Molecular analysis of episomal human papillomavirus type 16 DNA in a cervical carcinoma cell line

ELSEVIER

Virus Research 51 (1997) 183-195

Virus Research

Molecular analysis of episomal human papillomavirus type 16 DNA in a cervical carcinoma cell line Wendy Sears Hall a, Ryoko Goto-Mandeville a, Helen A. Shih a, Peter R. Shank b Lundy Braun a., a Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA b Department of Molecular Immunology and Microbiology, Brown University, Providence, RI 02912, USA

Received 5 May 1997; received in revised form 29 July 1997; accepted 11 August 1997

Abstract Integration of human papillomavirus type 16 DNA sequences into host DNA is a frequent event in cervical carcinogenesis. However, recent studies showing that HPV16 is present exclusively in an episomal form in many primary cervical cancers suggest that HPV16 can transform target cells by mechanisms that do not require viral integration. We have established a cervical carcinoma cell line that harbors episomal copies of HPV16 D N A of approximately 10 kb. Restriction enzyme and two-dimensional gel analysis confirmed that HPV16 DNA was extrachromosomal with both monomeric and multimeric forms present. HPV16 was maintained as episomes with passage both in culture and after subcutaneous growth in nude mice. The 10 kb viral genome, consisting of a full-length copy of HPV16 and a partial duplication of the long control region and the L1 open reading frame, exhibited transforming activity comparable to prototype HPV16. This cell line should provide a useful model system for studying the biological significance of the physical state of the HPV16 genome in cervical carcinoma cells. © 1997 Elsevier Science B.V. Keywords: Human papillomavirus type 16 (HPV16); Episomal; Physical state; Transformation; Cervical carcinogenesis

1. Introduction H u m a n papillomaviruses (HPVs) belong to a family o f small D N A viruses that usually produce

* Corresponding author. Fax: + 1 401 8639008.

benign lesions on cutaneous and mucosal surfaces. O f the m o r e than 100 H P V types described to date, only a subset are consistently f o u n d in h u m a n cancer (zur Hausen, 1996). H P V D N A , including that f r o m types 16, 18, 31, 33 and 35, has been detected in over 90% o f cervical cancers (Bosch et al., 1995). H P V type 16 (HPV16) is the

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most common HPV type found in cervical cancers, detected in over half of malignant lesions. The etiologic role of HPV in cervical neoplasia has been firmly established, although the precise mechanisms of transformation have yet to be elucidated. Initial studies of cervical biopsies reported that premalignant lesions contained extrachromosomal viral DNA whereas HPV DNA was integrated in cancer tissue (Diirst et al., 1985). Integration usually disrupts the E1 and/or the E2 open reading frames (ORFs) (Matsukura et al., 1986; Baker et al., 1987; Choo et al., 1987a), two genes whose products are important in regulation of virus replication and transcription (Romanczuk et al., 1990; Del Vecchio et al., 1992; Berumen et al., 1994). It has been reported that integration may also disrupt a potential m R N A instability element in the 3' untranslated region of E6 and E7 mRNA, resulting in increased stability and steady state levels of E6 and E7 transcripts (Jeon and Lambert, 1995). Thus, integration of viral DNA with subsequent disruption of regulatory DNA sequences provides a plausible mechanism for the progression of HPVinduced malignancy. Several investigators have shown that a significant proportion of HPV16-positive primary cervical cancers contain both episomal as well as integrated HPV sequences and that, in approximately 20-30% of tumors, HPV16 DNA is found exclusively in an extrachromosomal form (Choo et al., 1987b; Matsukura et al., 1989; Cullen et al., 1991; Das et al., 1992). This suggests that, without the integration-mediated disruption of virus regulatory elements, other changes in the viral genome may be necessary for transformation to occur. Although the mechanisms by which extrachromosomal HPV contributes to transformation are not known, studies suggest that mutations in the binding sites for the cellular transcription factor YYI may be important (May et al., 1994). HPVs do not infect cells in monolayer culture. Therefore, studies aimed at understanding the molecular mechanisms by which HPV16 and HPV18 transform epithelial cells have relied on permanent cell lines established by transfection

