JOURNAL OF VIROLOGY, Mar. 1976, p. 824-831 Copyright 0 1976 American Society for Microbiology
Vol. 17, No. 3 Printed in U.S.A.
Quantitation of Bovine Papilloma Viral DNA in Viral-Induced Tumors WAYNE D. LANCASTER,* CARL OLSON, AND WILLIAM MEINKE Department of Microbiology, Scripps Clinic and Research Foundation, La Jolla, California 92037,* and Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 22 September 1975
Bovine papilloma virus (BPV) DNA was labeled in vitro under conditions of repair synthesis and subsequently used as a "probe" in DNA-DNA reassociation studies to detect BPV-specific DNA sequences in a viral-induced calf meningioma and hamster fibroma. In vitro labeled BPV DNA had denaturation characteristics expected for duplex DNA and denatured DNA reassociated with apparent second-order kinetics. Analysis of in vitro labeled BPV DNA reassociation rates in the presence of excess tumor DNA revealed that the calf meningioma contained approximately 700 to 800 BPV genome equivalents per diploid cell whereas the hamster fibroma contained about 150 incomplete BPV genome equivalents per diploid cell. Thermal denaturation of in vitro labeled BPV DNA which reassociated in the presence of the two tumor DNA preparations indicated less than 1.5% base pair mismatching. The papilloma viruses, members of the papovavirus group (29), have not been intensively investigated. The major experimental drawback is the lack of suitable cell lines for virus replication. However, virus can be obtained in relatively large amounts from papillomas, and recent in vitro DNA labeling techniques (21, 24, 31) have allowed for the study of virus-cell interactions (21, 24). Papilloma viruses are the only DNA-containing viruses whose mode of replication leads to the production of tumors (warts) in their natural hosts. Although these tumors are considered benign and self-limiting, they can progress into malignancies. One example is the Shope papilloma virus, which produces papillomas in wild cottontail rabbits from which virus can be recovered. However, papillomas induced in domestic rabbits contain little or no virus and often develop into carcinomas (23, 37). Another member of the papilloma virus group is the bovine papilloma virus (BPV). BPV is the only papilloma virus shown to cross the species barrier. It can induce fibroblastic neoplasia in horses, hamsters, and mice as well as cattle (2, 6, 32). Both BPV and BPV DNA have been shown to transform bovine and mouse cells in culture (1, 3, 39). BPV readily induces fibromatous tumors of the meninges in brains of susceptible calves when injected intracranially and can produce tumors at other sites of inoculation (17). The Syrian hamster is also susceptible to the oncogenic action of BPV regardless of the
site of injection or age of the animal. These tumors can metastasize and may result in the death of the host (36). We report the detection and quantitation of BPV DNA in a calf meningioma and a hamster fibroma induced by this virus using DNA-DNA reassociation kinetics. The data suggest that there are a large number of BPV genome equivalents per cell present in the calf meningioma whereas there appears to be only a portion of the BPV genome in the hamster fibroma. Electron microscope investigation of the tumors did not reveal the presence of virus particles. MATERIALS AND METHODS Virus and viral DNA. BPV was isolated from papillomas experimentally induced in calves (7). Papillomas were excised and stored in 50% glycerolnormal saline at 4 C. Virus was obtained from 3 to 5 g of minced tissue suspended in 50 ml of 0.1 M Tris-hydrochloride (pH 7.5) and homogenized in a Virtis homogenizer at 45,000 rpm for 15 min. The homogenate was centrifuged at 12,000 x g for 10 min and the pellet was resuspended in 25 ml of 0.1 M Tris-hydrochloride (pH 7.5), containing 0.1% Sarkosyl and 25 ml of 1,1,2-trichloro-trifluoroethane (16). The mixture was further homogenized at 15,000 rpm for 10 min and centrifuged at 12,000 x g. The aqueous phase was recovered and combined with the previous supernatant fluid and the mixture was brought to 0.1% with Sarkosyl and centrifuged at 12,000 x g for 10 min. The resultant supernatant fluid was subjected to high-speed centrifugation in a Spinco type 30 rotor at 25,000 rpm for 3 h 824
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QUANTITATION OF BPV DNA IN TUMORS
at 4 C. Pellets were resuspended in CsCl (p = 1.33) in 0.01 M Tris-hydrochloride (pH 7.5) and centrifuged in a Spinco SW50.1 rotor at 32,000 rpm for 24 h at 25 C. The visible virus bands were collected and dialyzed against 0.2 M Tris-hydrochloride (pH 8.0), 0.001 M EDTA. BPV was ruptured by addition of sodium dodecyl sulfate (SDS) to 1% and heating to 50 C for 15 min. The mixture was deproteinated by two extractions with freshly distilled phenol saturated with 0.2 M Tris-hydrochloride (pH 8.0), 0.01 M EDTA. DNA was precipitated by addition of 2.5 volumes of ethanol at -20 C and subsequently dissolved in CsCl (p = 1.59) in 0.02 M Tris-hydrochloride (pH 8.0), 0.001 M EDTA containing ethidium bromide (33) and centrifuged in an SW50.1 rotor at 32,000 rpm for 48 h at 25 C. Supercoiled viral DNA was collected and ethidium bromide was removed by isopropanol extraction (25). DNA was dialyzed first against 4x SSC (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) followed by 0.1 x SSC and stored at - 20 C. The small-plaque variant of simian virus (SV40) was grown in TC-7 cell cultures (subline of CV-1) (35) and labeled with [3H]thymidine and virus, and viral DNA was isolated as described elsewhere (28). Induction of tumors. A partially purified suspension of BPV (17) was injected intracranially into a papilloma-free calf at 21 weeks of age. After 228 days, clinical signs of intracranial pressure developed and the animal was killed. The histology and characteristics of this BPV-induced calf meningioma have been described (17). Tumor induction in hamsters was by subcutaneous injection of a partially purified suspension of BPV. The tumor used in this study was passaged six times in hamsters prior to extraction of DNA. The characteristics and histology of similar hamster tumors have been described (36). Isolation of cellular DNA. Tumors (1 to 2 g) were minced and homogenized in 30 ml of 8 M urea, 0.24 M sodium phosphate (pH 6.8), 1% SDS, 0.001 EDTA, and 2% isoamyl alcohol in a Waring Blendor at maximum rpm for 30 s. The mixture was clarified by low-speed centrifugation, the supernatant fluid was applied to a column of hydroxyapatite, and the DNA was isolated as described previously (27). Calf thymus DNA was purified by the same regimen. Purified DNA preparations were sheared to an average singlestrand length of 570 nucleotides (26). In vitro labeling of BPV DNA. The methods of labeling DNA in vitro were similar to those described previously (24). Supercoiled viral DNA was treated with pancreatic DNase I in 0.05 M Tris-hydrochloride (pH 7.4), 0.05 M KCl, 0.005 M MgCl2 at a ratio of 500:1 (wt/wt) at 37 C for 15 min. DNase was inactivated by heating to 65 C for 10 min. DNase-treated DNA was then labeled in vitro with Escherichia coli DNA polymerase I under conditions of repair synthesis (22). Viral DNA (0.1 Ag) was suspended in a volume of 0.L ml containing 6.6 ,mol of potassium phosphate (pH 7.4), 0.66 limol of MgCl,, 0.1 gtmol of 2-mercaptoethanol, and 0.005 jgmol of each of the necessary unlabeled deoxynucleotide triphosphates (dNTP). Depending on the experi-
825
ment, DNA
was labeled with either [3H JTTP (54 Ci(mmol) at 500 iACilig of DNA or [a-32PJdCTP + [a-32PfTTP (116 Ci/mmol) at 2 mCi/ug of DNA or [a-"2P]dATP + [a-_2PJTTP (112 Ci/mmol) at 2 mCi/ug of DNA. Synthesis was initiated by ad-
dition of 20 units of E. coli DNA polymerase I per microgram of DNA and incubated at 15 C for 30 to 60 min. Reactions were terminated by addition of Sarkosyl to 1%. Labeled DNA was passed through a Sephadex G-50 column (0.7 by 20 cm) equilibrated with 0.02 M Tris-hydrochloride (pH 8.0), 0.1% Sarkosyl, 0.001 M EDTA. Fractions containing radiolabeled DNA were pooled and stored over chloroform at 4 C. Incorporation of radioactivity into DNA was determined by trichloroacetic acid precipitation with 100 gg of tRNA as carrier. Specific activities ranged from 1.8 x 106 to 4.7 x 10" counts/min per gsg of DNA for ['HJTTP-labeled DNA and 15.5 x 101 to 16.4 x 10' counts/min per ;g of DNA for [32P]dNTP-labeled
DNA. DNA-DNA reassociation. DNA solutions were denatured in 0.48 M sodium phosphate (pH 6.8), 0.05% SDS, 0.001 M EDTA by heating to 109 C for 5 min in screw-capped vials and quenched in an ice bath. Denatured DNA was allowed to reassociate at 68 C. At various times, samples were removed and diluted to 2.0 ml of 0.14 M sodium phosphate (pH 6.8), 0.05% SDS. Single-strand DNA was separated from reassociated duplex DNA by hydroxyapatite column chromatography (10 by 15 mm) at 60 C (5). Single-stranded DNA was eluted from the column with five 2.0-ml washes of 0.14 M sodium phosphate (pH 6.8), 0.05% SDS, and reassociated DNA was subsequently eluted with four 2.0-ml washes of 0.48 M sodium phosphate (pH 6.8), 0.05% SDS. Radioactivity was determined for each wash by addition of 15 ml of a toluene-Triton X-100 (2:1, vol/vol) scintillation fluid. Immediately after denaturation, about 8.0 to 8.3% of in vitro labeled DNA was eluted as doublestranded DNA on hydroxyapatite columns. These values were not used to normalize the reassociation data. Thermal denaturation. DNA preparations in 0.14 M sodium phosphate (pH 6.8), 0.05% SDS (eluting buffer) were applied to a column of hydroxyapatite at 60 C. After five 2.0-ml washes of eluting buffer, the column temperature was increased by approximately four-degree increments, single-stranded DNA was eluted with five 2.0-ml washes of prewarmed buffer, and radioactivity in each wash was determined as described above. Electron microscopy. Small portions of tumor were fixed in 3.5% glutaraldehyde in phosphate buffer (pH 7.2) for 1 h and then postfixed with 1% osmium tetroxide in phosphate buffer (pH 7.2) for 1 h. Preparations were dehydrated with ethanol, transferred to propylene oxide and embedded in epoxy resin.
