffer (MOPS) and separated on a 0.8 percent agarose gel containing 0.66 M formaldehyde. The RNA was transferred to Hybond N membranes (Amersham Buchler) and immobilized for 2 hours at 80°C in vacuo. A 2.1 kb 3-actin cDNA in pUC-8, kindly provided by Dr.H.Arnold, University of Hamburg, a 1975 bp DNA fragment coding for 18 S rRNA cloned in pBR 322 (21) and a 2.2 kb fragment of human topoisomerase I cDNA cloned in pUC-8 (22) were used in this study. All DNA probes were nick-translated with 32P-dATP to a specific activity of 108 cpm / Zg DNA. Hybridization was performed in 5 x SSC, 50 percent formamide, 0.5 percent sodium dodecyl sulfate at 42°C. After washing in 0.2 x SSC at 600C, the filters were blotted dry with 3MM paper (Whatman) and exposed to X-ray film with intensifying screen. Filters were dehybridized in 5 mM Tris-HCl pH 8.0, 2 mM EDTA for one hour at 65°C and were re-used with different radioactive probes. The amount of specific mRNA was measured by scanning autoradiograms with a LKB laser densitometer. The amount of polyA-mRNA was determined according to Harley (23). Nuclear run on transcription Nuclear run on transcription was performed as described (24) with some modifications. Briefly, nuclei were isolated from infected or mock-infected HeLa cells and frozen in a solution containing 50 mM Tris-HCl pH 8.3, 5 mM magnesium chloride, 0.1 mM EDTA and 40 percent glycerol at a concentration of x 108 nuclei per ml. In vitro transcription reactions were carried out at 30°C for 20 minutes in a solution containing one volume of isolated nuclei (corresponding to 107 cells) and one volume of a twofold concentrated reaction buffer with 10 mM Tris-HCl pH 8.0, 5 mM magnesium chloride, 300 mM potassiun chloride, 1 mM each of ATP, CTP,GTP and 100 4Ci of 32plabeled UTP. After the in vitro reaction, RNA was prepared from the nuclei by the guanidinium isothiocyanate method as described above. 1-2 x 106 cpm of labeled RNA was then hybridized to 2 4g of isolated topoisomerase I cDNA immobilized on Hybond N membranes.
DNA blot hybridization Adenovirus DNA was purified from Hirt supernatants, digested with HindIll and separated on 1 percent agarose gels. After transfer to nitrocellulose the filters were hybridized with radioactively labeled pAdS-XhoC DNA (25). pAd5-XhoC contains the adenovirus Xho-C fragment and was kindly provided by T.Shenk.
Preparation and analysis of nuclear extracts For this purpose we adapted a quantitative extraction procedure developed for the purification of topoisomerases from eukaryotic cells (26). Cells from one plate (2x107) were harvested and lysed in 0.25 percent Triton-X-100, 10 mM EDTA, 10 mM mercaptoethanol, 10 mM Tris-HCl pH 7.5. Nuclei were pelleted by a 5 minute centrifugation at 500xg and washed two times in the same buffer. The washed nuclei were then extracted into 500 /A of lysis buffer containing 650 mM sodium chloride. After
30 minutes on ice, nuclei were pelleted for 30 min at 20000 x g and 0°C (Sorvall centrifuge, SS34 rotor) and the supernatant was recovered. For Western analysis, nuclear extracts were prepared as described above. Sodium dodecylsulfate polyacrylamide gel electrophoresis (27) and immunoblotting analysis with antibodies against topoisomerase I was performed essentially as described by Towbin et al. (28) and Oddou et al. (22). The signals were quantitated by scanning of the stained filters with a LKB laser densitometer.
Determination of the topoisomerase I activity Nuclear extracts were prepared as described above. Nuclear extracts were serially diluted into 20 mM Tris-HCl pH 7.5, 10 mM 3-mercaptoethanol, 50 mM sodium chloride, 0.1 mM EDTA and 5 percent glycerol. Topoisomerase I activity was then determined by incubation of superhelical SV40 DNA with the diluted extracts at 37°C as described (22).
