- Email: [email protected]
Integration and methylation of shope papilloma virus DNA in the transplantable V×2 and V×7 rabbit carcinomas
131, 88-99 (1983)
VIROLOGY
Integration and Methylation of Shope Papilloma Virus DNA in the Transplantable Vx2 and Vx7 Rabbit Carcinomas KENJI
SUGAWARA,**’
KEI FUJINAGA,? TOSHIHARU AND YOHEI ITO*
YAMASHITA,?
‘Department of Microbi&cgy, Faculty of Medicine, Kyoto Univers-ity, Kyoto 606, and iCancer Research Institute, Sapporo Medical College, Suppuro 060, Japan Received May 20, 19X.9;accepted July 27, 1983 The Shope papilloma virus (SPV) DNA present in SPV-induced benign and malignant rabbit tumors, particularly in the transplantable carcinoma Vx2 and Vx7, was examined with regard to physical states and extent of methylation. Vx2 and Vx7 carcinomas contained 10-22 viral genomes per diploid cell, and domestic and cottontail rabbit papillomas 40460 and 1000-8000, respectively. The digestion of Vx2 and Vx7 DNA with the restriction enzyme KpnI, which does not cleave SPV DNA, yielded a single virus-specific DNA band about nine t.imes larger than the genome length, but EcoRI, which cuts the circular SPV DNA once, cleaved this DNA to the genome-size fragments. However, three or four weak bands which may contain viral segments linked to cellular sequences were also identified, and at least two were shared by both Vx2 and Vx7 carcinomas. The analysis with a set of MapI and HpoII, which discriminates the methylated DNA sequence -CC*GG-, showed that lo-40% of the sites of viral DNA are methylated in papillomas, 30-802 in primary carcinomas, and more than 90% in the transplantable carcinomas.
(Stevens and Wettstein, 1979). However, in papillomas, most of the viral DNA fraction is present in the form of episomes with the normal genome-length, while in primary and metastatic carcinomas, a considerable fraction is found in free oligomers consisting of several genome-length viral DNA molecules (Wettstein and Stevens, 1982; Sugawara et uL, unpublished observation). The transplantable carcinomas Vx2 and Vx7 were established from carcinomas induced by SPV in domestic rabbits more than 40 and 30 years ago, respectively (Kidd and ROUS, 1940; Rogers et ab, 1960), and have been used as the only available strains representing SPV-induced carcinomas. In these tumors, free genome-size viral DNA molecules do not exist, but multiple viral genomes are present in highmolecular-weight forms (McVay et d, 1982; Favre et aZ., 1982). McVay et al. (1982) have shown that the Vx7 carcinoma and the cell line derived from it contain lo-30 viral genomes per diploid cell and suggested that these viral DNA sequences are integrated into the host cell DNA. However, Favre et
INTRODUCTION
Shope papilloma virus (SPV) isolated from papillomas of wild cottontail rabbits also induces papillomas in domestic rabbits. When the rabbits carrying papillomas are maintained over 6 months, epidermoid carcinomas frequently develop at the same sites (for reviews, see Ito, 1975; Kreider and Bartlett, 1981). Although infectious virus is rarely recovered from the papillomas and carcinomas, viral genetic information is conserved in both types of tumors (Noyes and Mellors, 1957; Mellors, 1960; Ito and Evans, 1961; Ito, 1963; Ito and Evans, 1965; Osato and Ito, 1967), suggesting that this virus might play some role in the ensuing malignant conversion. Recent work has demonstrated that all these tumors contain multiple viral genomes (lo-400 copies per diploid cell), but quantitative differences between benign and malignant tumors were not apparent
’ Author addressed.
to whom requests for reprints
0042-6822/83 $3.00 Copyright All riyhts
G 19X3 hy Academic Press. Inc. of rcprociuction in an)’ form renewed.
should be
88
Vx2 AND
Vx7 CARCINOMAS
al. (1982) reported that 444 viral DNA copies are present in the Vx’Z carcinoma and suggested that these viral sequences as well as those in the Vx2 carcinoma, which contain 42 viral DNA copies, are not integrated. In benign and malignant tumors, viral transcripts complementary to about 10% of the genome are found in a low quantity (Wettstein and Stevens, 1981; McVay et al.. 1982). Recently, two species of 1.3 and 2.0 kilobases (kb) were identified (Nasseri et a.Z.,1982). Although no qualitative diffetence was detected between both types, increased amounts of 1.3-kb species were detected in carcinomas. Although little is known of the mechanisms of malignant eonversion of SPV-induced papillomas, attempts to detect the distinctive features between benign and malignant tumors have to be made. In the present study, mainly, Vx2 and Vx7 carcinomas but, for comparison, some SPV.indueed papjllomas and primary carcinomas of domestic and cottontail rabbits were analyzed for the physical state and the extent of methylation of persisting viral DNA. The results suggested the viral DNA integration in the Vx2 and Vx7 carcinomas. Data on the existence of a common viral DNA integration site between Vx2 and Qx7 carcinomas and extensive methylation of the integrated viral DNA sequences are also presented. UATERIALS
AND
METHODS
Virus and unimak The SPV originated from papillomas of wild cottontail rabbits trapped in Kansas. The glycerinated material was a generous gift from Dr. C. A. Evans. Eastern cottontail rabbits were brought to Japan and propagated on a small island. Tumors. Initial source of the Vx2 and Vx7 carcinomas has been described previously (Ito et al, 1968). These tumors were maintained by serial transfers through domestic rabbits. Twenty percent suspension (0.5 ml) of packed tumor cells was intramuscularly inoculated once every 4050 days. At the beginning of 1980, the passage numbers exceeded ??.A0 for the Vx2 and
89
350 for the Vx7 carcinoma. Tumor tissues collected during 1980 to 1982 were used for the experiments. Papillomas and primary carcinomas of domestic and cottontail rabbits were obtained by inoculating 10% tissue extracts of the original stock into the scarified skin of respective rabbits. Preparation of viral DN-4. SPV DNA was isolated from cottontail rabbit papillomas prepared in the laboratory. The tissues were cut and ground with sand in a mortar. Vjrus particles were extracted with 0.02 M Tris-HCl (pH 7.5) buffer and pelleted by centrifugation at 29,000 rpm for 1 hr. Virus DNA was released by incubation with 0.5% SDS and 0.5 my/ml Pronase E (Kaken Kagaku Co., predigested) and extracted with phenol and chloroform-isoamylalcohol (24:1). After treatment with 50 rg/ ml of RNase A (Sigma Chemical Co., preheated at 90” for 10 min), the DNA solution was prepared to contain 0.5 g/ml ethidiurn bromide and CsCl (density = 1.587 g/ml) and was centrjfuged at 3’1,000 rpm for 72 hr. Supercoiled (form I) SPV DNA was collected. For further purification, the viral DN,A was treated with ATP-dependent DNase to eliminate linear DNA molecules (Mukai et ~1.. 1973). ATP-dependent DNase was prepared from Micrococ~us luteus, as described by Anai & al. (:970).
Restrkkn en&mu&eases and physzmt mapping ajr the cleavage s&s. The restriction endonucleases EcaU and Hind111 were prepared from E. cxdi RY13 (Greene et al, 1974) and Haemophilus in$uenzae Rd (Takanami, 1974), respectively. HpaI, BarnHI, &XII, IwspI, and H&I weye purchased from Bethesda Research Labs., and KpI and SaJI from Takara Shuzo Company. Labeling of EcuRI-cleaved SPV DNA at 5’-termini with [Y-~~PJATP and T4 po!ynur?eotide kinsse was performed by the method of Maxam and Gilbert (1980). The cleavage sites of restriction endonucleases (Fig. 1) were determined by partial digestion of the two subfragments with one 92P-labeled terminus, generated by San digestion from the EcoRI-cleaved, 3”P-labeled SPV DNA. DNA fragments were separated on l.O-1.4% agarose or 5% polyacrylamide gels. Electrophoresis was carried out at 50 V for 6-24 hr. The buffer
90
SUGAWARA
ET AL. EcoRl I
EcoRl Sal1
I I
’ I ,
Hpal BamHl Hindlll
1
Hpall
1 I 0
I I
I
i I 1
Hincll
I I
I I
I I
, I
I I
I I ,I I,
I I
I I
I
25
50 MAP
75
I I, I, ,I ,I
Hi+ 8 100
UNITS
FIG. 1. Cleavage map of SPV DNA. A unique EcuRI site was taken as O/100 map units. The restriction endonuclease KpnI has no cleavage site on SPV DNA.