Chemicals. [a-32P]dATP, [a-32PJTTP, and [a'2P]dCTP (specific activities ranging from 112 Ci/ mmol to 116 Ci/mmol) were purchased from New England Nuclear Corp. [3H]TTP (specific activity,
54 Ci/mmol) was obtained from Schwarz-Mann. E. coli DNA polymerase I was obtained from Grand
826
LANCASTER, OLSON, AND MEINKE
Island Biological Co. or Boehringer Mannheim Corp. DNase I (electrophoretically pure) was purchased from Sigma Chemical Corp. Calf thymus DNA was from Calbiochem.
RESULTS Size distribution of in vitro labeled DNA. Viral DNA preparations to be labeled in vitro with [a-"2PJdNTP were first "nicked" with DNase and then labeled with ['H ]TP to estimate piece size. The size of single-stranded DNA was determined by co-sedimentation through 5 to 20% alkaline sucrose gradients with marker DNA which had an average length of 570 nucleotides (26). One preparation of in vitro 'H-labeled BPV DNA had an average length of 435 nucleotides (Fig. 1A) whereas another preparation, which was more heterogeneous, was about 775 nucleotides in length (Fig. 1B). Differences have not been noted in piece sizes of DNA labeled with either [3H ]TTP or [a"2P ]dNTP. In vitro labeled DNA preparations reassociated to >90% when denatured and allowed to reanneal in the presence of "nicked" unlabeled BPV DNA. Labeled DNA (0.002 gg/ml) (Fig. 1A) reassociated to 50% in 187 h (0.48 M sodium phosphate, pH 6.8, 68 C) whereas DNA shown in Fig. 1B was 50% reannealed by 101 h. BPV DNA from Fig. 1A reassociated 28% slower A
/200
400
600
200
FIG. 1. Sedimentation profiles of in vitro labeled BPV DNA in alkaline sucrose gradients. 'H-labeled BPV DNA and 14C-labeled marker DNA were sedimented in alkaline 5 to 20% linear sucrose (wt/wt) gradients (0.3 N NaOH, 1.0 M NaCl, 0.001 M EDTA, 0.015% Sarkosyl) in an SW56 rotor at 15 C for 6.5 to 7 h at 49,000 rpm. Letters A and B refer to two separate preparations of DNase-treated BPV DNA labeled in vitro. Symbols: (0) 'H-labeled BPV DNA; (0) 14Clabeled marker DNA 570 nucleotides in length.
J.VIROL.
than expected based on the reassociation rate of the BPV DNA preparation in Fig. 1B (40). This discrepancy could be due to inaccuracy of piece size determinations. However, the faster rate of BPV DNA reassociation was expected since the DNA preparation shown in Fig. 1B contained molecules with unit lengths about 44% greater than those of Fig. 1A. Thermal denaturation. The thermal denaturation profile of 32P-labeled BPV DNA in the presence of 'H-labeled SV40 DNA is shown in Fig. 2A. The temperature at which 50% of the DNA was denatured (Tm) for SV40 DNA, BPV DNA, and E. coli DNA was 90.3 C, 91.8 C, and 93.4 C, respectively (Table 1). The Tm for BPV DNA indicated it contained 45.4% guanine plus cytosine (G+C) based on G+C values of 41% for SV40 DNA (9) and 50% for E. coli DNA (Fig. 3). Others have reported that BPV DNA has a G+C value of 45.5% (10). These two values of G +C for BPV DNA are in excellent agreement. The Tm of 32P-labeled BPV DNA formed in the presence of either hamster fibroma (HT) or calf meningioma (CT) DNA (Fig. 2B) also was determined. The small decrease in the Tm indicated that there was only slight base pair mismatching (approximately 1 to 1.5%) (Table 1). This value is based on the observation by others that a mismatching of 1% of the base pairs will lower the Tm by 0.7 to 1.5 degrees C (4, 34). Similar base pair mismatching has been noted for in vitro labeled human papilloma viral DNA allowed to reassociate to completion in the presence of unlabeled viral DNA (W. D. Lancaster and W. Meinke, unpublished data). DNA-DNA reassociation. Reassociation of 32Plabeled BPV DNA in the presence of either calf thymus DNA, CT DNA or HT DNA is shown in Fig. 4. The rate of reassociation of 2P-labeled BPV DNA in the positive control (calf thymus DNA plus unlabeled "nicked" BPV DNA) was increased over that of the control (calf thymus DNA alone). The reassociation rate of 32P-labeled BPV DNA in the presence of CT DNA was very rapid. Also, the reassociation rate of 32P-labeled BPV DNA in the presence of HT DNA was rapid initially but later deviated from second-order kinetics. In another experiment in which the cellular DNA concentrations were reduced 10-fold, similar curves were generated (Fig. 5). "2P-labeled BPV DNA in the positive control reassociated faster than in the control reaction as expected. In both reactions "2P-labeled BPV DNA reassociated with second-order kinetics. Also 3P_ labeled BPV DNA reassociated linearly in the presence of CT DNA to >90%. However, 2Plabeled BPV DNA reassociating in the presence
QUANTITATION OF BPV DNA IN TUMORS
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TABLE 1. Thermal denaturation of DNA by hydroxyapatite chromatography DNA
['H ]simian virus 40 [14C ] E. coli [P2p]BPV [32P ]BPV-CT PBPV-HT [32p
.% associated
TM
ATm0
86.6 81.9
90.3 93.4 91.8 90.9 90.6
0.90 1.20
aThe difference between the Tm of in vitro labeled ['3PJBPV DNA and the Tm of "2P-labeled BPV DNA reassociated in the presence of either CT DNA or HT
Sq
DNA.
Cs I
I
t
'4
501-
48k 4.
461
441
42hF
TenpwutUre (C) FIG. 2. Thermal denaturation profiles of in vitro labeled BPVDNA and denatured reassociated in vitro labeled BPV DNA. (A) "2P-labeled BPV DNA was mixed with 'H-labeled SV40 DNA and applied to a column of hydroxyapatite and single-stranded DNA eluted at various temperatures as described in Materials and Methods. (B) Thermal stability
of
"2p_
labeled BPV DNA which reassociated in the presence of HT DNA or CT DNA.
of HT DNA again deviated significantly from second-order kinetics. The results of the two experiments are summarized in Table 2. In experiment 1, the reassociation rate of "2P-labeled BPV DNA in the positive control was 3.1 times faster than in the control (expected value was 4.0). 32P-labeled DNA in the presence of CT DNA reassociated approximately 485 times faster than the control reaction. The reannealing rate of 32P-labeled BPV DNA in the presence of HT DNA was 170 times that of the control reaction based on the initial rate of reassociation.
I
I
I
9/
92 Tm (C)
93
FIG. 3. Relationship between G+C content and Tm for DNA from E. coli, BPV, and SV40. Fragmented DNA preparations were thermally denatured on hydroxyapatite columns.