RESULTS Adenovirus infection increases the amount of topisomerase I mRNA Human type I DNA topoisomerase specific cDNA hybridized to one single mRNA species in agreement with the data published by D'Arpa et al. (29). This mRNA migrated under denaturing conditions in agarose gels as an RNA species of 4.2 kb relative to 28 S and 18 S ribosomal RNA (Fig. lA). Using the same cDNA probe, we examined the effect of adenovirus-5 infection on the amount of the topoisomerase I mRNA throughout the infection cycle up to sixteen hours after viral infection. For these experiments, total cellular RNA was isolated at various times after infection, fractionated on denaturing agarose gels, transferred to membranes and hybridized to a set of different radioactively labeled DNA probes. As shown in Figure lB, infection of HeLa cells with adenovirus-5 resulted in an increase in mRNA coding for topoisomerase I. The amount of the mRNA increased progressively starting already two hours after infection. A maximum value was reached four hours after adenovirus infection. At that time, the level of topoisomerase I mRNA was three- to fivefold higher compared to uninfected HeLa cells or to HeLa cells four hours after a 'mock' infection (not shown). Thereafter, the amount of topoisomerase I mRNA continuously decreased, reaching the basal level at 12 hours after infection. After sixteen hours, less than 30 percent of topoisomerase I mRNA was present compared to uninfected cells. As a control we determined the relative amount of 3-actin mRNA (Fig. IC), ribosomal RNA (Fig. ID) and of total poly (A)RNA (Fig. lE) at various times after adenovirus infection. Our results are summarized in Figure 2, demonstrating that the level of total poly(A)-RNA as well as the level of 3-actin mRNA remained constant during the early phase of the infection but declined later, reaching a value corresponding to 50 percent and 20 percent of the amount detectable in uninfected cells 16 hours after infection, respectively. The amount of ribosomal RNA remained constant throughout the entire infection cycle.
Adenovirus infection increases the rate of transcription of the topoisomerase I gene The adenovirus induced increase of topoisomerase I specific mRNA may be due to an altered stability of the topoisomerase I mRNA or to an increased rate of transcription of the
Nucleic Acids Research 803 M
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Fig. 1: Effect of adenovirus infection on the expression of cellular genes. (A) Northern analysis of total cellular RNA prepared from uninfected HeLa cells with a topoisomerase I cDNA probe (3.2.4). Linearized pUC3.2.4 DNA, containing the topoisomerase I cDNA (4.9 kb) or isolated 3.2.4 cDNA (2.2 kb) were run in a parallel lane and served as size markers (M). 28 S and 18 S shows the migration of 28 S and 18 S rRNA. kb = kilobases. (B-D) HeLa S3 cells were infected with adenovirus 5. At the times indicated total cellular RNA was prepared and 30 jg were separated on agarose gels. After transfer of RNA the Nylon membranes were hybridized with topoisomerase I cDNA (B), (3-actin cDNA (C) and a cloned rRNA gene fragment (D) successively. For analysis of poly(A)-RNA lig of total cellular RNA was spotted onto nitrocellulose filters and hybridized to end-labeled oligo-dTI8 (E).
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hours post Infection Fig. 2: Increase of the steady state level of topoisomerase I mRNA during adenovirus infection. Three independent experiments performed as described in the legend of Fig. 1 are summarized. After hybridization the radioactive bands were excised from the filters and quantitated by liquid scintillation counting. One arbitrary unit corresponds to an average of 229 counts per minute (topo I), 3400 counts per minute (,B-actin) and 590 counts per minute (poly(A)-RNA).
topoisomerase I gene. To distinguish between these possibilities, we performed run on transcription experiments with nuclei isolated from HeLa cells at different times after infection. For these experiments, identical amounts of in vitro labeled radioactive total nuclear RNA prepared from 107 nuclei were hybridized with 2 ug of the isolated topoisomerase I cDNA fragment immobilized on nitrocellulose filters. The same amount of pUC-8 DNA was used as a control. As shown in Figure 3, already two hours after adenovirus-5 infection we detected a fourfold higher amount of hybridized RNA compared to the signal obtained with RNA from uninfected cells. This value increased to approximately 15-fold four hours after infection. At later times, the amount of hybridized RNA decreased drastically. Eight hours after infection the radioactive signal was as low as in uninfected
cells. 12 hours after infection, when still considerable amounts of topoisomerase I mRNA are present in the cells, the signal further declined below the level of detection under our experimental conditions. The results shown in Figure 3 suggest, that the increase in the total amount of topoisomerase I mRNA observed early after adenovirus infection, is mainly due to newly synthesized mRNA and not to an increase in the stability of the topoisomerase I mRNA already present in the cells. This also means, that the topoisomerase I gene is specifically induced after adenovirus infection, a phenomenon previously observed for the cellular 3tubulin gene (12) or the heat shock protein 70 gene (13). Furthermore, there are striking similarities between the activation pattern observed for the expression of the topoisomerase I gene
804 Nucleic Acids Research
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Fig. 3: Transcriptional activation of the topoisomerase I gene. (A) Nuclear run on transcription experiments were performed as described in Material and Methods with nuclei isolated at the times indicated after adenovirus infection of HeLa cells. 1 -2 x 106 counts per minute were hybridized to 2 /kg of isolated topoisomerase I cDNA fragment. 2 j4g of pUC-8 DNA was immobilized on the filter and used as a control (lane C). The radioactive signals were quantitated by scanning the autoradiogram depicted in (A) with a laser densitometer (B).