contained 0.036 M Tris, 0.032 &f KHzPO,, and 0.001 M EDTA, pH 7.8, EcoRI digests and Hind111 digests of adenovirus 2 DNA served as size markers. Isolation of ceU DNA. Total cell DNA was isolated as described by Wold et al (1978). The minced tissue was lysed in 0.1 M Tris-HCI (pH 7.9), 0.1 M NaCI, 0.01 M EDTA, and 0.5% SDS and digested with 0.2 mg/ml protease K(Sigma Chemical Co.) for lo-12 hr at 37”. The lysate was extracted with an equal volume of phenol and a half volume of chloroform-isoamylalcohol. After dialysis, the DNA solution was treated with 50 pg/ml of RNase A for 2 hr at 37”, then with 100 pg/ml protease K for 3 hr and extracted with phenol and chloroform-isoamylalcohol. Nick-translation of viral DNA and reassociation kinetics. SPV DNA was =P-labeled by nick-translation, as described by Sawada et al (1979). Reassociation was performed in 0.4 M sodium phosphate buffer (pH 6.8) at 67”. Aliquots, 20 ~1, of the reaction mixture were sealed in glass capillaries and denatured by heating in boiling water for 10 min and then cooling in ice water. Batchwise elution from hydroxyapatite was used for the separation of single- and double-stranded DNA(Fujinaga et d, 1975). Blotting and hybridization procedure. DNA, 2-10 pg, extracted from tumors was digested with 2-10 units of endonucleases for 4-6 hr. DNA fragments were resolved by electrophoresis on 0.9% agarose slab gels, denatured in situ, and transferred to a nitrocellulose filter (type HAWP, Millipore Corp.) by modification of the tech-
nique of Southern (1975), as previously described (Fujinaga et al, 1979). The filter containing DNA was hybridized with qlabeled SPV DNA in solution containing 0.6 M NaCl, 0.2 M Tris-HCl(pH 7.9), 0.02 M EDTA, 0.5% SDS, and 50% formamide for 40 hr at 37”, washed, and exposed to Kodak RPX-Omat film using intensifying screens. The size of the large viral DNA molecules was determined by the blotting of 0.7% agarose gels. Lambda phage DNA and adenovirus 2 DNA were used as size markers. RESULTS
f&anti& of viral DNA in the transpluntable carc&onms and pap&mus. For the quantitation of viral DNA, total DNAs from four Vx2 and four Vx7 tumors, four and two papillomas of domestic and cottontail rabbits, and three normal tissues, all which were obtained from individual rabbits, were analyzed by reassociation kinetics, using =P-labeled SPV DNA. The reassociation rate(Cds/Css) was plotted against the Cot, and the amount of viral DNA was determined from the slope of the line, as shown in Fig. 2. The results are summarized in Table 1. The Vx2 and Vx7 tumors contained 15-19 and lo-22 copies of the genome per diploid cell DNA equivalent, respectively. The amounts of viral DNA in the four domestic rabbit papillomas varied considerably, but were significantly higher than those of the transplantable carcinomas. The amounts of viral DNA in the papillomas of cottontail rabbits were 10-100 times higher than those of domestic rabbits.
Vx2 AND
91
Vx7 CARCINOMAS
same DNA was digested with EcoRI which cleave circular SPV DNA at a single site, most of these sequences corn&rated with the form III SPV DNA of about 7.7 kb, but three weak DNA bands of 21.5, 15.4, and 10.8 kb and a faint band of 6.4 kb were also detected. Except for the 15.4-kb band, the other two weak and one faint bands did not comigrate with the form III SPV DNA or any of its oligomers. Complete digestion of the DNA with BamHI resulted in two major and three minor bands hybridizable with %P-labeled SPV DNA. The two major DNA bands coincided with the subfragTABLE FIG. 2. Kinetics of reassociation of .--labeled SPV DNA in the presence of total cell DNA extracted from benign and malignant tumors. The reaction mixture contained 15.1 rig/ml =P-labeled SPV DNA (3.8 X lo7 cpm/pg) and the renaturation rate was determined in the presence of 1.66 mg/ml calf thymus DNA (0); 1.63 mg/ml Vx2 DNA (0); 1.66 mg/ml Vx’7 DNA (A); 0.66 mg/ml domestic rabbit papilloma DNA (A); 0.14 mg/ml cottontail rabbit papilloma DNA (0). In all the cases, final DNA concentration was adjusted to 1.66 mg/ml with calf thymus DNA. Cds/css is the molar ratio of single- to double-stranded, labeled viral DNA and Cot is the product of the concentration of total labeled viral DNA and the reaction time (Sawada et ul., 1979).
Physical state of viral DNA in the transplantable carciw. To investigate the organization of viral DNA in the Vx2 and Vx’7 carcinomas, total DNAs extracted from Vx2 and Vx7 tumors were digested with several restriction endonucleases, electrophoresed on agarose gels, and transferred to nitrocellulose filters by the method of Southern (1975). The viral sequences were detected by hybridization with q-labeled SPV DNA. One typical example of these experiments is shown in Fig. 3. When DNA from a Vx2 tumor was completely digested with KpnI, which does not cleave SPV DNA, viral sequences were observed as a single DNA band of about 70,000 base pairs (70 kb). A faint band which moved slightly faster than the form III SPV DNA was also detected. When the
1
THE NUMBER OF SPV DNA COPIES PRESENT IN THE TRANSPLANTABLE CARCINOMAS Vx2 AND Vx? AND IN PAPILLOMAS INDUCED IN DOMESTIC AND COTTONTAIL RABBITS
Tissue Vx2 carcinoma Sob Sl Sll Nl Vx7 carcinoma s2 s3 s9 SlO Papilloma, domestic s4 S6 N3 N6 Papilloma, cottontail s5 S7 Normal kidney domestic cottontail
Number of viral DNA copies per diploid cell”
15.4 18.9 17.5 17.8 22.4 10.5 10.3 16.4 189 348 45.7 58.7 1200 7641
’ The number of viral DNA copies per diploid cell was calculated from the Cot% values obtained from Fig. 2 and the same plots for others. The amount of DNA per diploid rabbit cell was assumed to be 6.5 pg and the molecular weight of viral DNA was assumed to be 5 X lo6 Da. ’ Each tissue was obtained from an individual rabbit.
92
SIJGAWARA
ET AL.
but at least two minor bands which did not coincide with any incomplete digestion fragments were also detected. The other three DNA preparations from Vx2 tumors were analyzed with EcoRI as well. The results were the same as shown in Fig. 3. DNAs extracted from four Vx’7 tumors were analyzed by the same method. The results were analogous to those of Vx2 tumors, as shown in Fig. 4. However, there were differences. A band which would correspond to the faint band generated by KpnI from the Vx2 DNA was not evident.
Kbp
-50 -20.5
-7.7
-4.3
-2.8 FIG. 3. Analysis of the SPV DNA sequences present in a Vx2 tumor DNA. Total DNA (10 pg) was digested with the restriction endonucleases, and the fragments were separated by electrophoresis on a 0.9% agarose gel and transferred to a nitrocellulosc filter by the technique of Southern (1975). Virus-specific sequences were identified by hybridization with q-labeled SPV DNA. On the extreme left lane, viral DNA (2 X lo-’ pg in KB cell DNA solution) treated with KpnI was analyzed. H&II E-fragment is too small to be detectable clearly.
ments of circular SPV DNA and one of the minor bands, the fastest moving one had a similar mobility with the form III SPV DNA. However, two other minor bands were much larger than the subfragments. When the Vx2 DNA was digested with HincII, most of the viral sequences comigrated with the five subfragments (A-E),
-1.8
FIG. 4. Analysis of the SPV DNA sequences present in a Vx7 tumor. Total DNA (10 rg) was digested and analyzed, as described in Fig. 3. The left two lanes represent the viral DNA (3 X 10m4M:) treated with KpaI and EcoRI, respectively.
Vx2 AND
Vx’7 CARCINOMAS
Instead, a faint band slightly larger than the BamHI B-subfragment was sometimes apparent with BamHI digestion.
93
ferent mobilities. When the three minor bands yielded by BamHI were compared, the larger two bands comigrated, respecCompation of restricts fragments be- tively, but not the smallest bands, as shown tween Vx2 and Vx7 c4xrcinomu.s. Since some in Fig. 5. This difference is also obvious in fragments detected in the Vx2 and Vx7 Figs. 3 and 4 (lanes designated as BamHI). DNA digests seemed to have the same Methyl&ion of viral DNA in the transeleetrophoretic mobilities, both DNA di- plantable carcinoma. 9, primary mrci-, gests were coelectrophoresed. As seen in and domxxtic rabbit papiUumas. To compare Fig. 5, the two thick bands yielded by Km1 the extent of methylation of the viral DNA revealed distinct mobilities. Among the present in the three types of tumors, a set three weak bands yielded by EcoRI from of the restriction endonucleases, MspI, each tumor DNA, two bands, the largest HpaII, and SalI was used. MspI and HpuII and the smallest, comigrated, respectively, are isoschizomers, and both cleave SPV but the middle ones revealed slightly dif- DNA at least at 15 sites (see Fig. 1). MspI Kpn I
FIG. 5. Coelectrophoresis digested with KpnI, EcoRI, and analyzed as described arrows indicate two species that do not comigrate.
EcoRl
a
of the Vx2 and Vx’7 DNA digests. DNAs (10 pg) from both tumors were or BarnHI, coelectrophoresed for 10 hr at 50 V in a 0.9% agarose gel. in Fig. 3. (a) KpnI and EcoRI digests; (b) BumHI digests. The larger of the comigrating minor bands and the shorter ones the minor bands
94
SUGAWARA
cleaves both unmethylated and methylated DNA sequences -CC*GG-, but HpuII does not cleave the methylated sequence (Waalwijk and Flavell, 1978a). SuJI cleaves SPV DNA at a unique site (see Fig. 1) but does not cleave the recognition sequence containing methylated internal cytosine. DNAs extracted from a Vx2 and a Vx7 tumor, two primary carcinomas and two papillomas as well as virus DNA from virions were digested with either MspI or HpaII. The cleaved DNA was separated on an agarose gel, transferred to a nitrocellulose filter and hybridized to q-labeled SPV DNA. When the DNAs from seven different sources were digested with MspI,
ET AL.
in all the cases, viral DNA was completely digested to the subfragments (lanes A, C, E, G, and I in Fig. 6 and lanes A, C, and E in Fig. 7). In each of the Vx2 and Vx7 DNA digests, two or three weak off-size bands were detected, but these are not incomplete digestion bands and probably originated from the joint sequences between viral and cellular DNA. HpaJ.1 digested virion DNA almost completely but sometimes there remained a single, partially digested band with a faint density (lane B in Fig. 6), indicating that one of the cleavage sites might be methylated in a small fraction of virion DNA. In one papilloma, more than 50% of the viral DNA
ABCDEF
., ; y’ ‘; 1, 1,; .-~r-r-r,y,~“.‘,‘-P~~~‘” I .‘ L ; ,’.: t ,’ ‘,. .I-.:‘i’ ,,,,‘- .,r.,,,-: .;: ,‘, . ‘,.,’ ,, ,. ‘,‘;;‘.’ ,’ -.,’ I’ ,’ : ,, iI,-, :,’ 1: : ,’ ,.q I,:‘, I,;:.,: ‘,,“I :, I- .’ I,, ‘,’ ,/1 /, I /:F1, i :) ,y.-, ,1.& 1 ‘,‘I, ,
G Kbp
H
I
J
‘; ,;.; (:. -’ ,, ,; ‘-I’, ! ,. ,I,, L -,, ‘, -f .,,-, “, i ,. ,‘, ‘.; ,‘, ,; -I,‘. ‘- ‘, ‘.