Since 32P-labeled BPV DNA unexpectedly was reassociating at extremely fast rates in the presence of either CT or HT DNA, cellular DNA
concentrations were reduced and experiments repeated to obtain a more accurate estimation of reassociation rates. In experiment 2, 32P-labeled BPV DNA in the positive control reassociated 5.6 times faster than the control (expected value was 6.6). The rate of reassociation of 3P-labeled BPV DNA in the presence of CT DNA was 92 times the rate observed for the control reaction. This increased rate of reaswere
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LANCASTER, OLSON, AND MEINKE
J. VIROL.
3.0
2.5
d. 2.0 0
'.5
25 /5 Hours Incubetion FIG. 4. Reassociation of in vitro labeled BPVDNA in the presence of unlabeled DNA. The results are plotted as the ratio (Co/C) of the total 32P-labeled BPV DNA concentration (Co) to the concentration of single-stranded 32P-labeled BPV DNA (C) at various incubation times. 32P-labeled BPV DNA (0.002 fg/ mt) was allowed to reassociate in the presence of either 2 mg of denatured calf thymus DNA per ml (0); 2 mg of calf thymus DNA per ml plus 0.0062 gg of unlabeled BPV DNA per ml (0); 2 mg of hamster fibroma (HT) DNA per ml (0); or 2 mg of calf meningioma (CT) DNA per ml (A). 5
sociation corresponds to about 726 genome equivalents per CT cell. This value for the number of BPV genome equivalents in CT DNA is probably the more accurate value of the two since the rate of reassociation of 32P-labeled BPV DNA in the presence of the smaller amount of CT DNA could be more precisely determined. The initial rate of reassociation of 32P-labeled BPV DNA with HT DNA was 21.5 times that of the control and this corresponds to about 164 BPV genome equivalents per cell. The values obtained for the number of BPV genome equivalents per HT cell (based on the initial rate of reassociation) are in close agreement in both experiments. Electron microscopy. Since a large number of BPV genome equivalents per cell were detected in tumor DNA, tissues were examined for the presence of virus particles. Approximately 100 cells in each tumor were examined and no particles resembling papovaviruses were seen.
15 25 Hours Incubation FIG. 5. Reassociation of in vitro labeled BPV DNA in the presence of unlabeled DNA. The results are plotted as described for Fig. 4. "2P-labeled BPV DNA (0.002 ,g/ml) was allowed to reassociate in the presence of either 200 Mg of calf thymus DNA per ml (0); 200 Mg of calf thymus DNA per ml and 0.0112 Ag of BPV DNA per ml (0); 200 MLg of HT DNA per ml (0); or 200 Mg of CTDNA per ml (A). Analysis of tumor DNA by CsCi-ethidium bromide centrifugation. To determine if some of the BPV DNA was existing as free supercoiled DNA in the tumor cells, tumors were homogenized and DNA was isolated as described in Materials and Methods. A 1- to 0.7-mg amount of tumor DNA was analyzed by 5
dye buoyant density equilibrium centrifugation in an attempt to isolate BPV supercoiled DNA molecules. In HT and CT DNA preparations, no band corresponding to supercoiled DNA was observed. Since HT contains about 700 to 800 copies of BPV DNA per diploid cell, those fractions of the gradients corresponding to the density of supercoiled DNA, including a small portion (5 to 10%) of the cellular DNA peak, were pooled and the DNA was analyzed for BPV DNA sequences by DNA-DNA reassociation kinetics. The results indicated that about 800 BPV genome equivalents per diploid cell were present. If BPV supercoiled DNA was present, the number of BPV genome equivalents per cell should have been increased significantly.
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QUANTITATION OF BPV DNA IN TUMORS
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TABLE 2. Reassociation of 32P-labeled BPV DNA in the presence of unlabeled DNA Concn (gg/ml)
Expt 1
2
no.
Source of DNA
Unlabeled cellular
No. of genome
Factor of
equivalents! divlells
Unlabeled BPV DNA
increased rate
0.002 0.002 0.002 0.002
0.0062
3.1 170C 467
2 135 373
0.002 0.002 0.002 0.002
0.0112
5.6 21.5c 92
37 164 726
DNA
'P-labeled BPV DNA
Calf thymus Calf thymus HT CT
2,000 2,000 2,000 2,000
Calf thymus Calf thymus HT CT
200 200 200 200
diploid cell"
Factor of increased rate of reassociation over that of the control. bCalculated by the method of Gelb et al. (15) with molecular weight of BPV DNA of 5 x 106 (8); also as determined by restriction endonuclease cleavage of BPV DNA (Lancaster and Meinke, unpublished data). cEstimated from the initial rate of reassociation. a
DISCUSSION As previously demonstrated, human papilloma viral DNA could be labeled in vitro and used as a probe to determine the absence or presence of viral DNA in infected cell cultures (24). The results of this study also indicate that BPV DNA can be labeled in vitro and will similarly function as probe in DNA-DNA reassociation studies. Our results indicated that in vitro labeled BPV DNA had a Tm expected for DNA of 45.5% G+C as compared to in vivo labeled SV40 DNA and in vivo labeled E. coli DNA (Fig. 3). Denatured fragmented 32P-labeled BPV DNA reannealed with second-order kinetics and the rates of reassociation were approximately those expected for a genome with a genetic complexity of 5 x 10. daltons "nicked" to the piece sizes described (40). Furthermore, the results demonstrate that BPV DNA is present in a calf meningioma and hamster tumor, both considered to be induced by BPV, and that the tumors used in this study vary greatly in their content of BPV DNA per cell. The rate of reassociation of 82P-labeled BPV DNA in the presence of HT DNA deviated from second-order kinetics (Fig. 4 and 5); however, the initial rate of reassociation was linear. By comparing the increase in the initial reassociation rate of 32P-labeled BPV DNA in the absence and presence of HT DNA there were calculated to be between 135 and 164 BPV genome equivalents per diploid cell (Table 2). Since the reassociation rate deviated from second-order kinetics later in the reaction, the estimated number of genome equivalents per cell may be in error. The results also suggest that
the majority of BPV DNA present in HT DNA does not represent the complete viral genome. Sharp and co-workers found deviations in second-order kinetics when approximately 50% or more of the viral genome was absent in adenovirus 2-transformed cellular DNA (38). Taking this into account, our results would suggest that a considerable portion of BPV DNA is absent in DNA isolated from HT cells. Restriction endonuclease cleavage fragments of BPV DNA would be useful in determining the amount of the BPV genome present in HT cells. Human papilloma viral DNA contains adenine plus thymine (A+T)-rich regions (12) and adenovirus 5 DNA has A+T-rich and G+C-rich halves (11). Therefore, 32P-labeled BPV DNA reassociated in the presence of HT DNA was analyzed by thermal denaturation to determine if perhaps the portion of the BPV genome present in HT cellular DNA was possibly rich in A+T or G+C. The results (Fig. 2B, Table 1) indicate that either there was no preferential retainment of A+T- or G+C-rich regions or alternatively that BPV DNA does not contain such regions since the Tm for the duplexes between 3"P-labeled BPV-HT DNA were within experimental error of the Tm for 32P-labeled BPV-CT DNA. Thermal denaturation data also indicate that the DNA of the virus employed to induce the tumors is the same viral DNA present in the tumors and not from a virus partially related to BPV since there was almost perfect base pair matching in reassociated duplex molecules. The reassociation of 2P -labeled BPV DNA in the presence of HT DNA does not rule out the possibility that a small fraction of the viral DNA is present in the form of a complete
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J. VIROL.