early in infection and the activation of the early viral genes. This may be due to an effect of the viral ElA products. If this assumption is correct, we would expect to find a higher topoisomerase I mRNA level in human 293 cells which express the adenovirus EIA and E1B products constitutively. Indeed, when identical amounts of total RNA isolated from uninfected HeLa cells or from adenotransformed 293 cells were analysed by hybridization, we found an fourfold higher amount of mRNA specific for topoisomerase I in the adenotransformed 293 cells compared to HeLa cells (Fig.4). The amount of topoisomerase I mRNA present in 293 cells corresponded approximately to the amount of mRNA present in HeLa cells four hours after infection
with adenovirus-5.
Infection of HeLa cells with adenovirus wild type but not with the mutant virus dL312 increases the amount of topoisomerase I mRNA To further confirm this conclusion we infected HeLa cells with Ad d1312, an adenovirus mutant were the region coding for ElA/E1B has been deleted (18). Four hours after infection with the mutant virus we then superinfected the cells with wild type virus. The amount of topoisomerase I mRNA was determined after northern blotting of total cellular RNA. Figure 5A shows the result of such an experiment. After infection of HeLa cells with the deletion mutant d1312, the amount topoisomerase I mRNA remains approximately constant (lane 1 and 3: 1- vs 1.3-fold; in two other experiments this value for d1312 was 1). However, in cells superinfected with the adenovirus wild type the amount of topoisomerase I mRNA increased 3.8-fold four
Fig. 4: Comparision of the amount of topoisomerase I mRNA in HeLa and 293 cells. Total cellular RNA was prepared from HeLa and 293 cells. 30 mg each was subjected to agarose gel electrophoresis, blotted to nylon membranes and visualized after hybridization against topoisomerase I cDNA.
hours later (lane 4). This is a topoisomerase I mRNA level comparable to the level reached four hours after infection of HeLa cells with adenovirus wild type alone (lane 2; 3.4-fold). The same results were obtained when the cells were superinfected with wild type eight hours instead of four hours after infection with the mutant virus (data not shown). Similar to the situation after infection with wild type virus alone, eight hours after superinfection the amount of topoisomerase I mRNA again declined. Thus, the kinetics of topoisomerase I mRNA expression are the same in cells solely infected with the wild type virus, or infected with d1312 and wild type. As a control, we determined the amounts of viral DNA present in the infected cells. Adenovirus DNA was isolated from Hirt supernatants, purified and digested with restriction enzyme Hind mI. After separation of the DNA on agarose gels and transfer to nitrocellulose membranes, the filters were hybridized with the Xho-C fragment of pAdXhoC. Due to the deleted EIA region in the mutant DNA we expect one hybridizing fragment 1.8 kbp in length in samples containing d1312 DNA and, after superinfection a second fragment, 2.8 kbp long, derived from the adenovirus wild type. Fig. 5B clearly shows, that comparable amounts of DNA derived from mutant and wild type virus were present in the cells. Obviously, infection with the mutant virus d1312, were the E1A region is deleted, did not affect topoisomerase I expression.