of HPV16 or HPV18 DNA into cervical and cutaneous keratinocytes or cell lines derived from premalignant and malignant cervical tissue. In the majority of these lines, HPV DNA is integrated into the cellular genome (for review, see DiPaolo et al., 1993). There are a few reports in the literature of cell lines with episomal HPV16 established from a premalignant cervical lesion (Stanley et al., 1989; Jeon et al., 1995) or a cervical carcinoma (Choo et al., 1989). However, with the exception of two clones described by Jeon et al. (1995), these lines contain integrated as well as episomal copies of HPV DNA. Moreover, extrachromosomal HPV is rarely stable, disappearing after repeated passage in culture (DiLorenzo et al., 1992; Jeon et al., 1995). We have established a cell line, designated 140/7, from a moderately differentiated cervical carcinoma (Braun et al., 1993). Southern blot analysis indicated that the 140/7 line harbored approximately 100-200 episomal copies of HPV16 DNA of approximately 10 kb in size. The goals of the present study were: (i) to determine whether episomal HPV16 is maintained in an episomal form with passage in vitro and in vivo; (ii) to characterize the 10-kb viral genome of the 140/7 line, in particular the additional 2 kb of HPV16 DNA present and (iii) to analyze the transforming activity of the episomal HPV16. We found that HPV16 from the 140/7 line (designated HPV16-140/7) was maintained in an extrachromosomal form with passage in culture and in nude mice with no evidence of integration. HPV16-140/7 DNA contained a partial duplication of the long control region (LCR) and L 1 0 R F in addition to a full length copy of HPVI6. HPV16-140/7 had transforming capacity, dependent on the presence of the LCR duplication, that was similar to prototype HPVI6. The 140/7 line represents the first cervical carcinoma-derived cell line with exclusively episomal HPV16 and the availability of these cells will help elucidate the importance of the physical state of the viral genome in HPV-mediated cervical carcinogenesis.

W. Sears Hall et al. / Virus Research 51 (1997) 183-195

2. Material and methods

2.1. Cells The 140/5 and 140/7 cell lines were established from a moderately differentiated keratinizing cervical carcinoma (Braun et al., 1993). The 140/7 line has also been referred to as TC-140 (Mark et al., 1995). The 140/7 cells harbor extrachromosomal copies of HPV16 whereas 140/5 cells contain integrated HPV16 DNA. The cells were maintained in Dulbecco's modified medium(DMEM): F12 (2:1), supplemented with 10 ng/ml epidermal growth factor, 5 pg/ml insulin, 12.5 pg/ml gentamicin, 0.4 /tg/ml hydrocortisone, 18.2 pg/ml adenine, 8.4 ng/ml cholera toxin, and 5% fetal bovine serum (FBS). For in vivo studies, cells grown in tissue culture were trypsinized, washed, and resuspended in Hank's Balanced Salt Solution. The cell suspension, containing 5-10 x 10 6 cells, was injected subcutaneously into 4 - 5 week old female nude mice. Individual nodules were harvested after 2-3 weeks and frozen in liquid nitrogen for subsequent DNA extraction or fixed in formalin for histological examination. 2.2. DNA isolation and Southern blot analysis DNA was extracted from cultured cells using the Qiagen DNA purification kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. For isolation of DNA from nude mice nodules, frozen tissue was homogenized and digested with proteinase K (20 mg/ml) followed by phenol extraction (Maniatis et al., 1982). Southern blot analysis of genomic DNA (5 /~g) digested with the indicated restriction enzymes was performed as previously described (Braun et al., 1993). Full-length HPVI6 cloned into pBR322 was provided by Dr zur Hausen (Heidelberg, Germany). Two-dimensional gel electrophoresis was used to further analyze the physical state of HPVI6 DNA in 140/7 cells (Johnson and Grossmann, 1977). DNA (5/~g) was digested with either BgllI or HindlII and electrophoresed in a 0.4% agarose gel for 6 h at 50 V. Each lane was cut out, rotated 90 °, and recast in 1% agarose for the second