genome or that different populations of cells Health Service Research Grant CA-17244 to one of us from the National Cancer Institute and in part by the contain different segments of the BPV genome. (W.M.) College of Agricultural and Life Sciences, University of It is difficult to reconcile the fact that a tumor Wisconsin. W.D.L. was a recipient of a California Divisioninduced by a virus would result in the apparent American Cancer Society Junior Fellowship, No. J-292. lack of a consistent portion of the viral genome. LITERATURE CITED However, it is possible that BPV transformed 1. Black, P. H., J. W. Hartley, W. P. Rowe, and R. J. only a single cell which subsequently eliminated Huebner. 1963. Transformation of bovine tissue culportions of viral DNA and then repeated the ture cells by bovine papilloma virus. Nature (London) 199:1016-1018. fraction of the genome remaining. It is of M. Thomas, J. C. Friedmann, interest to note that there is evidence which 2. Boiron, M., J. P. Levy, 1964. Some properties of the bovine and J. Bernard. suggests that papillomas induced by the human papilloma virus. Nature (London) 201:423-424. wart virus are clonal in origin (30). Fujinaga et 3. Boiron, M., M. Thomas, and P. H. Chenaille. 1965. A biological property of deoxyribonucleic acid extracted al. (13) have shown that a line of adenovirus from bovine papilloma virus. Virology 26:150-153. 7-transformed hamster cells contains only 10 to 4. Bonner, T. I., D. J. Brenner, B. R. Neufeld, and R. J. 20% of the viral genome, of which there are Britten. 1973. Reduction in the rate of DNA reassociabetween 200 and 400 copies present. Also, most tion by sequence divergence. J. Mol. Biol. 81:123-135. adenovirus 2-transformed rat cells have been 5. Britten, R. J., and D. E. Kohne. 1968. Repeated sequences in DNA. Science 161:529-540. shown to contain only a given portion of the 6. Cheville, N. F. 1966. Studies on connective tissue tumors virus genome (14%) with 2.9 to 6.9 copies in the hamster produced by bovine papilloma virus. present (14). Cancer Res. 26:2334-2339. From the kinetics of reassociation of 32P- 7. Cheville, N. F., and C. Olson. 1964. Epithelial and fibroblastic proliferation in bovine cutaneous papillabeled BPV DNA in the presence of CT cellular lomatosis. Pathol. Vet. 1:248-257. DNA (Fig. 5), there appears to be about 700 to 8. Crawford, L. V. 1969. Nucleic acids of tumor viruses, p. 800 copies of BPV DNA in the calf meningioma 89-152. In K. M. Smith and M. A. Lauffer (ed.), Advances in virus research, vol. 14. Academic Press, cellular DNA (assuming diploid cells). The data Inc., New York. also suggest that the genome is complete since 9. Crawford, L. V., and P. H. Black. 1964. The nucleic the probe in the presence of CT DNA reanacid of simian virus 40. Virology 24:388-392. nealed to >90% with apparent second-order 10. Crawford, L. V., and E. M. Crawford. 1963. A comparative study of polyoma and papilloma viruses. kinetics. The number of genome equivalents per Virology 21:258-263. cell was substantially higher than expected 11. Ellens, D. J., J. S. Sussenbach, and H. S. Janz. 1974. since SV40-transformed cells contain from 1 to Studies on the mechanism of replication of adenovirus 20 genomes per cell (18). However, BPV may be DNA. III. Electron microscopy of replicating DNA. Virology 61:427-442. undergoing a lytic infection in a small proporE. A. C., and L. V. Crawford. 1967. Electron tion of cells, thus accounting for the large 12. Follett, microscopic study of the denaturation of human papilnumber of genome equivalents since cells lytiloma virus DNA. II. The specific location of denatured cally infected with SV40 may contain 20,000 regions. J. Mol. Biol. 28:461-467. genome equivalents per cell (20). The possibil- 13. Fujinaga, K., K. Sekikawa, H. Yamazaki, and M. Green. 1974. Analysis of multiple viral genome fragity also exists that BPV DNA in the calf tumor ments in adenovirus 7-transformed hamster cells. Cold may represent autonomously replicating DNA Spring Harbor Symp. Quant. Biol. 39:633-636. much like bacterial plasmid DNA. Attempts to 14. Gallimore, P. H., P. A. Sharp, and J. Sambrook. 1974. Viral DNA in transformed cells. II. A study of the isolate supercoiled BPV DNA from CT were sequences of adenovirus 2 DNA in nine lines of transBPV that indicates unsuccessful. This superformed rat cells using specific fragments of the viral coiled DNA may not be present in CT cells. genome. J. Mol. Biol. 89:49-72. However, the possibility exists that shearing 15. Gelb, L. D., D. E. Kohne, and M. A. Martin. 1971. Quantitation of simian virus 40 sequences in African forces generated in extraction of DNA from green monkey, mouse and virus transformed cell gesolid tumors may have relaxed supercoiled nomes. J. Mol. Biol. 57:129-145. DNA molecules. Determination of the physical 16. Girardi, A. J. 1959. The use of fluorocarbon for "unstate of BPV DNA in viral-induced tumors may masking" polyoma virus hemagglutinin. Virology 9:488-489. require the establishment of tumor cells in D. E., and C. Olson. 1968. Meningiomas and culture and use of a selective extraction proce- 17. Gordon, fibroblastic neoplasia in calves induced with bovine dure such as that described by Hirt (19). papilloma virus. Cancer Res. 28:2423-2431. Experiments are currently in progress to estab- 18. Hirai, K., and V. Defendi. 1974. Factors affecting the process and extent of integration of the viral genome. lish BPV-transformed cells in culture and isolaCold Spring Harbor Symp. Quant. Biol. 39:325-333. tion of BPV DNA restriction endonuclease frag- 19. Hirt, B. 1967. Selective extraction of polyoma DNA from ments. infected mouse cell cultures. J. Mol. Biol. 26:365-369. ACKNOWLEDGMENTS We wish to acknowledge the excellent assistance of Stephen Parry. This investigation was supported by Public
20. Holzel, F., and F. Sokol. 1974. Integration of progeny simian virus 40 DNA into the host cell genome. J. Mol. Biol. 84:423-444. 21. Jaenisch, R., and B. Mintz. 1974. Simian virus 40 DNA
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QUANTITATION OF BPV DNA IN TUMORS
sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc. Natl. Acad. Sci. U.S.A. 71:1250-1254. 22. Kelly, R. B., N. R. Cozzarelli, M. P. Deutscher, I. R. Lehman, and A. Kornberg. 1970. Enzymatic synthesis of deoxyribonucleic acid. XXXII. Replication of duplex deoxyribonucleic acid by polymerase at a single strand break. J. Biol. Chem. 245:39-45. 23. Kidd, J. G., J. W. Beard, and P. Rous. 1936. Serological reactions with a virus causing rabbit papillomas which become cancerous. I. Tests of the blood of animals carrying the papilloma. J. Exp. Med. 64:63-77. 24. Lancaster, W. D., and W. Meinke. 1975. Persistence of viral DNA in human cell cultures infected with human papilloma virus. Nature (London) 256:434-436. 25. Meinke, W., and D. A. Goldstein. 1971. Studies on the structure and formation of polyoma DNA replicative intermediates. J. Mol. Biol. 78:43-56. 26. Meinke, W., and D. A. Goldstein. 1974. Reassociation and dissociation of cytoplasmic membrane-associated DNA. J. Mol. Biol. 86:757-773. 27. Meinke, W., D. A. Goldstein, and M. R. Hail. 1974. Rapid isolation of mouse DNA from cells in tissue culture. Anal. Biochem. 52:82-88. 28. Meinke, W., M. R. Hall, and D. A. Goldstein. 1975. Proteins in intracellular simian virus 40 nucleoprotein complexes: comparison with simian virus 40 core proteins. J. Virol. 15:439-448. 29. Melnick, J. L., A. C. Allison, J. S. Butel, W. Eckhart, B. E. Eddy, S. Kit, A. J. Levine, J. A. R. Miles, J. S. Pagano, L. Sachs, and V. Vonka. 1974. Papovaviridae. Intervirology 3:106-120. 30. Murray, R. F., Jr., J. Hobbs, and B. Payne. 1971. Possible clonal origin of common warts (Verruca vulgaris). Nature (London) 232:51-52. 31. Nonoyama, M., and J. S. Pagano. 1973. Homology
32. 33.
34.
35.
36. 37.
38.
39.
40.
831
between Epstein-Barr virus DNA and viral DNA from Burkitt's lymphoma and nasopharyngeal carcinoma determined by DNA-DNA reassociation kinetics. Nature (London) 242:44-47. Olson, C., and R. H. Cook. 1951. Cutaneous sarcomalike lesions of the horse caused by the agent of bovine papilloma. Proc. Soc. Exp. Biol. Med. 77:281-284. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521. Raskas, H. J., and M. Green. 1971. DNA-RNA and DNA-DNA hybridization in virus research, p. 247-269. In K. Marmorosch and H. Koprowski (ed.), Methods in virology, vol. 5. Academic Press, Inc., New York. Robb, J. A., and R. G. Martin. 1972. Genetic analysis of simian virus 40. III. Characterization of a temperaturesensitive mutant blocked at an early stage of productive infection in monkey cells. J. Virol. 9:956-968. Robl, M. G., and C. Olson. 1968. Oncogenic action of bovine papilloma virus in hamsters. Cancer Res. 28:1596-1604. Rous, P., and J. W. Beard. 1935. The progression to carcinoma of virus-induced rabbit papillomas (Shope). J. Exp. Med. 62:528-548. Sharp, P. A., U. Pettersson, and J. Sambrook. 1974. Viral DNA in transformed cells. I. A study of the sequences of adenovirus 2 DNA in a line of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 86:709-726. Thomas, M., M. Boiron, J. Tanzer, J. P. Levy, and J. Bernard. 1964. In vitro transformation of mice cells by bovine papilloma virus. Nature (London) 202:709-710. Wetmur, J. G., and N. Davidson. 1968. Kinetics of renaturation of DNA. J. Mol. Biol. 31:349-370.