Nucleic Acids Research 805 A
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Fig. 5: The level of topoisomerase I mRNA in HeLa cells infected with the ad- d1312. (A) HeLa cells were either mock infected (lane 1), infected with wild type virus (lane 2), the mutant d1312 (lane 3 and 4). Total cellular RNA was prepared from the cells four hours later (lane 1, 2, 3). At that time, cells infected with d1312 were superinfected with wild type virus and after four hours total cellular RNA was prepared from these cells (lane 4). Topoisomerase I mRNA was then visulalized by northern blotting experiments. The relative amounts of topoisomerase I mRNA were determined by scintillation counting of the excised bands and were 1 (lane 1; 158 counts per minute), 3.4 (lane 2), 1.3 (lane 3) and 3.8 (lane 4) arbitrary units. (B) In a parallel experiment adenovirus DNA was prepared from the cells, digested with Hind III and separated on agarose gels. The adenovirus DNA was identified by hybridization to the nick-translated adenovirus Xho-C fragment DNA.
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Fig. 6: Analysis of the amount of topoisomerase I present in adenovirus infected cells. (A) HeLa cells were fractionated into cytosol (C), nuclear extract (NE) and residual pellet (P) as described in the text. An equal amount of total protein was then separated on polyacrylamide gels, transferred to filters and stained with topoisomerase I specific antibodies. (B) High-salt nuclear extracts were prepared at different times after adenovirus infection. 150 ug (lane 1) and 30 jig (lane 2) of extract were separated on denaturing polyacrylamide gels, transferred to filters and stained with topoisomerase I specific antibodies. As a control, 25 ng of purified topoisomerase I from calf thymus was applied onto the same gel (lane T). The solid triangles mark the position of the adenovirus coded 110 kDa protein. h = hours post infection. (-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa) ovalbumin (45 kDa) and carbonic anhydrase (29 kDa) were used as molecular weight markers.
The amount of topoisomerase I protein and the specific activity of the enzyme remain constant upon adenovirus infection We have further analysed, whether the elevated level of topoisomerase I mRNA in HeLa cells upon adenovirus infection is accompanied by an increase in the amount of topoisomerase I protein in infected cells. For this purpose nuclei were pelleted, washed several times in lysis buffer (see Material and Methods) and then extracted into the same buffer containing additionally 700 mM sodium chloride. After centrifugation, the supernatant
and the residual nuclear structures were collected. As shown in Figure 6A, this method resulted in an almost quantitative recovery of topoisomerase I in the salt extract when analysed in Western blots. Less than two percent of the topoisomerase I protein are found in the cytosol fraction or in the residual nuclear pellet. The same distribution of topoisomerase I is obtained when adenovirus infected HeLa cells are fractionated by this procedure (data not shown). This method therefore seems to be reliable to quantitatively assess changes of type I topoisomerase during infection.
806 Nucleic Acids Research The relative amounts of topoisomerase I protein were determined by Western analysis. For this purpose, nuclear extracts prepared from HeLa cells at different times after infection were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and transferred to nitrocellulose membranes. For our studies we used polyclonal rabbit antibodies, directed against a topoisomerase I cDNA fragment expressed in E.coli or the monoclonal antibody B7 described by Oddou et al. (22). The filters were stained and the signals quantitated by laser densitometry. Comparing the signals from different dilution steps of the nuclear extracts, we were able to determine the relative amounts of topoisomerase I protein (30).
A typical result is shown in Figure 6B. In this experiment nuclear
extracts were prepared 0,
6, 12, 18 and 24 hours after infection. 50 ng of purified topoisomerase I from calf thymus was separated on the gel as a control (Fig.6B, lane T). With both topoisomerase I specific antibodies we detected one major crossreacting band, corresponding to a protein migrating with 100 kDa, the molecular weight of undegraded topoisomerase I. One additional signal was present in extracts prepared 18 or 24 hours after infection, migrating with a slightly reduced mobility in the gel, compared to topoisomerase I. This signal is probably derived from the adenovirus 110 kDa protein. This viral protein constitutes the major Coomassie blue stainable band at late times after infection and we do not know, whether this weak crossreactivity is of any significance. In
a
series of experiments of this type, we never observe
changes in the amount of topoisomerase I up to 24 hours after infection. We have also analysed the amount of topoisomerase I present in nuclear extracts in two hours intervalls, starting from four hours after infection, when the topoisomerase I gene is maximally expressed, up to 14 hours after infection, when the
amount of mRNA is reduced and the topoisomerase I gene is already repressed, compared to uninfected cells. Again, we did not find any variation in the amount of topoisomerase I.