FIG. 6. Analysis of the DNA from purified virions, two papillomas, and two primary carcinomas using the restriction endonucleases MspI and H&I. 10 a of cell DNA or 4 X lo-’ pg of virion DNA was cleaved with the MspI (lanes A, C, E, G, and I) or HpaII (lanes B, D, F, H, and J). The fragments were separated by electrophoresis on a 0.9% agarose gel and transferred to a nitrocellulose filter. Virus-specific sequences were detected by hybridization with 8ZP-labeled SPV DNA. Lanes A and B, virion DNA; lanes C-F, papilloma DNAs (the same DNA was analyzed in the lanes C and D, and E and F); lanes G-J, primary carcinoma DNAs (the same DNA was analyzed in the lanes G and H, and I and J).
Vx2 AND
95
Vx7 CARCINOMAS
ylated -CCGG- sequence per genomelength DNA which contain at least 15 -CCGG- sequences. This unmethylated site may be distributed uniformly on these -CCGG- sequences, because only one band, corresponding to the HpII E-subfragment (see Fig. l), is visible in the HpaII digests of both DNAs (lanes D and F in Fig. 7). Digestion of the Vx2 and Vx7 DNA with Sa,+?Iresulted in no change in the mobilities of the virus-specific sequences as compared with the undigested DNA (data not shown). This indicates that in these tumors, almost all of the SalI sites present on the viral DNA are methylated.
ABCDEF
DISCUSSION
FIG. 7. Analysis of the DNA from a Vx2 and a Vx7 tumor using MspI and HpdI. 10 ~g of cell DNA or 2 X lo-’ gg of virion DNA was cleaved with the MspI (lanes A, C and E) or HpaII (lanes ES,D and F). Lanes A and B, SPV DNA; lanes C and D, Vx2 DNA; lanes E and F, Vx7 DNA.
sequences were completely
digested
with
HpaII but in another papilloma, more than 50% of the viral sequences remained incompletely digested (lanes D and F in Fig. 6). In one primary carcinoma, approximately 50% of the viral sequences were incomplete when digested with HpaII, but in another primary carcinoma, most viral sequences remained incompletely digested (lanes H and J in Fig. 6). When the Vx2 and Vx7 DNAs were digested with HpuII, most viral sequences remained incompletely digested, and the partially digested fragments came together around the position of unit-length viral DNA. This result indicates that the high-molecular-weight viral DNA present in the transplantable carcinomas contains roughly one unmeth-
The persistence of viral genetic information in the transplantable carcinoma Vx2 and Vx7, and domestic rabbit papillomas has been demonstrated in several earlier studies (Noyes and Mellors, 1957; Rogers et d, 1960; Mellors, 1960; Ito and Evans, 1961; Osato and Ito, 1967; Ito and Evans, 196.5;Orth et a& 1978). The presence of multiple viral genomes was also reported (Stevens and Wettstein, 1979; McVay et al, 1982; Favre et d, 1982). The results of our
analysis of these tumors with reassociation kinetics are in good agreement with these earlier and recent findings; however, there were minor differences. In the Vx2 and Vx7 carcinomas, viral DNA is present in high-molecular-weight forms, and almost all fractions of this DNA are digested by EcoRI, which cleaves SPV DNA at a single site, into linear genomesize DNA molecules (McVay et aL, 1982; Favre et d, 1982). This indicates that most fractions of this DNA consist of oligomeric forms of the unit-length viral DNA molecules. The results of our analysis with .the Southern blotting are consistent with this conclusion McVay et al. (1982) reported that in the cell line derived from the Vx7 carcinoma, five weak bands which do not correspond to the form III SPV DNA were also detected by EcoRI digestion. Since four contained only part of the viral genome, they concluded that at least the four are viral
96
SUGAWARA
segments covalently linked with cellular sequences. The four weak bands we found here in the EcoRI-restricted Vx’7 DNA are probably identical with those reported by McVay et a& although we did not detect a band corresponding to the fastest-moving one. Four similar bands were identified also in the Vx2 DNA digests and at least two (21.5- and 10.8-kb bands) revealed identical mobilities to those from the Vxi’ DNA and, therefore, to the two integration bands reported by McVay et al Two off-size species of virus-specific bands common to both Vx2 and Vx’7 tumors were also identified by BamHI and Hind1 digestion. One of the five weak bands detected by McVay et al. (1982) had a similar mobility to the dimer of linear genome-length viral DNA and contained all or almost all sequences of the viral genome. We also found a similar band in the Vx7 DSA digests, :but it revealed a slightly different mobility from the analogous band detected in the Vx2 DNA digests. Thus, it does not seem to be a simple dimer resulting from incomplete digestion, but may be a viral segment linked to a cellular sequence, or may originate from oligomeric viral DNA carrying a mutation or a small deletion at the EcoRI sites. All these results taken together indicate that in both Vx2 and Vx7 carcinomas, at least some fraction of viral DNA is integrated into the cell DNA. Since the viral DNA present, in the Vxi’ cell line moved together with cellular DNA in agarose gels, McVay et al. (1982) suggested that all the viral DNA sequences are integrated as tandem head-to-tail repeats of unit-length molecules. In the present study, the digestion of Vx2 and Vx7 DNA with KpnI, which does not cleave the SPV DNA, resulted in a single virusspecific DNA band of about 70 kb. If the viral sequences in this band are linear, these should be tandem ,head-to-tail repeats consisti,ng,of 9 to lO&nit-length viral DNA molecules. This is compatible with the present results and those obtained by McVay et al. (1982) that about 20 viral genomes per diploid cell DNA equivalent are present. We suggested here one or two viral DNA integration sites’ for both trans-
ET AL.
plantable tumors and McVay et al suggested two or three integration sites for the Vx7 cell line. The integration sites might be three, but major ones, where multiple viral genomes in oligomeric forms are integrated, are probably two, and therefore, the 70-kb viral band identified by KpI may contain two species of viral DNA fragments with similar electrophoretie mobilities. Favre et al. (1982) reported different results. They detected none of the integration bands but instead, found circular viral DNA molecules having a deletion of about 10% of the genome length. Since these deleted molecules were detectable only after EcoRI digestion, they suggested that the viral DNA are present as free catenated oligomers. This discrepancy may be due to the different maintenance carriers of tumors. The Vx7 carcinoma analyzed by Mcvay et ah, who reported almost identical results to ours, originated from the tumors recently sent from our laboratory; but the Vx2 and Vx7 carcinomas analyzed by Favre et al. had been independently maintained from ours. perhaps for more than 20 years (Ito et al, 1968; Favre et al, 1982). During this period, partial deletion or rearrangement of viral DNA, probably followed by cell selection, may have occurred in these tumors, depending on the passage number and individual variation of rabbits used for the maintenance. This explanation can be supported by the following: (1) At least Vx7 carcinomas we and Favre et al analyzed are extremely different in their passage numbers. They analyzed the tumors before the 200th passage but the tumors we used were those after the 350th passage. (2) From these tumors, at least free monomer-size viral DNA molecules would have been eliminated during the maintenance. There are data indicating that the Vx2 carcinoma produced virus particles until at least the 22nd transplant (Rous et aL, 1952) and the Vx7 carcinoma until recently (Mellors, 1960; Ito and Evans, 1965; Orth et aL, 1978). Whereas, McVay et al. (1982), Favre et al. (1982) and we, failed to detect free monomer-size viral DNA molecules, in both tumors;
97
Vx2 AND Vx'7 CARCINOMAS Another explanation of the discrepancy is that our Vx7 carcinoma was accidently replaced by the Vx2 carcinoma during the maintenance. While several reports have indicated that the Vx2 and Vx7 carcinomas differ in their ability to produce virus and viral antigens, and in the presence or absence of extractable infectious viral DNA (Rous et ah, 1952; Mellors, 1960; Ito and Evans, 1965; Osato and Ito, 1967), we found no significant difference in the quantities and physical states of viral DNA between both carcinomas. The result obtained by Favre et al. that the Vx7 tumor contains much higher numbers of viral genomes then we detected, seems more compatible with the data previously reported. However, it seems improbable that the Vx7 tumors we analyzed were Vx2 carcinomas, because: (1) The Vx2 and Vx7 tumors are not identical. The analysis with the Southern blotting revealed several minor differences in the electrophoretic patterns. (2) If the discrepancy is the consequence of a simple error of maintenance, our results and those obtained by Favre et al. (1982) on the Vx2 carcinoma should be the same, but they also found no integration band in their Vx2 tumors. (3) According to our recent pathological examination, our Vx2 and Vx7 carcinomas seem to conserve the original distinguishable features previously reported (Ito et al, 1968). (4) If the intcgration of viral DNA into a specific cellular DNA site has a role in keeping cells malignant or enhances cell growth, a certain cell type which carries inserted viral DNA at a specific site of cell DNA can be selected in tumors of different origins, as suggested recently by the studies of avian leukosis virus (Hayward et aL, 1981; Payne et uL, 1982). Two species of viral transcripts have been identified in both papillomas and carcinomas (Nasseri et uZ., 1982). While 2.0kb species are more common in benign tumors, in malignant tumors there is a larger amount of 1.3-kb species. We have shown here that the extent of methylation of viral DNA in primary and the transplantable carcinomas is higher than that in papillomas. Several recent studies suggested
that gene expression is inversely correlated with the level of DNA methylation (Waalwijk and Flavell, 1978b; Bird & aL, 1979; McGhee and Ginder, 1979; Cohen, 1980; Guntaka et al, 1980; Sutter and Doerfler, 1980). Therefore, the increased amount of 1.3-kb species in carcinomas might be due to the elevated level of viral DNA methylation. However, it is not known whether such alteration correlates with the development of malignant tumors. The present analysis with MspI and HpuII also indicates that the viral DNA in the Vx2 and Vx7 carcinomas is more extensively methylated than in the primary carcinomas. This could be a consequence of a long history of the maintenance, or otherwise, could relate to the integration of whole viral DNA into the cell DNA, because as evidenced in other viruscell systems, integrated viral DNA is extensively methylated (Cohen, 1980; Guntaka et al, 1980; Sutter and Doerfler, 1980), and, in primary carcinomas, all or almost all fractions of the viral DNA are present in episomal forms (Wettstein and Stevens, 1982; Sugawara et al, unpublished observation). ACKNOWLEDGMENTS We thank Drs. Yasuyuki Takagi and Motoaki Anai for pertinent advice and provision of a bacterial strain and M. Ohara for useful comments on the manuscript. This investigation was supported in part by grants from the Ministry of Education, Science, and Gulture, Japan.