Vol. 17, No. 3 Printed in U.S.A.
Quantitation of Bovine Papilloma Viral DNA in Viral-Induced Tumors WAYNE D. LANCASTER,* CARL OLSON, AND WILLIAM MEINKE Department of Microbiology, Scripps Clinic and Research Foundation, La Jolla, California 92037,* and Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 22 September 1975
Bovine papilloma virus (BPV) DNA was labeled in vitro under conditions of repair synthesis and subsequently used as a "probe" in DNA-DNA reassociation studies to detect BPV-specific DNA sequences in a viral-induced calf meningioma and hamster fibroma. In vitro labeled BPV DNA had denaturation characteristics expected for duplex DNA and denatured DNA reassociated with apparent second-order kinetics. Analysis of in vitro labeled BPV DNA reassociation rates in the presence of excess tumor DNA revealed that the calf meningioma contained approximately 700 to 800 BPV genome equivalents per diploid cell whereas the hamster fibroma contained about 150 incomplete BPV genome equivalents per diploid cell. Thermal denaturation of in vitro labeled BPV DNA which reassociated in the presence of the two tumor DNA preparations indicated less than 1.5% base pair mismatching. The papilloma viruses, members of the papovavirus group (29), have not been intensively investigated. The major experimental drawback is the lack of suitable cell lines for virus replication. However, virus can be obtained in relatively large amounts from papillomas, and recent in vitro DNA labeling techniques (21, 24, 31) have allowed for the study of virus-cell interactions (21, 24). Papilloma viruses are the only DNA-containing viruses whose mode of replication leads to the production of tumors (warts) in their natural hosts. Although these tumors are considered benign and self-limiting, they can progress into malignancies. One example is the Shope papilloma virus, which produces papillomas in wild cottontail rabbits from which virus can be recovered. However, papillomas induced in domestic rabbits contain little or no virus and often develop into carcinomas (23, 37). Another member of the papilloma virus group is the bovine papilloma virus (BPV). BPV is the only papilloma virus shown to cross the species barrier. It can induce fibroblastic neoplasia in horses, hamsters, and mice as well as cattle (2, 6, 32). Both BPV and BPV DNA have been shown to transform bovine and mouse cells in culture (1, 3, 39). BPV readily induces fibromatous tumors of the meninges in brains of susceptible calves when injected intracranially and can produce tumors at other sites of inoculation (17). The Syrian hamster is also susceptible to the oncogenic action of BPV regardless of the
site of injection or age of the animal. These tumors can metastasize and may result in the death of the host (36). We report the detection and quantitation of BPV DNA in a calf meningioma and a hamster fibroma induced by this virus using DNA-DNA reassociation kinetics. The data suggest that there are a large number of BPV genome equivalents per cell present in the calf meningioma whereas there appears to be only a portion of the BPV genome in the hamster fibroma. Electron microscope investigation of the tumors did not reveal the presence of virus particles. MATERIALS AND METHODS Virus and viral DNA. BPV was isolated from papillomas experimentally induced in calves (7). Papillomas were excised and stored in 50% glycerolnormal saline at 4 C. Virus was obtained from 3 to 5 g of minced tissue suspended in 50 ml of 0.1 M Tris-hydrochloride (pH 7.5) and homogenized in a Virtis homogenizer at 45,000 rpm for 15 min. The homogenate was centrifuged at 12,000 x g for 10 min and the pellet was resuspended in 25 ml of 0.1 M Tris-hydrochloride (pH 7.5), containing 0.1% Sarkosyl and 25 ml of 1,1,2-trichloro-trifluoroethane (16). The mixture was further homogenized at 15,000 rpm for 10 min and centrifuged at 12,000 x g. The aqueous phase was recovered and combined with the previous supernatant fluid and the mixture was brought to 0.1% with Sarkosyl and centrifuged at 12,000 x g for 10 min. The resultant supernatant fluid was subjected to high-speed centrifugation in a Spinco type 30 rotor at 25,000 rpm for 3 h 824
VOL. 17, 1976
QUANTITATION OF BPV DNA IN TUMORS
at 4 C. Pellets were resuspended in CsCl (p = 1.33) in 0.01 M Tris-hydrochloride (pH 7.5) and centrifuged in a Spinco SW50.1 rotor at 32,000 rpm for 24 h at 25 C. The visible virus bands were collected and dialyzed against 0.2 M Tris-hydrochloride (pH 8.0), 0.001 M EDTA. BPV was ruptured by addition of sodium dodecyl sulfate (SDS) to 1% and heating to 50 C for 15 min. The mixture was deproteinated by two extractions with freshly distilled phenol saturated with 0.2 M Tris-hydrochloride (pH 8.0), 0.01 M EDTA. DNA was precipitated by addition of 2.5 volumes of ethanol at -20 C and subsequently dissolved in CsCl (p = 1.59) in 0.02 M Tris-hydrochloride (pH 8.0), 0.001 M EDTA containing ethidium bromide (33) and centrifuged in an SW50.1 rotor at 32,000 rpm for 48 h at 25 C. Supercoiled viral DNA was collected and ethidium bromide was removed by isopropanol extraction (25). DNA was dialyzed first against 4x SSC (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) followed by 0.1 x SSC and stored at - 20 C. The small-plaque variant of simian virus (SV40) was grown in TC-7 cell cultures (subline of CV-1) (35) and labeled with [3H]thymidine and virus, and viral DNA was isolated as described elsewhere (28). Induction of tumors. A partially purified suspension of BPV (17) was injected intracranially into a papilloma-free calf at 21 weeks of age. After 228 days, clinical signs of intracranial pressure developed and the animal was killed. The histology and characteristics of this BPV-induced calf meningioma have been described (17). Tumor induction in hamsters was by subcutaneous injection of a partially purified suspension of BPV. The tumor used in this study was passaged six times in hamsters prior to extraction of DNA. The characteristics and histology of similar hamster tumors have been described (36). Isolation of cellular DNA. Tumors (1 to 2 g) were minced and homogenized in 30 ml of 8 M urea, 0.24 M sodium phosphate (pH 6.8), 1% SDS, 0.001 EDTA, and 2% isoamyl alcohol in a Waring Blendor at maximum rpm for 30 s. The mixture was clarified by low-speed centrifugation, the supernatant fluid was applied to a column of hydroxyapatite, and the DNA was isolated as described previously (27). Calf thymus DNA was purified by the same regimen. Purified DNA preparations were sheared to an average singlestrand length of 570 nucleotides (26). In vitro labeling of BPV DNA. The methods of labeling DNA in vitro were similar to those described previously (24). Supercoiled viral DNA was treated with pancreatic DNase I in 0.05 M Tris-hydrochloride (pH 7.4), 0.05 M KCl, 0.005 M MgCl2 at a ratio of 500:1 (wt/wt) at 37 C for 15 min. DNase was inactivated by heating to 65 C for 10 min. DNase-treated DNA was then labeled in vitro with Escherichia coli DNA polymerase I under conditions of repair synthesis (22). Viral DNA (0.1 Ag) was suspended in a volume of 0.L ml containing 6.6 ,mol of potassium phosphate (pH 7.4), 0.66 limol of MgCl,, 0.1 gtmol of 2-mercaptoethanol, and 0.005 jgmol of each of the necessary unlabeled deoxynucleotide triphosphates (dNTP). Depending on the experi-
825
ment, DNA
was labeled with either [3H JTTP (54 Ci(mmol) at 500 iACilig of DNA or [a-32PJdCTP + [a-32PfTTP (116 Ci/mmol) at 2 mCi/ug of DNA or [a-"2P]dATP + [a-_2PJTTP (112 Ci/mmol) at 2 mCi/ug of DNA. Synthesis was initiated by ad-
dition of 20 units of E. coli DNA polymerase I per microgram of DNA and incubated at 15 C for 30 to 60 min. Reactions were terminated by addition of Sarkosyl to 1%. Labeled DNA was passed through a Sephadex G-50 column (0.7 by 20 cm) equilibrated with 0.02 M Tris-hydrochloride (pH 8.0), 0.1% Sarkosyl, 0.001 M EDTA. Fractions containing radiolabeled DNA were pooled and stored over chloroform at 4 C. Incorporation of radioactivity into DNA was determined by trichloroacetic acid precipitation with 100 gg of tRNA as carrier. Specific activities ranged from 1.8 x 106 to 4.7 x 10" counts/min per gsg of DNA for ['HJTTP-labeled DNA and 15.5 x 101 to 16.4 x 10' counts/min per ;g of DNA for [32P]dNTP-labeled
DNA. DNA-DNA reassociation. DNA solutions were denatured in 0.48 M sodium phosphate (pH 6.8), 0.05% SDS, 0.001 M EDTA by heating to 109 C for 5 min in screw-capped vials and quenched in an ice bath. Denatured DNA was allowed to reassociate at 68 C. At various times, samples were removed and diluted to 2.0 ml of 0.14 M sodium phosphate (pH 6.8), 0.05% SDS. Single-strand DNA was separated from reassociated duplex DNA by hydroxyapatite column chromatography (10 by 15 mm) at 60 C (5). Single-stranded DNA was eluted from the column with five 2.0-ml washes of 0.14 M sodium phosphate (pH 6.8), 0.05% SDS, and reassociated DNA was subsequently eluted with four 2.0-ml washes of 0.48 M sodium phosphate (pH 6.8), 0.05% SDS. Radioactivity was determined for each wash by addition of 15 ml of a toluene-Triton X-100 (2:1, vol/vol) scintillation fluid. Immediately after denaturation, about 8.0 to 8.3% of in vitro labeled DNA was eluted as doublestranded DNA on hydroxyapatite columns. These values were not used to normalize the reassociation data. Thermal denaturation. DNA preparations in 0.14 M sodium phosphate (pH 6.8), 0.05% SDS (eluting buffer) were applied to a column of hydroxyapatite at 60 C. After five 2.0-ml washes of eluting buffer, the column temperature was increased by approximately four-degree increments, single-stranded DNA was eluted with five 2.0-ml washes of prewarmed buffer, and radioactivity in each wash was determined as described above. Electron microscopy. Small portions of tumor were fixed in 3.5% glutaraldehyde in phosphate buffer (pH 7.2) for 1 h and then postfixed with 1% osmium tetroxide in phosphate buffer (pH 7.2) for 1 h. Preparations were dehydrated with ethanol, transferred to propylene oxide and embedded in epoxy resin.