Fig. 7: Enzymatic activity of topoisomerase I throughout the infection cycle. Highsalt nuclear extracts were prepared at different times after adenovirus infection of HeLa cells. Nuclear extracts were diluted 1:40 and incubated for 30 minutes (lane 1) or 15 minutes (lane 2) with 0.5 pAg of supercoiled SV40 DNA. The DNA was purified from the samples and separated on 0.8% agarose gels in 40 mM Tris-acetate pH 8.0, 5 mM sodium acetate and 2 mM EDTA. F I and F IV marks the position of supercoiled and relaxed SV40 DNA respectively.
Finally, we have examined the enzymatic activity of topoisomerase I after adenovirus infection in extracts prepared as described above. First, the relaxation activity of topoisomerase I was determined in serial dilutions of these extracts prepared from uninfected cells. The dilution step, which resulted in the complete relaxation of 0.5 mg of supercoiled SV40 DNA in 30 minutes at 37°C, was then used to compare the topoisomerase I activities in extracts prepared at different times after infection. As shown in Figure 7 the relaxation activity was constant at all times after infection and not distinguishable from the enzyme activity present in uninfected cells. Essentially the same results were obtained, when the activity of the enzyme was monitored by a DNA binding assay (not shown) which measures the transfer
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hours post Infection Fig.8: Activation of the topoisomerase I gene during the early phase of adenovirus-5 infection. Fig.8 summarizes the data given in Fig. lB and Fig.3B in relation to the infection cycle. Adenovirus DNA synthesis was determined after in vivo incubation with tritiated thymidine in Hirt supematants prepared at different times
after infection.
Nucleic Acids Research 807 of a radioactively labeled oligonucleotide to topoisomerase I as described by Chow and Pearson (16). This indicates, that the relaxation activity as well as the DNA binding activity of topoisomerase I remained unaffected, throughout the infection cycle.
DISCUSSION The expression of most of the cellular genes remaines unaffected during the first ten hours after adenovirus infection (31, 32). As summarized in Figure 8 and in contrast to most of the known cellular genes, the transcriptional activity of the topoisomerase I gene was greatly stimulated upon infection. This stimulation resulted in a three- to fivefold increase in the steady state level of topoisomerase I mRNA compared to uninfected cells and is specific for the adenovirus system. Mock infection, or infection of african green monkey kidney cells with another DNA tumor virus, the Simian virus 40 (not shown), had no effect on the transcription of the topoisomerase I gene or the steady state level of the mRNA. The increase in gene activity as well as of the amount of topoisomerase I specific mRNA was restricted to a time period between two and four hours after infection. During this time, the transcription of early adenovirus genes is activated by the products of the ElA region. Thereafter, from 4 to 8 hours after infection, these early viral genes are repressed by an unknown mechanism and the transcription rate of these genes declines. This biphasic kinetics of transcription is typical for the regulation of early viral genes by the EIA products. The regulation of the cellular topoisomerase I gene followed the same kinetic parameters as that of the early adenoviral genes. In agreement with the data published by Berk (10) and Stein and Ziff (12), the amount of total cellular RNA, represented mainly by ribosomal RNA, and the amount of polyadenylated mRNA remained constant throughout this period of time. The same was also found for the expression of the 3-actin gene which may be representative for the majority of cellular genes. We have compared the expression of the topoisomerase I gene in HeLa cells with its expression in human adenotransformed embryonic kidney cells. These transformed cells, which constitutively express the EIA and E1B genes, showed a permanently elevated level of DNA topoisomerase I mRNA. The relative amounts of mRNA present in these cells corresponded exactly to the amount of mRNA found in HeLa cells at four hours after infection with adenovirus-5 (Fig. 4). Interestingly, the amount of topoisomerase I mRNA present in transformed HeLa cells is comparable to the amount of mRNA present in untransformed human fibroblast cells (Hel 299 cells; data not shown). This makes it rather unlikely that the transformed phenotype per se is responsible for the increase in topoisomerase I mRNA observed in the adenotransformed 293 cells. Furthermore, we could show that infection of HeLa cells with the adenovirus mutant d1312, in which the region coding for the EIA proteins had been deleted, did not result in an increase of the amount of topoisomerase I mRNA (Fig.5). Taken together, these results strongly suggest, that the increase of DNA topoisomerase I specific mRNA in infected HeLa cells and the elevated level in adenotransformed cells is a consequence of transactivation of the gene by the adenovirus ElA products. The increase in the steady state level of topoisomerase I mRNA was not accompanied by a comparable increase on the protein level. Instead we have found, that the amount of topoisomerase I remained constant after adenovirus infection of HeLa cells