185

dimension. This gel was then run for 12 h at 50 V. The two-dimensional gels and a duplicate lane of the first dimension were transferred to nitrocellulose and hybridized with full-length 32p-labeled HPV16 DNA. 2.3. Molecular cloning of HPVI6-140/7 DNA To clone the episomal form of HPV16 DNA from 140/7 cells, supercoiled HPV16 was first purified using an anion exchange column (Qiagen) according to the manufacturer's instructions for bacterial plasmid DNA purification. The yield of supercoiled episomal DNA was significantly higher with this method than with the conventional method of Hirt (1967). Cells (4 x 107) were trypsinized, pelleted and lysed and the supernatant was loaded onto the Qiagen anion exchange column. Supercoiled DNA was then eluted and precipitated with isopropanol. For cloning, purified episomal HPV16-140/7 DNA was cut with BamHI (Promega, Madison, WI), generating two fragments of 2 and 8 kb in length. These fragments were cloned by the method of Maniatis et al. (1982) into the multiple cloning site (MCS) of pBluescribe (pBS; Stratagene, La Jolla, CA). Transformants were selected by LacZ color selection and ampicillin resistance and screened for 2- and 8-kb inserts by Southern blot hybridization using 3ZP-labeled full-length HPV16. Two clones, designated pBS-B2 and pBSB8 (corresponding to the 2- and 8-kb BamHI fragments, respectively), were selected and expanded for restriction endonuclease analysis. An additional clone, pGEM-5Zf-HPV16-140/7, was generated which contained the full-length 10-kb HPV16 from the 140/7 line cloned into the ApaI site of the MCS of pGEM-5Zf (Promega). From this clone, pGEM-5Zf-HPV16-140/7-B8 was generated which contained the 8-kb Bam HI fragment in the ApaI site of pGEM-5Zf. 2.4. DNA sequence analysis Selected regions of the HPV16-140/7 BamHI fragments were sequenced by a modified chain termination reaction using the AmpliTaq cycle sequencing kit (Perkin Elmer Cetus, Norwalk,

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CT), according to the manufacturer's instructions. For sequencing the 2 and 8-kb fragments, a combination of M13 universal forward and reverse primers, virus-specific primers, and restriction digest-generated subclones of either pBS-B2 or pBSB8 was utilized. The subclones, which removed fragments from the ends of the pBS-B2 or pBS-B8 insert to sites in the polylinker, were sequenced with either the M13 universal forward or reverse primers. The D N A sequence generated was compared to the sequence of prototype HPV16 (Seedorf et al., 1985). 2.5. TransJormation assay in primary baby rat kidney cells

The transforming activity of the 10-kb HPVI6140/7 was determined by co-transfection with an activated ras oncogene into baby rat kidney (BRK) cells (Matlashewski et al., 1987). Primary cultures of kidney cells from 10-day old inbred Fischer rats were grown in 60-mm culture dishes in D M E M with 10% FBS until subconfluent. The cells were transfected using D O T A P reagent (Boehringer-Mannheim, Indianapolis, IN) with 5 /~g of pUC/EJ, which contains the 6.6-kb B a m H I fragment of the activated ras H oncogene (Shih and Weinberg, 1982), and 10 /~g of an HPV16containing plasmid. The following plasmids were used: pGEM-5Zf-HPV16 (prototype HPV16 in the A p a I site pGEM-5Zf); pGEM-5Zf-HPV16140/7 (10-kb HPVI6-140/7 in the A p a I site of pGEM-5Zf); pGEM-5Zf-HPVI6-140/7-B8 (8-kb B a m H I fragment of HPV16-140/7 in the A p a I site of pGEM-5Zf); pBS-B2 (2-kb B a m H I fragment of HPV16-140/7 in pBluescribe); or p G E M - 5 Z f control. The establishment of the various HPV16140/7 plasmids was described above in Section 2.3. pGEM-5Zf-HPV16 was derived by subcloning prototype HPV16 (Diirst et al., 1983), kindly provided by Stoler (Stoler and Broker, 1986), into the A p a I site of the MCS of p G E M 5Zf. After 5 h, the transfection media was replaced with fresh media and 24 h later, cells were trypsinized and replated into 100-mm culture dishes. Cells were maintained in D M E M , 10% FBS and 10 6M dexamethasone. After 3 weeks in

culture, cells were fixed in methanol:acetic acid and stained with Giemsa for colony counts.

3. Results

3.1. Cells contain episomal copies o f H P V 1 6 DNA

We previously reported that the 140/7 cell line contained approximately 100-200 copies of episomal HPV16 D N A (Braun et al., 1993). To determine if integrated viral sequences were present in this cell line, we performed additional restriction digests of 140/7 D N A with H i n d I I I and BglII, restriction endonucleases which do not cut HPV16. As shown in Fig. 1, low molecular weight bands corresponding to supercoiled, nicked circu-

A.