Chemicals. [a-32P]dATP, [a-32PJTTP, and [a'2P]dCTP (specific activities ranging from 112 Ci/ mmol to 116 Ci/mmol) were purchased from New England Nuclear Corp. [3H]TTP (specific activity,
54 Ci/mmol) was obtained from Schwarz-Mann. E. coli DNA polymerase I was obtained from Grand
826
LANCASTER, OLSON, AND MEINKE
Island Biological Co. or Boehringer Mannheim Corp. DNase I (electrophoretically pure) was purchased from Sigma Chemical Corp. Calf thymus DNA was from Calbiochem.
RESULTS Size distribution of in vitro labeled DNA. Viral DNA preparations to be labeled in vitro with [a-"2PJdNTP were first "nicked" with DNase and then labeled with ['H ]TP to estimate piece size. The size of single-stranded DNA was determined by co-sedimentation through 5 to 20% alkaline sucrose gradients with marker DNA which had an average length of 570 nucleotides (26). One preparation of in vitro 'H-labeled BPV DNA had an average length of 435 nucleotides (Fig. 1A) whereas another preparation, which was more heterogeneous, was about 775 nucleotides in length (Fig. 1B). Differences have not been noted in piece sizes of DNA labeled with either [3H ]TTP or [a"2P ]dNTP. In vitro labeled DNA preparations reassociated to >90% when denatured and allowed to reanneal in the presence of "nicked" unlabeled BPV DNA. Labeled DNA (0.002 gg/ml) (Fig. 1A) reassociated to 50% in 187 h (0.48 M sodium phosphate, pH 6.8, 68 C) whereas DNA shown in Fig. 1B was 50% reannealed by 101 h. BPV DNA from Fig. 1A reassociated 28% slower A
/200
400
600
200
FIG. 1. Sedimentation profiles of in vitro labeled BPV DNA in alkaline sucrose gradients. 'H-labeled BPV DNA and 14C-labeled marker DNA were sedimented in alkaline 5 to 20% linear sucrose (wt/wt) gradients (0.3 N NaOH, 1.0 M NaCl, 0.001 M EDTA, 0.015% Sarkosyl) in an SW56 rotor at 15 C for 6.5 to 7 h at 49,000 rpm. Letters A and B refer to two separate preparations of DNase-treated BPV DNA labeled in vitro. Symbols: (0) 'H-labeled BPV DNA; (0) 14Clabeled marker DNA 570 nucleotides in length.
J.VIROL.
than expected based on the reassociation rate of the BPV DNA preparation in Fig. 1B (40). This discrepancy could be due to inaccuracy of piece size determinations. However, the faster rate of BPV DNA reassociation was expected since the DNA preparation shown in Fig. 1B contained molecules with unit lengths about 44% greater than those of Fig. 1A. Thermal denaturation. The thermal denaturation profile of 32P-labeled BPV DNA in the presence of 'H-labeled SV40 DNA is shown in Fig. 2A. The temperature at which 50% of the DNA was denatured (Tm) for SV40 DNA, BPV DNA, and E. coli DNA was 90.3 C, 91.8 C, and 93.4 C, respectively (Table 1). The Tm for BPV DNA indicated it contained 45.4% guanine plus cytosine (G+C) based on G+C values of 41% for SV40 DNA (9) and 50% for E. coli DNA (Fig. 3). Others have reported that BPV DNA has a G+C value of 45.5% (10). These two values of G +C for BPV DNA are in excellent agreement. The Tm of 32P-labeled BPV DNA formed in the presence of either hamster fibroma (HT) or calf meningioma (CT) DNA (Fig. 2B) also was determined. The small decrease in the Tm indicated that there was only slight base pair mismatching (approximately 1 to 1.5%) (Table 1). This value is based on the observation by others that a mismatching of 1% of the base pairs will lower the Tm by 0.7 to 1.5 degrees C (4, 34). Similar base pair mismatching has been noted for in vitro labeled human papilloma viral DNA allowed to reassociate to completion in the presence of unlabeled viral DNA (W. D. Lancaster and W. Meinke, unpublished data). DNA-DNA reassociation. Reassociation of 32Plabeled BPV DNA in the presence of either calf thymus DNA, CT DNA or HT DNA is shown in Fig. 4. The rate of reassociation of 2P-labeled BPV DNA in the positive control (calf thymus DNA plus unlabeled "nicked" BPV DNA) was increased over that of the control (calf thymus DNA alone). The reassociation rate of 32P-labeled BPV DNA in the presence of CT DNA was very rapid. Also, the reassociation rate of 32P-labeled BPV DNA in the presence of HT DNA was rapid initially but later deviated from second-order kinetics. In another experiment in which the cellular DNA concentrations were reduced 10-fold, similar curves were generated (Fig. 5). "2P-labeled BPV DNA in the positive control reassociated faster than in the control reaction as expected. In both reactions "2P-labeled BPV DNA reassociated with second-order kinetics. Also 3P_ labeled BPV DNA reassociated linearly in the presence of CT DNA to >90%. However, 2Plabeled BPV DNA reassociating in the presence
QUANTITATION OF BPV DNA IN TUMORS
VOL. 17, 1976
827
TABLE 1. Thermal denaturation of DNA by hydroxyapatite chromatography DNA
['H ]simian virus 40 [14C ] E. coli [P2p]BPV [32P ]BPV-CT PBPV-HT [32p
.% associated
TM
ATm0
86.6 81.9
90.3 93.4 91.8 90.9 90.6
0.90 1.20
aThe difference between the Tm of in vitro labeled ['3PJBPV DNA and the Tm of "2P-labeled BPV DNA reassociated in the presence of either CT DNA or HT
Sq
DNA.
Cs I
I
t
'4
501-
48k 4.
461
441
42hF
TenpwutUre (C) FIG. 2. Thermal denaturation profiles of in vitro labeled BPVDNA and denatured reassociated in vitro labeled BPV DNA. (A) "2P-labeled BPV DNA was mixed with 'H-labeled SV40 DNA and applied to a column of hydroxyapatite and single-stranded DNA eluted at various temperatures as described in Materials and Methods. (B) Thermal stability
of
"2p_
labeled BPV DNA which reassociated in the presence of HT DNA or CT DNA.
of HT DNA again deviated significantly from second-order kinetics. The results of the two experiments are summarized in Table 2. In experiment 1, the reassociation rate of "2P-labeled BPV DNA in the positive control was 3.1 times faster than in the control (expected value was 4.0). 32P-labeled DNA in the presence of CT DNA reassociated approximately 485 times faster than the control reaction. The reannealing rate of 32P-labeled BPV DNA in the presence of HT DNA was 170 times that of the control reaction based on the initial rate of reassociation.
I
I
I
9/
92 Tm (C)
93
FIG. 3. Relationship between G+C content and Tm for DNA from E. coli, BPV, and SV40. Fragmented DNA preparations were thermally denatured on hydroxyapatite columns.