REFERENCES ANAI, M., HIHAHASHI, T., and TAKAGI, Y. (19’7Oj. A deoxyribonuclease which requires nucleoside triphosphate from Mtimnxxms l?~ltodeih&~~ I. Purification and characterization of the deoxyribonuclease activity. J. Bid Chem. 245, 76’7-774. BIRD, A. P., TAGGART, M. H., and SMITH, B. A. (lS79). Methylated and unmethylated DNA compartments in the sea urchin genome. CoU 17, 339-901. COHEN, J. C. (1980). Methylation of milk-borne and genetically transmitted mouse mammary tumor virus proviral DNA. CeU 19.653-662. FAVRE, M., JUURII, N., and ORTII, G. (1982). Restriction mapping and physical characterization of the cottontail rahbit papillomavirus genome in trans-
98
SUGAWARA
plantable Vx2 and Vx7 domestic rabbit carcinomas. Virology 119.298-309. FUJINAGA, K., SAWADA, Y., and SEKIKAWA, K. (1979). Three different classes of human adenovirus transforming DNA sequences; highly oncogenic subgroup A-, weakly oncogenic subgroup B- and subgroup Cspecific transforming DNA sequences. Virology 93. 578-581. F’UJINAGA, K., SEKIKAWA, K., and YAMAZAKI, H. (1975). Method for determination of nucleotide sequence homology between viral genomes by DNA reaasociation kinetics. .Z. Viral. 15, 466-470. GREENE, P. J., BETLACH, M. C., GOODMAN, H. M., and BOYER, H. W. (1974). The EcoRI restriction endonuciease. In “Methods in Molecular Biology” (R. Wecker, ed.), Vol. ‘7, pp. 8’7-111. Dekker, New York. GUNTAKA, R. V., RAO, P. Y., MITSIALIS, S. A., and KATZ, R. (1980). Modification of avian sarcoma proviral DNA sequences in monpermissive XC cells but not in permissive chicken cells. J. V&Z 34,569572. HAYWARD, W. S., NEEL, B. G., and ASTRIN, S. M. (1981). Activation of a cellular one gene by promoter insertion in ALV-induced lymphoid leukosis. Nature (Zmdm) 290,475-480. ITO, Y. (1963). Studies on subviral tumorigenesis: Carcinoma derived from nucleic acid-induced papillomas of rabbit skin. Acta Unti. Znt. Contra Canerum 19.280-283. ITo, Y. (1975). Papilloma-myxoma viruses. In “Cancer” (E. Becker, ed.), Vol. 2, pp. 323340. Plenum, New York. ITO, Y., and EVANS, C. A. (1961). Induction of tumors in domestic rabbits with nucleic acid preparations from partially purified Shape papilioma virus and from extracts of the papiilomas of domestic and cottontail rabbits. .Z. Exp. Med 114, 485-500. ITO, Y., and EVANS, C. A. (1965). Tumorigenic nucleic acid extracts from tissues of a transplantable carcinoma, Vx7. J. Nnt Cuncer Inst. 34, 431-437. ITO, Y., KIYURA, I., and MIYAKE, T. (1968). Comparative studies on pathogenesis of two strains of transplantable carcinomas of rabbits, Vx2 and Vx7. Proc Sot Exp Bid Med 127, 1106-1111. KIDD, J. G.. and Rous, P. (1940). A transplantable rabbit carcinoma originating in a virus-induced papilloma and containing the virus in masked or altered form. J. Exp Med 71, 813-838. KREIDER, J. W., and BARTLE?T, G. (1981). Tbe Shope papilloma-carcinoma complex of rabbits: A model system of neoplastie progression and spontaneous regression. Adv. Cancer Z&s. 35, 81-110. MAXAM, A. M., and GILBERT, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 65, pp. 499-560. Academic Press, New York.
ET AL. MCGHEE, J. D., and GINDER, G. D. (1979). Specific DNA methylation sites in the vicinity of the chicken 6-globin gene. Nature (Londa) 280.419-420. MCVAY, P., FRETZ, M., WETTSTEIN, F., STEVENS, J., and ITO, Y. (1982). Integrated Shope virus DNA is present and transcribed in the transplantable rabbit tumour Vx7. .Z. Ga Vilpl 60,2’S278. MELLORS, R. C. (1966). Tumor cell localization of the antigens of the Shope papilioma virus and the Rous sarcoma virus. Cancer Rtx 20. 744-746. MUKAI, T., MATSUBARA, K., and TAKAGI, Y. (1973). Isolation of circular DNA molecules from whole cellular DNA by use of ATP-dependent deoxyribonuclease. Pm Nat Auw! USA 70.2884-2887. NASSERI, M., W~TTSTEIN, F. O., and STEVENS, J. G. (1982). Two colinear and spliced viral transcripts are present in non-virus-producing benign and malignant neoplasm8 induced by the Shope (rabbit) papilloma virus. .Z. fir& 44,263-268. NOYES, W. F., and MELLORS, R. C. (1957). Fluorescent antibody detection of the antigens of the Shope papilloma virus in papillomas of the wild and domestic rabbit. J. Exp. Med 106, 555-562. ORTH, G., BREITBURD, F., and FAVRE, M. (1978). Evidence for antigenic determinants shared by the structural polypeptides of (Shope) rabbit papiilomavirus and human papillomavirus type 1. virology 91.243-255. OSATO, T., and ITO, Y. (1967). In vitro cultivation and immunofluorescent studies of transplantable carcinomas Vx2 and Vx7. J. Exp. Med 126,881~886. PAYNE, G. S., BISHOP, J. M., and VARMIJS, H. E. (1982). Multiple arrangements of viral DNA and an activated host oncogene in bursal lymphomas. Nature (Zmdon) 295,209-214. ROGERS, S., KLDD, J. G., and Rows, P. (1960). Relationship of the Shope papilloma virus to the cancer it determines in domestic rabbits. Acta Unio. Zti C?oniru Canmum 16, 129-130. Rows, P., KIDD, J. G., and SMITH, W. E. (1952). Experiments on the cause of the rabbit carcinomas derived from virus-induced papiilomaa. II. Loss by the Vx2 carcinoma of the power to immunize hosts against the papilloma virus. .Z. Exp. Meo! 96, 159174. SAW~A, Y., OJIMA, S., SHIMOJO, H., SHIROKI, K., and FUJINAGA, K. (1979). Transforming DNA sequences in rat cells transformed by DNA fragments of highly oncogenic human adenovirus type 12. .Z. VimL 32, 379-385. SOUTHERN.E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Md Bid 98,503-517. SEVENS. J. G., and WETT~TEIN, F. 0. (1979). Multiple copies of Shope virus DNA are present in cells of benign and malignant non-virus-producing neoplasms. .Z. ViroL 30, 891-898.
Vx2 AND
Vx’7 CARCINOMAS
SUMTER, D., and DOERFLER, W. (1980). Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression. Proc. Nat Acud Ski. 77, 253-256. TAKANAMI, M. (1974). Restriction endonucleases AP, GA, and H-I from three Haemophilus strains. In “Methods in Molecular Biology” (R. Wecker, ed.), Vol. 7, pp. 113-133. Dekker, New York. WAALWLIK, C., and FLAVEL.L, R. A. (1978a). MspI, an isoschizomer of ZZpaII which cleaves both unmethylated and methylated ZZpuII sites. Nucleic Acid Rex 5, 3231-3236. WAALWIJK, C., and FLAVELL, R. A. (1978b). DNA methylation at a CCGG sequence in the large intron of the rabbit @-globin gene: Tissue-specific variations. NucZeic Acid Rev. 5,4631-4641.
99
WETISTEIN, F. O., and STEVENS, J. G. (1981). Transcription of the viral genome in papillomas and carcinomas induced by the Shope virus. Virdogy 109,448451. WFITSTEIN, F. O., and STEVENS, J. G. (1982). Variable sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomea are detected in some. Proc. Nat. Acad Sci USA 79.790-794. WOLD, W. S. M., GREEN, M., and MACKEY, J. K. (1978). Methods and rationale for analysis of human tumors for nucleic acid sequences of oncogenic human DNA viruses. In “Methods in Cancer Research” (H. Busch, ed.), Vol. 15, pp. 70-161. Academic Press, New York.