140/7 U

H Bgl

140/5 U

H Bgl

.~aallt

81,

Fig. 1. Physical state of HPVI6 in 140/5 and 140/7 cervical carcinoma cells. A total of 5 /~g of undigested (U), Hindlll(H) or BglII-digested (Bgl) cellular DNA was electrophoresed in 0.8°/,, agarose, transferred to nitrocellulose and hybridized with 32p-labeled HPV16 DNA. Arrowheads: supercoiled (s), linear (1), and nicked circular (n) forms of the 10-kb episomal HPVI6-140/7.

W. Sears Hall et al. / Virus Research 51 (1997) 183-195

lar and linear viral DNA were present in both uncut and HindlII- or BgllI-digested 140/7 DNA. Southern analysis of the clonally-derived 140/5 line from the same primary tumor which contains integrated HPV16 DNA is shown for comparison. In addition to bands corresponding to monomeric forms of episomal HPV16, we noted a diffuse high molecular weight band in undigested DNA from 140/7 ceils that was converted to four discrete bands after digestion with either HindIII or BgllI. The identical banding pattern was also seen with EcoRV, another noncutting enzyme (data not shown.) A semilogarithmic plot of the electrophoretic mobilities of these upper molecular weight bands against the molecular weights of 10-kb unit multimers generates a smooth curve that is nearly linear for the upper molecular weight bands (data not shown.) This banding pattern is indicative of multimeric forms of extrachromosomal HPV16, corresponding to supercoiled episomes containing up to 5 copies of the 10-kb HPV16 DNA. In contrast, digestion of DNA from 140/5 cells with either HindlII or BgllI produced a restriction enzyme profile consistent with the presence of integrated HPV16. These results, together with previous PstI digests, suggest that the 140/7 line contains exclusively episomal HPV16 DNA with no detectable integrated viral sequences. Low levels of human-viral junction fragments from integrated HPV16 D N A in the 140/7 line could, however, be obscured by the higher episomal viral copy number. Therefore, as a further means of determining whether any integrated HPV16 DNA could be detected in the 140/7 cell line, we analyzed 140/7 DNA by two-dimensional gel electrophoresis after digestion with either HindlII or BgllI. HPV16 DNA appeared above the arc of linear DNA in positions expected for monomeric and multimeric episomal DNA molecules with supercoiled 10-kb HPV16 DNA as the fastest migrating species (Fig. 2). On the arc of linear DNA, a 10- and 20-kb species were present in addition to a slow-migrating species. These species most likely represent the linearized forms of monomeric, dimeric and larger multimeric episomal HPV-140/7 DNA molecules, respectively.

187

A. Bgl H

B.

Ist D I M E N S I O N

z

C.

1st DIMENSION

W,I

.... ,

iii~iii i

Fig. 2. Two-dimensional gel electrophoretic analysis of DNA isolated from 140/7 cells. A total of 5 #g of HindlII- or BgllI-digested DNA was electrophoresed in two-dimensional gels, Southern blotted and hybridized with 32p-labeled HPV16 DNA as described in Section 2.2 in the methods. (A) One-dimensional gel with ~.-HindlII molecular weight marker (top to bottom--23, 9.4, 6.6 and 4.4 kb). (B) Two-dimensional gel with HindlII-digested DNA. (C) Two-dimensional gel with BgllI-digested DNA and an interpretation of results. S~, $2, Sm indicate supercoiled monomers, dimers and multimeric forms; L1, L 2 and L m indicate linearized monomers, dimers and multimers, respectively.

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W. Sears Hall et al. / Virus Research 51 (1997) 183 195

A.

P3 U

~0

H

B U

H

~1 B

U

~

H

B

U

H

~0 B

U

H

~1 B

U

H

B

Sb

B

o

w

Q

B.

y/b

lb

S¢.

Fig. 3. Maintenance of episomal HPV16 DNA in 140/7 cells with passage in vitro and in vivo. (A) Cellular DNA isolated from cultured cells at passages 3, 10 and 31 and separated in 0.8% agarose gels, either undigested (U) or after digestion with HindIII (H) or BarnHI (B), transferred to nitrocellulose and hybridized with 32p-labeled HPVI6 DNA. (B) Undigested DNA from cells at passage 15 and nude mouse nodules harvested at 2 or 3 weeks was processed as indicated in A. Bars on the right correspond to X-HindIlI molecular weight markers (from top to bottom --23, 9.4, 6.6, 4.4, 2.3 and 2.0 kb). Arrowheads indicated supercoiled (s), linear (1), and nicked circular (n) forms of episomal HPV16-140/7 DNA. The ethidium bromide stained gels are shown in the right panels.