Since 32P-labeled BPV DNA unexpectedly was reassociating at extremely fast rates in the presence of either CT or HT DNA, cellular DNA
concentrations were reduced and experiments repeated to obtain a more accurate estimation of reassociation rates. In experiment 2, 32P-labeled BPV DNA in the positive control reassociated 5.6 times faster than the control (expected value was 6.6). The rate of reassociation of 3P-labeled BPV DNA in the presence of CT DNA was 92 times the rate observed for the control reaction. This increased rate of reaswere
828
LANCASTER, OLSON, AND MEINKE
J. VIROL.
3.0
2.5
d. 2.0 0
'.5
25 /5 Hours Incubetion FIG. 4. Reassociation of in vitro labeled BPVDNA in the presence of unlabeled DNA. The results are plotted as the ratio (Co/C) of the total 32P-labeled BPV DNA concentration (Co) to the concentration of single-stranded 32P-labeled BPV DNA (C) at various incubation times. 32P-labeled BPV DNA (0.002 fg/ mt) was allowed to reassociate in the presence of either 2 mg of denatured calf thymus DNA per ml (0); 2 mg of calf thymus DNA per ml plus 0.0062 gg of unlabeled BPV DNA per ml (0); 2 mg of hamster fibroma (HT) DNA per ml (0); or 2 mg of calf meningioma (CT) DNA per ml (A). 5
sociation corresponds to about 726 genome equivalents per CT cell. This value for the number of BPV genome equivalents in CT DNA is probably the more accurate value of the two since the rate of reassociation of 32P-labeled BPV DNA in the presence of the smaller amount of CT DNA could be more precisely determined. The initial rate of reassociation of 32P-labeled BPV DNA with HT DNA was 21.5 times that of the control and this corresponds to about 164 BPV genome equivalents per cell. The values obtained for the number of BPV genome equivalents per HT cell (based on the initial rate of reassociation) are in close agreement in both experiments. Electron microscopy. Since a large number of BPV genome equivalents per cell were detected in tumor DNA, tissues were examined for the presence of virus particles. Approximately 100 cells in each tumor were examined and no particles resembling papovaviruses were seen.
15 25 Hours Incubation FIG. 5. Reassociation of in vitro labeled BPV DNA in the presence of unlabeled DNA. The results are plotted as described for Fig. 4. "2P-labeled BPV DNA (0.002 ,g/ml) was allowed to reassociate in the presence of either 200 Mg of calf thymus DNA per ml (0); 200 Mg of calf thymus DNA per ml and 0.0112 Ag of BPV DNA per ml (0); 200 MLg of HT DNA per ml (0); or 200 Mg of CTDNA per ml (A). Analysis of tumor DNA by CsCi-ethidium bromide centrifugation. To determine if some of the BPV DNA was existing as free supercoiled DNA in the tumor cells, tumors were homogenized and DNA was isolated as described in Materials and Methods. A 1- to 0.7-mg amount of tumor DNA was analyzed by 5
dye buoyant density equilibrium centrifugation in an attempt to isolate BPV supercoiled DNA molecules. In HT and CT DNA preparations, no band corresponding to supercoiled DNA was observed. Since HT contains about 700 to 800 copies of BPV DNA per diploid cell, those fractions of the gradients corresponding to the density of supercoiled DNA, including a small portion (5 to 10%) of the cellular DNA peak, were pooled and the DNA was analyzed for BPV DNA sequences by DNA-DNA reassociation kinetics. The results indicated that about 800 BPV genome equivalents per diploid cell were present. If BPV supercoiled DNA was present, the number of BPV genome equivalents per cell should have been increased significantly.
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QUANTITATION OF BPV DNA IN TUMORS
829
TABLE 2. Reassociation of 32P-labeled BPV DNA in the presence of unlabeled DNA Concn (gg/ml)
Expt 1
2
no.
Source of DNA
Unlabeled cellular
No. of genome
Factor of
equivalents! divlells
Unlabeled BPV DNA
increased rate
0.002 0.002 0.002 0.002
0.0062
3.1 170C 467
2 135 373
0.002 0.002 0.002 0.002
0.0112
5.6 21.5c 92
37 164 726
DNA
'P-labeled BPV DNA
Calf thymus Calf thymus HT CT
2,000 2,000 2,000 2,000
Calf thymus Calf thymus HT CT
200 200 200 200
diploid cell"
Factor of increased rate of reassociation over that of the control. bCalculated by the method of Gelb et al. (15) with molecular weight of BPV DNA of 5 x 106 (8); also as determined by restriction endonuclease cleavage of BPV DNA (Lancaster and Meinke, unpublished data). cEstimated from the initial rate of reassociation. a
DISCUSSION As previously demonstrated, human papilloma viral DNA could be labeled in vitro and used as a probe to determine the absence or presence of viral DNA in infected cell cultures (24). The results of this study also indicate that BPV DNA can be labeled in vitro and will similarly function as probe in DNA-DNA reassociation studies. Our results indicated that in vitro labeled BPV DNA had a Tm expected for DNA of 45.5% G+C as compared to in vivo labeled SV40 DNA and in vivo labeled E. coli DNA (Fig. 3). Denatured fragmented 32P-labeled BPV DNA reannealed with second-order kinetics and the rates of reassociation were approximately those expected for a genome with a genetic complexity of 5 x 10. daltons "nicked" to the piece sizes described (40). Furthermore, the results demonstrate that BPV DNA is present in a calf meningioma and hamster tumor, both considered to be induced by BPV, and that the tumors used in this study vary greatly in their content of BPV DNA per cell. The rate of reassociation of 82P-labeled BPV DNA in the presence of HT DNA deviated from second-order kinetics (Fig. 4 and 5); however, the initial rate of reassociation was linear. By comparing the increase in the initial reassociation rate of 32P-labeled BPV DNA in the absence and presence of HT DNA there were calculated to be between 135 and 164 BPV genome equivalents per diploid cell (Table 2). Since the reassociation rate deviated from second-order kinetics later in the reaction, the estimated number of genome equivalents per cell may be in error. The results also suggest that
the majority of BPV DNA present in HT DNA does not represent the complete viral genome. Sharp and co-workers found deviations in second-order kinetics when approximately 50% or more of the viral genome was absent in adenovirus 2-transformed cellular DNA (38). Taking this into account, our results would suggest that a considerable portion of BPV DNA is absent in DNA isolated from HT cells. Restriction endonuclease cleavage fragments of BPV DNA would be useful in determining the amount of the BPV genome present in HT cells. Human papilloma viral DNA contains adenine plus thymine (A+T)-rich regions (12) and adenovirus 5 DNA has A+T-rich and G+C-rich halves (11). Therefore, 32P-labeled BPV DNA reassociated in the presence of HT DNA was analyzed by thermal denaturation to determine if perhaps the portion of the BPV genome present in HT cellular DNA was possibly rich in A+T or G+C. The results (Fig. 2B, Table 1) indicate that either there was no preferential retainment of A+T- or G+C-rich regions or alternatively that BPV DNA does not contain such regions since the Tm for the duplexes between 3"P-labeled BPV-HT DNA were within experimental error of the Tm for 32P-labeled BPV-CT DNA. Thermal denaturation data also indicate that the DNA of the virus employed to induce the tumors is the same viral DNA present in the tumors and not from a virus partially related to BPV since there was almost perfect base pair matching in reassociated duplex molecules. The reassociation of 2P -labeled BPV DNA in the presence of HT DNA does not rule out the possibility that a small fraction of the viral DNA is present in the form of a complete