VIROLOGY
Integration and Methylation of Shope Papilloma Virus DNA in the Transplantable Vx2 and Vx7 Rabbit Carcinomas KENJI
SUGAWARA,**’
KEI FUJINAGA,? TOSHIHARU AND YOHEI ITO*
YAMASHITA,?
‘Department of Microbi&cgy, Faculty of Medicine, Kyoto Univers-ity, Kyoto 606, and iCancer Research Institute, Sapporo Medical College, Suppuro 060, Japan Received May 20, 19X.9;accepted July 27, 1983 The Shope papilloma virus (SPV) DNA present in SPV-induced benign and malignant rabbit tumors, particularly in the transplantable carcinoma Vx2 and Vx7, was examined with regard to physical states and extent of methylation. Vx2 and Vx7 carcinomas contained 10-22 viral genomes per diploid cell, and domestic and cottontail rabbit papillomas 40460 and 1000-8000, respectively. The digestion of Vx2 and Vx7 DNA with the restriction enzyme KpnI, which does not cleave SPV DNA, yielded a single virus-specific DNA band about nine t.imes larger than the genome length, but EcoRI, which cuts the circular SPV DNA once, cleaved this DNA to the genome-size fragments. However, three or four weak bands which may contain viral segments linked to cellular sequences were also identified, and at least two were shared by both Vx2 and Vx7 carcinomas. The analysis with a set of MapI and HpoII, which discriminates the methylated DNA sequence -CC*GG-, showed that lo-40% of the sites of viral DNA are methylated in papillomas, 30-802 in primary carcinomas, and more than 90% in the transplantable carcinomas.
(Stevens and Wettstein, 1979). However, in papillomas, most of the viral DNA fraction is present in the form of episomes with the normal genome-length, while in primary and metastatic carcinomas, a considerable fraction is found in free oligomers consisting of several genome-length viral DNA molecules (Wettstein and Stevens, 1982; Sugawara et uL, unpublished observation). The transplantable carcinomas Vx2 and Vx7 were established from carcinomas induced by SPV in domestic rabbits more than 40 and 30 years ago, respectively (Kidd and ROUS, 1940; Rogers et ab, 1960), and have been used as the only available strains representing SPV-induced carcinomas. In these tumors, free genome-size viral DNA molecules do not exist, but multiple viral genomes are present in highmolecular-weight forms (McVay et d, 1982; Favre et aZ., 1982). McVay et al. (1982) have shown that the Vx7 carcinoma and the cell line derived from it contain lo-30 viral genomes per diploid cell and suggested that these viral DNA sequences are integrated into the host cell DNA. However, Favre et
INTRODUCTION
Shope papilloma virus (SPV) isolated from papillomas of wild cottontail rabbits also induces papillomas in domestic rabbits. When the rabbits carrying papillomas are maintained over 6 months, epidermoid carcinomas frequently develop at the same sites (for reviews, see Ito, 1975; Kreider and Bartlett, 1981). Although infectious virus is rarely recovered from the papillomas and carcinomas, viral genetic information is conserved in both types of tumors (Noyes and Mellors, 1957; Mellors, 1960; Ito and Evans, 1961; Ito, 1963; Ito and Evans, 1965; Osato and Ito, 1967), suggesting that this virus might play some role in the ensuing malignant conversion. Recent work has demonstrated that all these tumors contain multiple viral genomes (lo-400 copies per diploid cell), but quantitative differences between benign and malignant tumors were not apparent
’ Author addressed.
to whom requests for reprints
0042-6822/83 $3.00 Copyright All riyhts
G 19X3 hy Academic Press. Inc. of rcprociuction in an)’ form renewed.
should be
88
Vx2 AND
Vx7 CARCINOMAS
al. (1982) reported that 444 viral DNA copies are present in the Vx’Z carcinoma and suggested that these viral sequences as well as those in the Vx2 carcinoma, which contain 42 viral DNA copies, are not integrated. In benign and malignant tumors, viral transcripts complementary to about 10% of the genome are found in a low quantity (Wettstein and Stevens, 1981; McVay et al.. 1982). Recently, two species of 1.3 and 2.0 kilobases (kb) were identified (Nasseri et a.Z.,1982). Although no qualitative diffetence was detected between both types, increased amounts of 1.3-kb species were detected in carcinomas. Although little is known of the mechanisms of malignant eonversion of SPV-induced papillomas, attempts to detect the distinctive features between benign and malignant tumors have to be made. In the present study, mainly, Vx2 and Vx7 carcinomas but, for comparison, some SPV.indueed papjllomas and primary carcinomas of domestic and cottontail rabbits were analyzed for the physical state and the extent of methylation of persisting viral DNA. The results suggested the viral DNA integration in the Vx2 and Vx7 carcinomas. Data on the existence of a common viral DNA integration site between Vx2 and Qx7 carcinomas and extensive methylation of the integrated viral DNA sequences are also presented. UATERIALS
AND
METHODS
Virus and unimak The SPV originated from papillomas of wild cottontail rabbits trapped in Kansas. The glycerinated material was a generous gift from Dr. C. A. Evans. Eastern cottontail rabbits were brought to Japan and propagated on a small island. Tumors. Initial source of the Vx2 and Vx7 carcinomas has been described previously (Ito et al, 1968). These tumors were maintained by serial transfers through domestic rabbits. Twenty percent suspension (0.5 ml) of packed tumor cells was intramuscularly inoculated once every 4050 days. At the beginning of 1980, the passage numbers exceeded ??.A0 for the Vx2 and
89
350 for the Vx7 carcinoma. Tumor tissues collected during 1980 to 1982 were used for the experiments. Papillomas and primary carcinomas of domestic and cottontail rabbits were obtained by inoculating 10% tissue extracts of the original stock into the scarified skin of respective rabbits. Preparation of viral DN-4. SPV DNA was isolated from cottontail rabbit papillomas prepared in the laboratory. The tissues were cut and ground with sand in a mortar. Vjrus particles were extracted with 0.02 M Tris-HCl (pH 7.5) buffer and pelleted by centrifugation at 29,000 rpm for 1 hr. Virus DNA was released by incubation with 0.5% SDS and 0.5 my/ml Pronase E (Kaken Kagaku Co., predigested) and extracted with phenol and chloroform-isoamylalcohol (24:1). After treatment with 50 rg/ ml of RNase A (Sigma Chemical Co., preheated at 90” for 10 min), the DNA solution was prepared to contain 0.5 g/ml ethidiurn bromide and CsCl (density = 1.587 g/ml) and was centrjfuged at 3’1,000 rpm for 72 hr. Supercoiled (form I) SPV DNA was collected. For further purification, the viral DN,A was treated with ATP-dependent DNase to eliminate linear DNA molecules (Mukai et ~1.. 1973). ATP-dependent DNase was prepared from Micrococ~us luteus, as described by Anai & al. (:970).
Restrkkn en&mu&eases and physzmt mapping ajr the cleavage s&s. The restriction endonucleases EcaU and Hind111 were prepared from E. cxdi RY13 (Greene et al, 1974) and Haemophilus in$uenzae Rd (Takanami, 1974), respectively. HpaI, BarnHI, &XII, IwspI, and H&I weye purchased from Bethesda Research Labs., and KpI and SaJI from Takara Shuzo Company. Labeling of EcuRI-cleaved SPV DNA at 5’-termini with [Y-~~PJATP and T4 po!ynur?eotide kinsse was performed by the method of Maxam and Gilbert (1980). The cleavage sites of restriction endonucleases (Fig. 1) were determined by partial digestion of the two subfragments with one 92P-labeled terminus, generated by San digestion from the EcoRI-cleaved, 3”P-labeled SPV DNA. DNA fragments were separated on l.O-1.4% agarose or 5% polyacrylamide gels. Electrophoresis was carried out at 50 V for 6-24 hr. The buffer
90
SUGAWARA
ET AL. EcoRl I
EcoRl Sal1
I I
’ I ,
Hpal BamHl Hindlll
1
Hpall
1 I 0
I I
I
i I 1
Hincll
I I
I I
I I
, I
I I
I I ,I I,
I I
I I
I
25
50 MAP
75
I I, I, ,I ,I
Hi+ 8 100
UNITS
FIG. 1. Cleavage map of SPV DNA. A unique EcuRI site was taken as O/100 map units. The restriction endonuclease KpnI has no cleavage site on SPV DNA.