(Fig.6). This was surprising, as Chow and Pearson have reported
increase in the amount of enzyme as well as of topoisomerase I activity upon adenovirus infection of HeLa cells (16). We could not detect an net increase in the enzyme activity in an relaxation assay (Fig. 7) or in an assay measuring the covalent complex formation (data not shown). The reason for this discrepancy between our results and the results published by Chow and Pearson may be due to the different preparation procedures used. With our procedure, topoisomerase I is extracted quantitatively in one high salt fraction from purified nuclei. In contrast, Chow and Pearson have used a buffer with moderately low salt concentrations (33). The observed increase of topoisomerase I in their extracts could therefore result from an altered distribution of the enzyme upon adenovirus infection between different nuclear compartments, rendering part of the enzyme extractable at low salt concentrations. Given an 3- to 5-fold increase of the amount of topoisomerase I mRNA over a period of several hours, one might expect to find a significant increase in the amount of topoisomerase protein as well. Obviously this was not the case. This finding may be, however, less surprising if we realize the consequence of the increase of topoisomerase I mRNA after serum stimulation of quiescent human fibroblast cells on the level of topoisomerase I protein. In this cells we observed an continuous increase in topoisomerase I mRNA over a period of 25 hours. After that time, serum stimulated cells contained the 6-fold amount of topoisomerase I mRNA compared to resting cells. During the same period of time the amount of topoisomerase I protein and the enzyme activity increased only twofold (H.Romig and A.Richter, submitted). This finding may indicate that topoisomerase I protein synthesis is regulated at the level of translation as well. Assuming a similar relationship between mRNA level and translation in adenovirus infected HeLa cells, one would not expect that the increase in the amount of topoisomerase I mRNA over a period of 3-4 hour in these cells would result in a drastic increase of topoisomerase I protein and enzyme activity. Infection of human cells with adenovirus result in the shut-off of host protein synthesis late in infection (for review see 34). This is caused by the blockade of the transport of mRNA from the nucleus to the cytoplasm by proteins encoded by the virus (35) and the inhibition of host mRNA translation. The availability of host proteins essential for viral metabolism therefore greatly depends on the half-life of these proteins and the amount of their mRNA synthesized early in infection. Heck et al. (36) have determined the half-live of topoisomerase I in transformed chicken hepatoma cells and primary chicken embryo fibroblast cells. The half-life of topoisomerase I was found to be 23 hours in these primary cells and 16 hours in the transformed cells. Providing the half-live of HeLa topoisomerase I is within the same range, a considerable amount of enzyme would have decayed late in infection after host shut-off. At this time maximal adenovirus DNA synthesis and transcription of late viral genes takes place. The constancy in the level of topoisomerase I protein observed late in the infection cycle may be a consequence of the transcriptional activation of the gene. The increased amount of topoisomerase I mRNA may compensate for a depressed synthesis of protein later in infection thus providing enough enzyme to substitute those topoisomerase I molecules which decay after host shut-off. The transcriptional activation of the topoisomerase I gene may be a useful reaction, if the virus needs an active topoisomerase I for its replication and/or transcription. The participation of an
808 Nucleic Acids Research topoisomerase I in transcription of cellular genes has been shown in several systems (4, 5, 38). Though a direct experimental evidence is not available, a function of the enzyme in transcription of adenovirus DNA cannot be excluded. More data have been presented which strongly argue in favour of a participation of topoisomerase I in adenovirus replication. Nagata et al. (8) have purified a cellular factor (nuclear factor II; NF II) essential for the replication of adenovirus DNA in vitro which is most probably topoisomerase I. Furthermore, in a recent publication, Schaack et al. (37) have shown that topoisomerase I (together with topoisomerase II) is involved in adenovirus replication in vivo as well. Thus, the biological function of the transcriptional activation of the topoisomerase I gene may be, to provide an excess of transcripts, needed for a constant supply of active topoisomerase I necessary for the propagation of the virus during late stages after viral infection.
ACKNOWLEDGEMENT The author wish to thank Drs. T.Shenk, J.L.Bos and E.Fanning for providing plasmids harbouring ElA/ElB and a stock lysate of the adenovirus mutant d1312. We further thank Dr. R.Knippers for helpful discussions and critical reading of the manuscript and M.Mollerbernd for preparation of plasmids and virus. The work was supported by the Deutsche Forschungsgemeinschaft through SFB 156.
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