830
LANCASTER, OLSON, AND MEINKE
J. VIROL.
genome or that different populations of cells Health Service Research Grant CA-17244 to one of us from the National Cancer Institute and in part by the contain different segments of the BPV genome. (W.M.) College of Agricultural and Life Sciences, University of It is difficult to reconcile the fact that a tumor Wisconsin. W.D.L. was a recipient of a California Divisioninduced by a virus would result in the apparent American Cancer Society Junior Fellowship, No. J-292. lack of a consistent portion of the viral genome. LITERATURE CITED However, it is possible that BPV transformed 1. Black, P. H., J. W. Hartley, W. P. Rowe, and R. J. only a single cell which subsequently eliminated Huebner. 1963. Transformation of bovine tissue culportions of viral DNA and then repeated the ture cells by bovine papilloma virus. Nature (London) 199:1016-1018. fraction of the genome remaining. It is of M. Thomas, J. C. Friedmann, interest to note that there is evidence which 2. Boiron, M., J. P. Levy, 1964. Some properties of the bovine and J. Bernard. suggests that papillomas induced by the human papilloma virus. Nature (London) 201:423-424. wart virus are clonal in origin (30). Fujinaga et 3. Boiron, M., M. Thomas, and P. H. Chenaille. 1965. A biological property of deoxyribonucleic acid extracted al. (13) have shown that a line of adenovirus from bovine papilloma virus. Virology 26:150-153. 7-transformed hamster cells contains only 10 to 4. Bonner, T. I., D. J. Brenner, B. R. Neufeld, and R. J. 20% of the viral genome, of which there are Britten. 1973. Reduction in the rate of DNA reassociabetween 200 and 400 copies present. Also, most tion by sequence divergence. J. Mol. Biol. 81:123-135. adenovirus 2-transformed rat cells have been 5. Britten, R. J., and D. E. Kohne. 1968. Repeated sequences in DNA. Science 161:529-540. shown to contain only a given portion of the 6. Cheville, N. F. 1966. Studies on connective tissue tumors virus genome (14%) with 2.9 to 6.9 copies in the hamster produced by bovine papilloma virus. present (14). Cancer Res. 26:2334-2339. From the kinetics of reassociation of 32P- 7. Cheville, N. F., and C. Olson. 1964. Epithelial and fibroblastic proliferation in bovine cutaneous papillabeled BPV DNA in the presence of CT cellular lomatosis. Pathol. Vet. 1:248-257. DNA (Fig. 5), there appears to be about 700 to 8. Crawford, L. V. 1969. Nucleic acids of tumor viruses, p. 800 copies of BPV DNA in the calf meningioma 89-152. In K. M. Smith and M. A. Lauffer (ed.), Advances in virus research, vol. 14. Academic Press, cellular DNA (assuming diploid cells). The data Inc., New York. also suggest that the genome is complete since 9. Crawford, L. V., and P. H. Black. 1964. The nucleic the probe in the presence of CT DNA reanacid of simian virus 40. Virology 24:388-392. nealed to >90% with apparent second-order 10. Crawford, L. V., and E. M. Crawford. 1963. A comparative study of polyoma and papilloma viruses. kinetics. The number of genome equivalents per Virology 21:258-263. cell was substantially higher than expected 11. Ellens, D. J., J. S. Sussenbach, and H. S. Janz. 1974. since SV40-transformed cells contain from 1 to Studies on the mechanism of replication of adenovirus 20 genomes per cell (18). However, BPV may be DNA. III. Electron microscopy of replicating DNA. Virology 61:427-442. undergoing a lytic infection in a small proporE. A. C., and L. V. Crawford. 1967. Electron tion of cells, thus accounting for the large 12. Follett, microscopic study of the denaturation of human papilnumber of genome equivalents since cells lytiloma virus DNA. II. The specific location of denatured cally infected with SV40 may contain 20,000 regions. J. Mol. Biol. 28:461-467. genome equivalents per cell (20). The possibil- 13. Fujinaga, K., K. Sekikawa, H. Yamazaki, and M. Green. 1974. Analysis of multiple viral genome fragity also exists that BPV DNA in the calf tumor ments in adenovirus 7-transformed hamster cells. Cold may represent autonomously replicating DNA Spring Harbor Symp. Quant. Biol. 39:633-636. much like bacterial plasmid DNA. Attempts to 14. Gallimore, P. H., P. A. Sharp, and J. Sambrook. 1974. Viral DNA in transformed cells. II. A study of the isolate supercoiled BPV DNA from CT were sequences of adenovirus 2 DNA in nine lines of transBPV that indicates unsuccessful. This superformed rat cells using specific fragments of the viral coiled DNA may not be present in CT cells. genome. J. Mol. Biol. 89:49-72. However, the possibility exists that shearing 15. Gelb, L. D., D. E. Kohne, and M. A. Martin. 1971. Quantitation of simian virus 40 sequences in African forces generated in extraction of DNA from green monkey, mouse and virus transformed cell gesolid tumors may have relaxed supercoiled nomes. J. Mol. Biol. 57:129-145. DNA molecules. Determination of the physical 16. Girardi, A. J. 1959. The use of fluorocarbon for "unstate of BPV DNA in viral-induced tumors may masking" polyoma virus hemagglutinin. Virology 9:488-489. require the establishment of tumor cells in D. E., and C. Olson. 1968. Meningiomas and culture and use of a selective extraction proce- 17. Gordon, fibroblastic neoplasia in calves induced with bovine dure such as that described by Hirt (19). papilloma virus. Cancer Res. 28:2423-2431. Experiments are currently in progress to estab- 18. Hirai, K., and V. Defendi. 1974. Factors affecting the process and extent of integration of the viral genome. lish BPV-transformed cells in culture and isolaCold Spring Harbor Symp. Quant. Biol. 39:325-333. tion of BPV DNA restriction endonuclease frag- 19. Hirt, B. 1967. Selective extraction of polyoma DNA from ments. infected mouse cell cultures. J. Mol. Biol. 26:365-369. ACKNOWLEDGMENTS We wish to acknowledge the excellent assistance of Stephen Parry. This investigation was supported by Public
20. Holzel, F., and F. Sokol. 1974. Integration of progeny simian virus 40 DNA into the host cell genome. J. Mol. Biol. 84:423-444. 21. Jaenisch, R., and B. Mintz. 1974. Simian virus 40 DNA
VOL. 17, 1976
QUANTITATION OF BPV DNA IN TUMORS
sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc. Natl. Acad. Sci. U.S.A. 71:1250-1254. 22. Kelly, R. B., N. R. Cozzarelli, M. P. Deutscher, I. R. Lehman, and A. Kornberg. 1970. Enzymatic synthesis of deoxyribonucleic acid. XXXII. Replication of duplex deoxyribonucleic acid by polymerase at a single strand break. J. Biol. Chem. 245:39-45. 23. Kidd, J. G., J. W. Beard, and P. Rous. 1936. Serological reactions with a virus causing rabbit papillomas which become cancerous. I. Tests of the blood of animals carrying the papilloma. J. Exp. Med. 64:63-77. 24. Lancaster, W. D., and W. Meinke. 1975. Persistence of viral DNA in human cell cultures infected with human papilloma virus. Nature (London) 256:434-436. 25. Meinke, W., and D. A. Goldstein. 1971. Studies on the structure and formation of polyoma DNA replicative intermediates. J. Mol. Biol. 78:43-56. 26. Meinke, W., and D. A. Goldstein. 1974. Reassociation and dissociation of cytoplasmic membrane-associated DNA. J. Mol. Biol. 86:757-773. 27. Meinke, W., D. A. Goldstein, and M. R. Hail. 1974. Rapid isolation of mouse DNA from cells in tissue culture. Anal. Biochem. 52:82-88. 28. Meinke, W., M. R. Hall, and D. A. Goldstein. 1975. Proteins in intracellular simian virus 40 nucleoprotein complexes: comparison with simian virus 40 core proteins. J. Virol. 15:439-448. 29. Melnick, J. L., A. C. Allison, J. S. Butel, W. Eckhart, B. E. Eddy, S. Kit, A. J. Levine, J. A. R. Miles, J. S. Pagano, L. Sachs, and V. Vonka. 1974. Papovaviridae. Intervirology 3:106-120. 30. Murray, R. F., Jr., J. Hobbs, and B. Payne. 1971. Possible clonal origin of common warts (Verruca vulgaris). Nature (London) 232:51-52. 31. Nonoyama, M., and J. S. Pagano. 1973. Homology
32. 33.
34.
35.
36. 37.
38.
39.
40.
831
between Epstein-Barr virus DNA and viral DNA from Burkitt's lymphoma and nasopharyngeal carcinoma determined by DNA-DNA reassociation kinetics. Nature (London) 242:44-47. Olson, C., and R. H. Cook. 1951. Cutaneous sarcomalike lesions of the horse caused by the agent of bovine papilloma. Proc. Soc. Exp. Biol. Med. 77:281-284. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521. Raskas, H. J., and M. Green. 1971. DNA-RNA and DNA-DNA hybridization in virus research, p. 247-269. In K. Marmorosch and H. Koprowski (ed.), Methods in virology, vol. 5. Academic Press, Inc., New York. Robb, J. A., and R. G. Martin. 1972. Genetic analysis of simian virus 40. III. Characterization of a temperaturesensitive mutant blocked at an early stage of productive infection in monkey cells. J. Virol. 9:956-968. Robl, M. G., and C. Olson. 1968. Oncogenic action of bovine papilloma virus in hamsters. Cancer Res. 28:1596-1604. Rous, P., and J. W. Beard. 1935. The progression to carcinoma of virus-induced rabbit papillomas (Shope). J. Exp. Med. 62:528-548. Sharp, P. A., U. Pettersson, and J. Sambrook. 1974. Viral DNA in transformed cells. I. A study of the sequences of adenovirus 2 DNA in a line of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 86:709-726. Thomas, M., M. Boiron, J. Tanzer, J. P. Levy, and J. Bernard. 1964. In vitro transformation of mice cells by bovine papilloma virus. Nature (London) 202:709-710. Wetmur, J. G., and N. Davidson. 1968. Kinetics of renaturation of DNA. J. Mol. Biol. 31:349-370.