contained 0.036 M Tris, 0.032 &f KHzPO,, and 0.001 M EDTA, pH 7.8, EcoRI digests and Hind111 digests of adenovirus 2 DNA served as size markers. Isolation of ceU DNA. Total cell DNA was isolated as described by Wold et al (1978). The minced tissue was lysed in 0.1 M Tris-HCI (pH 7.9), 0.1 M NaCI, 0.01 M EDTA, and 0.5% SDS and digested with 0.2 mg/ml protease K(Sigma Chemical Co.) for lo-12 hr at 37”. The lysate was extracted with an equal volume of phenol and a half volume of chloroform-isoamylalcohol. After dialysis, the DNA solution was treated with 50 pg/ml of RNase A for 2 hr at 37”, then with 100 pg/ml protease K for 3 hr and extracted with phenol and chloroform-isoamylalcohol. Nick-translation of viral DNA and reassociation kinetics. SPV DNA was =P-labeled by nick-translation, as described by Sawada et al (1979). Reassociation was performed in 0.4 M sodium phosphate buffer (pH 6.8) at 67”. Aliquots, 20 ~1, of the reaction mixture were sealed in glass capillaries and denatured by heating in boiling water for 10 min and then cooling in ice water. Batchwise elution from hydroxyapatite was used for the separation of single- and double-stranded DNA(Fujinaga et d, 1975). Blotting and hybridization procedure. DNA, 2-10 pg, extracted from tumors was digested with 2-10 units of endonucleases for 4-6 hr. DNA fragments were resolved by electrophoresis on 0.9% agarose slab gels, denatured in situ, and transferred to a nitrocellulose filter (type HAWP, Millipore Corp.) by modification of the tech-
nique of Southern (1975), as previously described (Fujinaga et al, 1979). The filter containing DNA was hybridized with qlabeled SPV DNA in solution containing 0.6 M NaCl, 0.2 M Tris-HCl(pH 7.9), 0.02 M EDTA, 0.5% SDS, and 50% formamide for 40 hr at 37”, washed, and exposed to Kodak RPX-Omat film using intensifying screens. The size of the large viral DNA molecules was determined by the blotting of 0.7% agarose gels. Lambda phage DNA and adenovirus 2 DNA were used as size markers. RESULTS
f&anti& of viral DNA in the transpluntable carc&onms and pap&mus. For the quantitation of viral DNA, total DNAs from four Vx2 and four Vx7 tumors, four and two papillomas of domestic and cottontail rabbits, and three normal tissues, all which were obtained from individual rabbits, were analyzed by reassociation kinetics, using =P-labeled SPV DNA. The reassociation rate(Cds/Css) was plotted against the Cot, and the amount of viral DNA was determined from the slope of the line, as shown in Fig. 2. The results are summarized in Table 1. The Vx2 and Vx7 tumors contained 15-19 and lo-22 copies of the genome per diploid cell DNA equivalent, respectively. The amounts of viral DNA in the four domestic rabbit papillomas varied considerably, but were significantly higher than those of the transplantable carcinomas. The amounts of viral DNA in the papillomas of cottontail rabbits were 10-100 times higher than those of domestic rabbits.
Vx2 AND
91
Vx7 CARCINOMAS
same DNA was digested with EcoRI which cleave circular SPV DNA at a single site, most of these sequences corn&rated with the form III SPV DNA of about 7.7 kb, but three weak DNA bands of 21.5, 15.4, and 10.8 kb and a faint band of 6.4 kb were also detected. Except for the 15.4-kb band, the other two weak and one faint bands did not comigrate with the form III SPV DNA or any of its oligomers. Complete digestion of the DNA with BamHI resulted in two major and three minor bands hybridizable with %P-labeled SPV DNA. The two major DNA bands coincided with the subfragTABLE FIG. 2. Kinetics of reassociation of .--labeled SPV DNA in the presence of total cell DNA extracted from benign and malignant tumors. The reaction mixture contained 15.1 rig/ml =P-labeled SPV DNA (3.8 X lo7 cpm/pg) and the renaturation rate was determined in the presence of 1.66 mg/ml calf thymus DNA (0); 1.63 mg/ml Vx2 DNA (0); 1.66 mg/ml Vx’7 DNA (A); 0.66 mg/ml domestic rabbit papilloma DNA (A); 0.14 mg/ml cottontail rabbit papilloma DNA (0). In all the cases, final DNA concentration was adjusted to 1.66 mg/ml with calf thymus DNA. Cds/css is the molar ratio of single- to double-stranded, labeled viral DNA and Cot is the product of the concentration of total labeled viral DNA and the reaction time (Sawada et ul., 1979).
Physical state of viral DNA in the transplantable carciw. To investigate the organization of viral DNA in the Vx2 and Vx’7 carcinomas, total DNAs extracted from Vx2 and Vx7 tumors were digested with several restriction endonucleases, electrophoresed on agarose gels, and transferred to nitrocellulose filters by the method of Southern (1975). The viral sequences were detected by hybridization with q-labeled SPV DNA. One typical example of these experiments is shown in Fig. 3. When DNA from a Vx2 tumor was completely digested with KpnI, which does not cleave SPV DNA, viral sequences were observed as a single DNA band of about 70,000 base pairs (70 kb). A faint band which moved slightly faster than the form III SPV DNA was also detected. When the
1
THE NUMBER OF SPV DNA COPIES PRESENT IN THE TRANSPLANTABLE CARCINOMAS Vx2 AND Vx? AND IN PAPILLOMAS INDUCED IN DOMESTIC AND COTTONTAIL RABBITS
Tissue Vx2 carcinoma Sob Sl Sll Nl Vx7 carcinoma s2 s3 s9 SlO Papilloma, domestic s4 S6 N3 N6 Papilloma, cottontail s5 S7 Normal kidney domestic cottontail
Number of viral DNA copies per diploid cell”
15.4 18.9 17.5 17.8 22.4 10.5 10.3 16.4 189 348 45.7 58.7 1200 7641
’ The number of viral DNA copies per diploid cell was calculated from the Cot% values obtained from Fig. 2 and the same plots for others. The amount of DNA per diploid rabbit cell was assumed to be 6.5 pg and the molecular weight of viral DNA was assumed to be 5 X lo6 Da. ’ Each tissue was obtained from an individual rabbit.
92
SIJGAWARA
ET AL.
but at least two minor bands which did not coincide with any incomplete digestion fragments were also detected. The other three DNA preparations from Vx2 tumors were analyzed with EcoRI as well. The results were the same as shown in Fig. 3. DNAs extracted from four Vx’7 tumors were analyzed by the same method. The results were analogous to those of Vx2 tumors, as shown in Fig. 4. However, there were differences. A band which would correspond to the faint band generated by KpnI from the Vx2 DNA was not evident.
Kbp
-50 -20.5
-7.7
-4.3
-2.8 FIG. 3. Analysis of the SPV DNA sequences present in a Vx2 tumor DNA. Total DNA (10 pg) was digested with the restriction endonucleases, and the fragments were separated by electrophoresis on a 0.9% agarose gel and transferred to a nitrocellulosc filter by the technique of Southern (1975). Virus-specific sequences were identified by hybridization with q-labeled SPV DNA. On the extreme left lane, viral DNA (2 X lo-’ pg in KB cell DNA solution) treated with KpnI was analyzed. H&II E-fragment is too small to be detectable clearly.
ments of circular SPV DNA and one of the minor bands, the fastest moving one had a similar mobility with the form III SPV DNA. However, two other minor bands were much larger than the subfragments. When the Vx2 DNA was digested with HincII, most of the viral sequences comigrated with the five subfragments (A-E),
-1.8
FIG. 4. Analysis of the SPV DNA sequences present in a Vx7 tumor. Total DNA (10 rg) was digested and analyzed, as described in Fig. 3. The left two lanes represent the viral DNA (3 X 10m4M:) treated with KpaI and EcoRI, respectively.
Vx2 AND
Vx’7 CARCINOMAS
Instead, a faint band slightly larger than the BamHI B-subfragment was sometimes apparent with BamHI digestion.
93
ferent mobilities. When the three minor bands yielded by BamHI were compared, the larger two bands comigrated, respecCompation of restricts fragments be- tively, but not the smallest bands, as shown tween Vx2 and Vx7 c4xrcinomu.s. Since some in Fig. 5. This difference is also obvious in fragments detected in the Vx2 and Vx7 Figs. 3 and 4 (lanes designated as BamHI). DNA digests seemed to have the same Methyl&ion of viral DNA in the transeleetrophoretic mobilities, both DNA di- plantable carcinoma. 9, primary mrci-, gests were coelectrophoresed. As seen in and domxxtic rabbit papiUumas. To compare Fig. 5, the two thick bands yielded by Km1 the extent of methylation of the viral DNA revealed distinct mobilities. Among the present in the three types of tumors, a set three weak bands yielded by EcoRI from of the restriction endonucleases, MspI, each tumor DNA, two bands, the largest HpaII, and SalI was used. MspI and HpuII and the smallest, comigrated, respectively, are isoschizomers, and both cleave SPV but the middle ones revealed slightly dif- DNA at least at 15 sites (see Fig. 1). MspI Kpn I
FIG. 5. Coelectrophoresis digested with KpnI, EcoRI, and analyzed as described arrows indicate two species that do not comigrate.
EcoRl
a
of the Vx2 and Vx’7 DNA digests. DNAs (10 pg) from both tumors were or BarnHI, coelectrophoresed for 10 hr at 50 V in a 0.9% agarose gel. in Fig. 3. (a) KpnI and EcoRI digests; (b) BumHI digests. The larger of the comigrating minor bands and the shorter ones the minor bands
94
SUGAWARA
cleaves both unmethylated and methylated DNA sequences -CC*GG-, but HpuII does not cleave the methylated sequence (Waalwijk and Flavell, 1978a). SuJI cleaves SPV DNA at a unique site (see Fig. 1) but does not cleave the recognition sequence containing methylated internal cytosine. DNAs extracted from a Vx2 and a Vx7 tumor, two primary carcinomas and two papillomas as well as virus DNA from virions were digested with either MspI or HpaII. The cleaved DNA was separated on an agarose gel, transferred to a nitrocellulose filter and hybridized to q-labeled SPV DNA. When the DNAs from seven different sources were digested with MspI,
ET AL.
in all the cases, viral DNA was completely digested to the subfragments (lanes A, C, E, G, and I in Fig. 6 and lanes A, C, and E in Fig. 7). In each of the Vx2 and Vx7 DNA digests, two or three weak off-size bands were detected, but these are not incomplete digestion bands and probably originated from the joint sequences between viral and cellular DNA. HpaJ.1 digested virion DNA almost completely but sometimes there remained a single, partially digested band with a faint density (lane B in Fig. 6), indicating that one of the cleavage sites might be methylated in a small fraction of virion DNA. In one papilloma, more than 50% of the viral DNA
ABCDEF
., ; y’ ‘; 1, 1,; .-~r-r-r,y,~“.‘,‘-P~~~‘” I .‘ L ; ,’.: t ,’ ‘,. .I-.:‘i’ ,,,,‘- .,r.,,,-: .;: ,‘, . ‘,.,’ ,, ,. ‘,‘;;‘.’ ,’ -.,’ I’ ,’ : ,, iI,-, :,’ 1: : ,’ ,.q I,:‘, I,;:.,: ‘,,“I :, I- .’ I,, ‘,’ ,/1 /, I /:F1, i :) ,y.-, ,1.& 1 ‘,‘I, ,
G Kbp
H
I
J
‘; ,;.; (:. -’ ,, ,; ‘-I’, ! ,. ,I,, L -,, ‘, -f .,,-, “, i ,. ,‘, ‘.; ,‘, ,; -I,‘. ‘- ‘, ‘.