3.2. H P V 1 6 D N A is stably maintained in an episomal f o r m in vitro and in vivo Replicating H P V D N A in established cell lines is generally lost ( T a i c h m a n a n d LaPorta, 1987; D i L o r e n z o et al., 1992) or becomes integrated into cellular D N A with repeated passage in culture ( C r o o k et al., 1990; Bedell et al., 1991). T o

determine if H P V 1 6 was retained in episomal form with passage in culture, we analyzed D N A isolated from 140/7 cells at passage 3, 10 a n d 31 by S o u t h e r n blot hybridization. As s h o w n in Fig. 3A, low molecular weight b a n d s c o r r e s p o n d i n g to supercoiled m o n o m e r i c HPV16 are visible in u n cut a n d HindIII-digested D N A at each passage. Distinct multimeric forms were also detected in all

w. Sears Hall et al. / Virus Research 51 (1997) 183-195 samples. Scanning densitometry revealed a 1-1.5fold increase in the monomeric and dimeric forms and a 2-5-fold increase in the multimeric forms of HPV16 in later passage cells. We also examined the physical state of the HPV16 genome in nodules produced by 140/7 cells in nude mice. Histological analysis of the nodules revealed cystic structures composed of well-differentiated epithelium, similar to that previously described for this cell line (Braun et al., 1993). As shown in Fig. 3B, episomal HPV16 DNA was present in each of five nodules isolated at 2 and 3 weeks. These results suggest that the majority of HPV16 DNA in the 140/7 line is maintained as monomeric and multimeric episomes in vivo as well as in vitro.

3.3. Molecular cloning, restriction enzyme analysis and DNA sequencing of HPV16-140/7 DNA To characterize episomal HPV16 DNA from 140/7 cells in more detail, we cloned the supercoiled viral DNA. BamHI digestion of episomal HPV16 DNA from 140/7 cells produced two fragments of approximately 2 and 8 kb, each of which was cloned into pBS, generating the plasmids pBS-B2 and pBS-B8, respectively. HPV16 DNA was extracted from each plasmid, digested with an extensive panel of restriction endonucleases, and hybridized with full-length HPV16 DNA as probe. Cleavage of pBS-B8 with KpnI, StuI, NcoI, TaqI, SphI, HinclI, EcoRI, PvulI, and PstI generated a restriction enzyme pattern nearly identical to that of prototype HPV16 (data not shown.) The only difference from prototype HPV16 detected by this analysis was an additional HinclI site that mapped to the viral LCR. Only SphI, EcoRI, and PstI from the same panel of restriction enzymes cleaved pBS-B2 DNA, indicating that the 2-kb fragment contained DNA corresponding to sequences surrounding the BamHI restriction site in prototype. HPV16 (in the viral L 1 0 R F and LCR). The additional HinclI site detected in the 8-kb fragment was also found in the 2-kb fragment.

189

Based on the restriction mapping results which indicated a duplication of sequences including the LCR and L1 ORF, we sequenced the complete LCR and sequences in the L1 ORF surrounding the BamHI site of the 8-kb BamHI fra~gnent and approximately 1.8 kb (over 90%) of the 2-kb BamHI fragment of HPV16-140/7. The LCR in the 8-kb fragment was identical to that of prototype HPV16 with the exception of several single nucleotide changes (Table 1), including an A to C transition at nt 7173 that generated the HinclI site. In addition to three previously reported mutations at nts 7432, 7727, and 7861 (Bavin et al., 1993; Fang et al., 1993; May et al., 1994), we noted a G to A transition at nt 7839 in one of the four YY1 binding sites in the HPV16 LCR. Only minor nucleotide alterations were detected within the L 1 0 R F of the 8-kb fragment, none of which disrupted the reading frame. Sequencing of the 2-kb Bam HI fragment of HPV16-140/7 revealed a continuous L 1 0 R F into the viral LCR at the 5' end as well as another continuous L 1 0 R F at the 3' end of the fragment. The LCR of the 2-kb fragment ended prematurely at nt 7690, producing a truncated LCR (referred to as LCR*); the L 1 0 R F at the 3' end began at nt 5733, resulting in a truncated L 1 0 R F (LI*). All nucleotide mutations within the L 1 0 R F s of the 2-kb fragment were also found in the 8-kb fragment as were several of the alterations in the LCR* compared to the full-length LCR (Table 1). In addition, some mutations unique to the LCR* were detected, including a C to T transition at nt 7268 and an A to C transition at nt 7287. Based on our restriction endonuclease and DNA sequence analysis, we constructed a map of episomal HPV16-140/7 DNA (Fig. 4A). HPV16140/7 contains a complete HPV16 genome with an intact LCR as well as a duplication of sequences consisting of the LCR* (approximately 1.4 kb upstream of the full-length LCR) and the L l* ORF. No major deletions were found at the site of the duplication or elsewhere in HPV16-140/7. A detailed map of the LCRs of HPV16-140/7 is shown in Fig. 4B. Several important transcription regulatory domains were duplicated within the LCR*, including part of the enhancer region with

W. Sears' Hall et al.

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Virus Research 51 (1997) 183 195

Table 1 Summary of base changes detected in cloned 140/7 BamHI fragments ORF

L1

LCR

8-kb Fragment

2-kb Fragment

Map position

Base change

Map position

Base change

6178 6237 6240 6245 6432

A ---,C G deleted CATACT-~GATT A -, G

6900

A ---,TCAT

6178 6237 6240 6245 6432 6613 6615 6900

A--,C G deleted CATACT --, TTGATT A~G GTA deleted A ~ TCAT

7173 7175

A ~C T-*C

7432-7433 7518 7727 7839 7861 24

GC~CGG G~A A --*C G-~A A deleted C~T

7173 7175 7268 7287 7432 7433 7518

A-~C T~C C--,T A--,C GC-~CGG G ---,A

all of the cell-type specific response element (Taniguchi et al., 1993) and approximately 75% of the keratinocyte-dependent enhancer (Cripe et al., 1987), as well as the glucocorticoid response element (Chan et al., 1989), four nuclear factor-I (NF-1) transcription enhancer binding sites (not shown--Gloss et al., 1989), two AP-1 binding motifs (Chong et al., 1991), and one viral E2 regulatory protein binding site (Romanczuk et al., 1990). 3.4. Transforming activity oJ' HPV16-140/7 compared to prototype HPV16 To determine whether the transforming activity of the 10-kb HPV16-140/7 genome differed from prototype HPV16, we assayed the transformation potential of cloned HPV16-140/7 DNA in primary cultures of BRK cells. Prototype HPVI6 was derived from a cervical cancer which contained both episomal and integrated HPVI6 (Dtirst et al., 1983, 1985). This viral DNA genome contains a frameshift mutation in the E1 gene not found in wild-type HPVI6 that significantly enhances the immortalizing capacity of HPV16 in

human keratinocytes (Romanczuk and Howley, 1992). As shown in Table 2, the 10-kb HPVI6-140/7 transformed primary BRK cells when co-transfected with an activated ras oncogene, producing an average of 361 colonies per 100-mm culture dish. This was approximately 71% as many transformed colonies as prototype HPV16, which produced 505 colonies on average, although given the variation in the experimental replicates, this difference was not statistically significant. In contrast to full-length HPV16-140/7, the 8-kb BamHI fragment of HPV16-140/7 produced an average of only 8 colonies per experiment, less than 2% of the transforming capacity of prototype HPV16. The 2-kb BamHI fragment of HPV16-140/7 on its own did not have any transformation capacity above the control plasmid, pGEM-5Zf (both averaging 1 colony per experiment). These results demonstrate that the 10-kb HPV16-140/7 was capable of transforming primary cultures of BRK cells at levels slightly below prototype HPV16, whereas the 8- and 2-kb BamHI fragments of HPV16-140/7 did not show significant transforming capacity.

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W. Sears Hall et al. / Virus Research 51 (1997) 183-195

A.