FIG. 6. Analysis of the DNA from purified virions, two papillomas, and two primary carcinomas using the restriction endonucleases MspI and H&I. 10 a of cell DNA or 4 X lo-’ pg of virion DNA was cleaved with the MspI (lanes A, C, E, G, and I) or HpaII (lanes B, D, F, H, and J). The fragments were separated by electrophoresis on a 0.9% agarose gel and transferred to a nitrocellulose filter. Virus-specific sequences were detected by hybridization with 8ZP-labeled SPV DNA. Lanes A and B, virion DNA; lanes C-F, papilloma DNAs (the same DNA was analyzed in the lanes C and D, and E and F); lanes G-J, primary carcinoma DNAs (the same DNA was analyzed in the lanes G and H, and I and J).
Vx2 AND
95
Vx7 CARCINOMAS
ylated -CCGG- sequence per genomelength DNA which contain at least 15 -CCGG- sequences. This unmethylated site may be distributed uniformly on these -CCGG- sequences, because only one band, corresponding to the HpII E-subfragment (see Fig. l), is visible in the HpaII digests of both DNAs (lanes D and F in Fig. 7). Digestion of the Vx2 and Vx7 DNA with Sa,+?Iresulted in no change in the mobilities of the virus-specific sequences as compared with the undigested DNA (data not shown). This indicates that in these tumors, almost all of the SalI sites present on the viral DNA are methylated.
ABCDEF
DISCUSSION
FIG. 7. Analysis of the DNA from a Vx2 and a Vx7 tumor using MspI and HpdI. 10 ~g of cell DNA or 2 X lo-’ gg of virion DNA was cleaved with the MspI (lanes A, C and E) or HpaII (lanes ES,D and F). Lanes A and B, SPV DNA; lanes C and D, Vx2 DNA; lanes E and F, Vx7 DNA.
sequences were completely
digested
with
HpaII but in another papilloma, more than 50% of the viral sequences remained incompletely digested (lanes D and F in Fig. 6). In one primary carcinoma, approximately 50% of the viral sequences were incomplete when digested with HpaII, but in another primary carcinoma, most viral sequences remained incompletely digested (lanes H and J in Fig. 6). When the Vx2 and Vx7 DNAs were digested with HpuII, most viral sequences remained incompletely digested, and the partially digested fragments came together around the position of unit-length viral DNA. This result indicates that the high-molecular-weight viral DNA present in the transplantable carcinomas contains roughly one unmeth-
The persistence of viral genetic information in the transplantable carcinoma Vx2 and Vx7, and domestic rabbit papillomas has been demonstrated in several earlier studies (Noyes and Mellors, 1957; Rogers et d, 1960; Mellors, 1960; Ito and Evans, 1961; Osato and Ito, 1967; Ito and Evans, 196.5;Orth et a& 1978). The presence of multiple viral genomes was also reported (Stevens and Wettstein, 1979; McVay et al, 1982; Favre et d, 1982). The results of our
analysis of these tumors with reassociation kinetics are in good agreement with these earlier and recent findings; however, there were minor differences. In the Vx2 and Vx7 carcinomas, viral DNA is present in high-molecular-weight forms, and almost all fractions of this DNA are digested by EcoRI, which cleaves SPV DNA at a single site, into linear genomesize DNA molecules (McVay et aL, 1982; Favre et d, 1982). This indicates that most fractions of this DNA consist of oligomeric forms of the unit-length viral DNA molecules. The results of our analysis with .the Southern blotting are consistent with this conclusion McVay et al. (1982) reported that in the cell line derived from the Vx7 carcinoma, five weak bands which do not correspond to the form III SPV DNA were also detected by EcoRI digestion. Since four contained only part of the viral genome, they concluded that at least the four are viral
96
SUGAWARA
segments covalently linked with cellular sequences. The four weak bands we found here in the EcoRI-restricted Vx’7 DNA are probably identical with those reported by McVay et a& although we did not detect a band corresponding to the fastest-moving one. Four similar bands were identified also in the Vx2 DNA digests and at least two (21.5- and 10.8-kb bands) revealed identical mobilities to those from the Vxi’ DNA and, therefore, to the two integration bands reported by McVay et al Two off-size species of virus-specific bands common to both Vx2 and Vx’7 tumors were also identified by BamHI and Hind1 digestion. One of the five weak bands detected by McVay et al. (1982) had a similar mobility to the dimer of linear genome-length viral DNA and contained all or almost all sequences of the viral genome. We also found a similar band in the Vx7 DSA digests, :but it revealed a slightly different mobility from the analogous band detected in the Vx2 DNA digests. Thus, it does not seem to be a simple dimer resulting from incomplete digestion, but may be a viral segment linked to a cellular sequence, or may originate from oligomeric viral DNA carrying a mutation or a small deletion at the EcoRI sites. All these results taken together indicate that in both Vx2 and Vx7 carcinomas, at least some fraction of viral DNA is integrated into the cell DNA. Since the viral DNA present, in the Vxi’ cell line moved together with cellular DNA in agarose gels, McVay et al. (1982) suggested that all the viral DNA sequences are integrated as tandem head-to-tail repeats of unit-length molecules. In the present study, the digestion of Vx2 and Vx7 DNA with KpnI, which does not cleave the SPV DNA, resulted in a single virusspecific DNA band of about 70 kb. If the viral sequences in this band are linear, these should be tandem ,head-to-tail repeats consisti,ng,of 9 to lO&nit-length viral DNA molecules. This is compatible with the present results and those obtained by McVay et al. (1982) that about 20 viral genomes per diploid cell DNA equivalent are present. We suggested here one or two viral DNA integration sites’ for both trans-
ET AL.
plantable tumors and McVay et al suggested two or three integration sites for the Vx7 cell line. The integration sites might be three, but major ones, where multiple viral genomes in oligomeric forms are integrated, are probably two, and therefore, the 70-kb viral band identified by KpI may contain two species of viral DNA fragments with similar electrophoretie mobilities. Favre et al. (1982) reported different results. They detected none of the integration bands but instead, found circular viral DNA molecules having a deletion of about 10% of the genome length. Since these deleted molecules were detectable only after EcoRI digestion, they suggested that the viral DNA are present as free catenated oligomers. This discrepancy may be due to the different maintenance carriers of tumors. The Vx7 carcinoma analyzed by Mcvay et ah, who reported almost identical results to ours, originated from the tumors recently sent from our laboratory; but the Vx2 and Vx7 carcinomas analyzed by Favre et al. had been independently maintained from ours. perhaps for more than 20 years (Ito et al, 1968; Favre et al, 1982). During this period, partial deletion or rearrangement of viral DNA, probably followed by cell selection, may have occurred in these tumors, depending on the passage number and individual variation of rabbits used for the maintenance. This explanation can be supported by the following: (1) At least Vx7 carcinomas we and Favre et al analyzed are extremely different in their passage numbers. They analyzed the tumors before the 200th passage but the tumors we used were those after the 350th passage. (2) From these tumors, at least free monomer-size viral DNA molecules would have been eliminated during the maintenance. There are data indicating that the Vx2 carcinoma produced virus particles until at least the 22nd transplant (Rous et aL, 1952) and the Vx7 carcinoma until recently (Mellors, 1960; Ito and Evans, 1965; Orth et aL, 1978). Whereas, McVay et al. (1982), Favre et al. (1982) and we, failed to detect free monomer-size viral DNA molecules, in both tumors;
97
Vx2 AND Vx'7 CARCINOMAS Another explanation of the discrepancy is that our Vx7 carcinoma was accidently replaced by the Vx2 carcinoma during the maintenance. While several reports have indicated that the Vx2 and Vx7 carcinomas differ in their ability to produce virus and viral antigens, and in the presence or absence of extractable infectious viral DNA (Rous et ah, 1952; Mellors, 1960; Ito and Evans, 1965; Osato and Ito, 1967), we found no significant difference in the quantities and physical states of viral DNA between both carcinomas. The result obtained by Favre et al. that the Vx7 tumor contains much higher numbers of viral genomes then we detected, seems more compatible with the data previously reported. However, it seems improbable that the Vx7 tumors we analyzed were Vx2 carcinomas, because: (1) The Vx2 and Vx7 tumors are not identical. The analysis with the Southern blotting revealed several minor differences in the electrophoretic patterns. (2) If the discrepancy is the consequence of a simple error of maintenance, our results and those obtained by Favre et al. (1982) on the Vx2 carcinoma should be the same, but they also found no integration band in their Vx2 tumors. (3) According to our recent pathological examination, our Vx2 and Vx7 carcinomas seem to conserve the original distinguishable features previously reported (Ito et al, 1968). (4) If the intcgration of viral DNA into a specific cellular DNA site has a role in keeping cells malignant or enhances cell growth, a certain cell type which carries inserted viral DNA at a specific site of cell DNA can be selected in tumors of different origins, as suggested recently by the studies of avian leukosis virus (Hayward et aL, 1981; Payne et uL, 1982). Two species of viral transcripts have been identified in both papillomas and carcinomas (Nasseri et uZ., 1982). While 2.0kb species are more common in benign tumors, in malignant tumors there is a larger amount of 1.3-kb species. We have shown here that the extent of methylation of viral DNA in primary and the transplantable carcinomas is higher than that in papillomas. Several recent studies suggested
that gene expression is inversely correlated with the level of DNA methylation (Waalwijk and Flavell, 1978b; Bird & aL, 1979; McGhee and Ginder, 1979; Cohen, 1980; Guntaka et al, 1980; Sutter and Doerfler, 1980). Therefore, the increased amount of 1.3-kb species in carcinomas might be due to the elevated level of viral DNA methylation. However, it is not known whether such alteration correlates with the development of malignant tumors. The present analysis with MspI and HpuII also indicates that the viral DNA in the Vx2 and Vx7 carcinomas is more extensively methylated than in the primary carcinomas. This could be a consequence of a long history of the maintenance, or otherwise, could relate to the integration of whole viral DNA into the cell DNA, because as evidenced in other viruscell systems, integrated viral DNA is extensively methylated (Cohen, 1980; Guntaka et al, 1980; Sutter and Doerfler, 1980), and, in primary carcinomas, all or almost all fractions of the viral DNA are present in episomal forms (Wettstein and Stevens, 1982; Sugawara et al, unpublished observation). ACKNOWLEDGMENTS We thank Drs. Yasuyuki Takagi and Motoaki Anai for pertinent advice and provision of a bacterial strain and M. Ohara for useful comments on the manuscript. This investigation was supported in part by grants from the Ministry of Education, Science, and Gulture, Japan.
REFERENCES ANAI, M., HIHAHASHI, T., and TAKAGI, Y. (19’7Oj. A deoxyribonuclease which requires nucleoside triphosphate from Mtimnxxms l?~ltodeih&~~ I. Purification and characterization of the deoxyribonuclease activity. J. Bid Chem. 245, 76’7-774. BIRD, A. P., TAGGART, M. H., and SMITH, B. A. (lS79). Methylated and unmethylated DNA compartments in the sea urchin genome. CoU 17, 339-901. COHEN, J. C. (1980). Methylation of milk-borne and genetically transmitted mouse mammary tumor virus proviral DNA. CeU 19.653-662. FAVRE, M., JUURII, N., and ORTII, G. (1982). Restriction mapping and physical characterization of the cottontail rahbit papillomavirus genome in trans-
98
SUGAWARA
plantable Vx2 and Vx7 domestic rabbit carcinomas. Virology 119.298-309. FUJINAGA, K., SAWADA, Y., and SEKIKAWA, K. (1979). Three different classes of human adenovirus transforming DNA sequences; highly oncogenic subgroup A-, weakly oncogenic subgroup B- and subgroup Cspecific transforming DNA sequences. Virology 93. 578-581. F’UJINAGA, K., SEKIKAWA, K., and YAMAZAKI, H. (1975). Method for determination of nucleotide sequence homology between viral genomes by DNA reaasociation kinetics. .Z. Viral. 15, 466-470. GREENE, P. J., BETLACH, M. C., GOODMAN, H. M., and BOYER, H. W. (1974). The EcoRI restriction endonuciease. In “Methods in Molecular Biology” (R. Wecker, ed.), Vol. ‘7, pp. 8’7-111. Dekker, New York. GUNTAKA, R. V., RAO, P. Y., MITSIALIS, S. A., and KATZ, R. (1980). Modification of avian sarcoma proviral DNA sequences in monpermissive XC cells but not in permissive chicken cells. J. V&Z 34,569572. HAYWARD, W. S., NEEL, B. G., and ASTRIN, S. M. (1981). Activation of a cellular one gene by promoter insertion in ALV-induced lymphoid leukosis. Nature (Zmdm) 290,475-480. ITO, Y. (1963). Studies on subviral tumorigenesis: Carcinoma derived from nucleic acid-induced papillomas of rabbit skin. Acta Unti. Znt. Contra Canerum 19.280-283. ITo, Y. (1975). Papilloma-myxoma viruses. In “Cancer” (E. Becker, ed.), Vol. 2, pp. 323340. Plenum, New York. ITO, Y., and EVANS, C. A. (1961). Induction of tumors in domestic rabbits with nucleic acid preparations from partially purified Shape papilioma virus and from extracts of the papiilomas of domestic and cottontail rabbits. .Z. Exp. Med 114, 485-500. ITO, Y., and EVANS, C. A. (1965). Tumorigenic nucleic acid extracts from tissues of a transplantable carcinoma, Vx7. J. Nnt Cuncer Inst. 34, 431-437. ITO, Y., KIYURA, I., and MIYAKE, T. (1968). Comparative studies on pathogenesis of two strains of transplantable carcinomas of rabbits, Vx2 and Vx7. Proc Sot Exp Bid Med 127, 1106-1111. KIDD, J. G.. and Rous, P. (1940). A transplantable rabbit carcinoma originating in a virus-induced papilloma and containing the virus in masked or altered form. J. Exp Med 71, 813-838. KREIDER, J. W., and BARTLE?T, G. (1981). Tbe Shope papilloma-carcinoma complex of rabbits: A model system of neoplastie progression and spontaneous regression. Adv. Cancer Z&s. 35, 81-110. MAXAM, A. M., and GILBERT, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 65, pp. 499-560. Academic Press, New York.
ET AL. MCGHEE, J. D., and GINDER, G. D. (1979). Specific DNA methylation sites in the vicinity of the chicken 6-globin gene. Nature (Londa) 280.419-420. MCVAY, P., FRETZ, M., WETTSTEIN, F., STEVENS, J., and ITO, Y. (1982). Integrated Shope virus DNA is present and transcribed in the transplantable rabbit tumour Vx7. .Z. Ga Vilpl 60,2’S278. MELLORS, R. C. (1966). Tumor cell localization of the antigens of the Shope papilioma virus and the Rous sarcoma virus. Cancer Rtx 20. 744-746. MUKAI, T., MATSUBARA, K., and TAKAGI, Y. (1973). Isolation of circular DNA molecules from whole cellular DNA by use of ATP-dependent deoxyribonuclease. Pm Nat Auw! USA 70.2884-2887. NASSERI, M., W~TTSTEIN, F. O., and STEVENS, J. G. (1982). Two colinear and spliced viral transcripts are present in non-virus-producing benign and malignant neoplasm8 induced by the Shope (rabbit) papilloma virus. .Z. fir& 44,263-268. NOYES, W. F., and MELLORS, R. C. (1957). Fluorescent antibody detection of the antigens of the Shope papilloma virus in papillomas of the wild and domestic rabbit. J. Exp. Med 106, 555-562. ORTH, G., BREITBURD, F., and FAVRE, M. (1978). Evidence for antigenic determinants shared by the structural polypeptides of (Shope) rabbit papiilomavirus and human papillomavirus type 1. virology 91.243-255. OSATO, T., and ITO, Y. (1967). In vitro cultivation and immunofluorescent studies of transplantable carcinomas Vx2 and Vx7. J. Exp. Med 126,881~886. PAYNE, G. S., BISHOP, J. M., and VARMIJS, H. E. (1982). Multiple arrangements of viral DNA and an activated host oncogene in bursal lymphomas. Nature (Zmdon) 295,209-214. ROGERS, S., KLDD, J. G., and Rows, P. (1960). Relationship of the Shope papilloma virus to the cancer it determines in domestic rabbits. Acta Unio. Zti C?oniru Canmum 16, 129-130. Rows, P., KIDD, J. G., and SMITH, W. E. (1952). Experiments on the cause of the rabbit carcinomas derived from virus-induced papiilomaa. II. Loss by the Vx2 carcinoma of the power to immunize hosts against the papilloma virus. .Z. Exp. Meo! 96, 159174. SAW~A, Y., OJIMA, S., SHIMOJO, H., SHIROKI, K., and FUJINAGA, K. (1979). Transforming DNA sequences in rat cells transformed by DNA fragments of highly oncogenic human adenovirus type 12. .Z. VimL 32, 379-385. SOUTHERN.E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Md Bid 98,503-517. SEVENS. J. G., and WETT~TEIN, F. 0. (1979). Multiple copies of Shope virus DNA are present in cells of benign and malignant non-virus-producing neoplasms. .Z. ViroL 30, 891-898.
Vx2 AND
Vx’7 CARCINOMAS
SUMTER, D., and DOERFLER, W. (1980). Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression. Proc. Nat Acud Ski. 77, 253-256. TAKANAMI, M. (1974). Restriction endonucleases AP, GA, and H-I from three Haemophilus strains. In “Methods in Molecular Biology” (R. Wecker, ed.), Vol. 7, pp. 113-133. Dekker, New York. WAALWLIK, C., and FLAVEL.L, R. A. (1978a). MspI, an isoschizomer of ZZpaII which cleaves both unmethylated and methylated ZZpuII sites. Nucleic Acid Rex 5, 3231-3236. WAALWIJK, C., and FLAVELL, R. A. (1978b). DNA methylation at a CCGG sequence in the large intron of the rabbit @-globin gene: Tissue-specific variations. NucZeic Acid Rev. 5,4631-4641.
99
WETISTEIN, F. O., and STEVENS, J. G. (1981). Transcription of the viral genome in papillomas and carcinomas induced by the Shope virus. Virdogy 109,448451. WFITSTEIN, F. O., and STEVENS, J. G. (1982). Variable sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomea are detected in some. Proc. Nat. Acad Sci USA 79.790-794. WOLD, W. S. M., GREEN, M., and MACKEY, J. K. (1978). Methods and rationale for analysis of human tumors for nucleic acid sequences of oncogenic human DNA viruses. In “Methods in Cancer Research” (H. Busch, ed.), Vol. 15, pp. 70-161. Academic Press, New York.