B, • HIIn¢# EcoRI EcoRi Pstl~ ~

P S l I ~

BamHI ~ / L I "

A^

I tL

0

HPV16"140/7

,Pstl

EI~ J/

Pstl

Pstl

7152*

7691 *

7152

p97 promoter

103

F

Cell-type enhancers:

7454 -7632 (CTRE) 7529-7752 (KD)

GlucocorlicoidRE:

7641-7655

II

AP-1 motifs:

7633, 7649

A

E2 bindingsites:

7450, 7859, 35, 50

•

YYI bindingsites:

7787 - 7779 7791 - 7799 7834 - 7842 7840 - 7848

O

Fig. 4. (A) The genomic map of HPV16-140/7 indicating the position of the HPV16 ORFs and the viral LCRs. LCR* represents a truncated long control region from nucleotides 7152 to 7691. LI* represents a truncated L 1 0 R F from nucleotides 5733 to 7152. Two additional HinclI sites in each LCR, which differ from prototype HPVI6, are also shown. (B) Schematic diagram of the HPVI6-140/7 LCRs with some of the important regulatory domains within the LCR indicated. The location of the mutated YY1 site found HPV16-140/7 is depicted by an open ovoid.

binding

in

4. Discussion

In this paper we reported a detailed analysis of the physical state of the HPV16 genome in the 140/7 cervical carcinoma-derived cell line. We found that HPV16 DNA in 140/7 cells exists as monomeric episomes of 10 kb in length as well as multimeric copies of increasing size. Discrete Table 2 Colony formation in BRK cultures Plasmida

Average number of coloniesb (S.E.M.)

pGEM-5Zf pGEM-5Zf-HPV16 pGEM-5Zf-HPV16140/7 pGEM-5Zf-HPVI6140/7-B8 pBS-B2

1 ( + 0.6) 505 ( + 250) 361 ( -I- 185) 8 ( -I- 5) 1 ( + 1)

a See Section 2.5 in the methods for a detailed description of the plasmids used. b The average number of colonies was derived from counting transformed foci that formed after 3 weeks in culture in 100-mm culture dishes in three separate experiments. S.E.M., standard error of the mean.

bands of identical electrophoretic mobilities in either one-dimensional or two-dimensional gels were generated after digestion with three no-cut restriction enzymes. Fluorescent in situ hybridization analysis of over 5000 140/7 cells with a HPV16 probe revealed no specific pattern of hybridization on any chromosome (Braun and Mark, unpublished data). Taken together, these results indicate that the 140/7 cell line contains exclusively extrachromosomal HPV DNA. Previous studies of established cell lines have found that episomal copies of HPV are lost or integrate into cellular DNA With time in culture (Crook et al., 1990; Bedell et al., 1991; Jeon et al., 1995). Jeon et al. (1995) recently reported that episomal HPV16 DNA sequences were rapidly lost from the parental W12 line, but could be maintained as stable episomes in two clonal derivatives of this line for at least 15 passages. In 140/7 cells, HPV16 DNA remained extrachromosomal for at least 50 passages in culture (data not shown) and after growth for 3 weeks in nude mice. These results indicate that the 140/7 cervical carcinoma cell line supports stable replication of episomal HPV16 in vivo and in vitro.

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The CC7T cervical carcinoma cell line described by Choo et al. (1989) contains both integrated and extrachromosomal HPV16, present as catenated molecules of interlocking rings. On the other hand, in the W12 line derived from a stage I cervical intraepithelial neoplasia, only unitlength episomes were detected (Stanley et al., 1989). In stable clones derived from the W12 line, no multimeric forms were described, even with viral copy number of approximately 1000 (Jeon and Lambert, 1995). Our studies indicate that HPV16 is present in 140/7 cells as multimeric molecules of increasing size, up to 5 unit-lengths. It is intriguing to speculate that the generation of large multimeric episomes occurs during the process of malignant transformation. Indeed, free episomes tended to be larger in Shope papillomavirus-induced carcinomas compared to benign papillomas (Wettstein and Stevens, 1982). Restriction enzyme and DNA sequence analysis of HPV16 cloned from 140/7 cells revealed a duplication of approximately 2 kb corresponding to sequences from the LCR and the L 1 0 R F . The duplicated region of the LCR resulted in the addition of several transcriptional regulatory elements, including two AP-1 binding sites, an E2 binding site, the glucocorticoid responsive element, the cell-type responsive element, and part of the keratinocyte-dependent enhancer. It is not known whether this duplication was present in the primary tumor or arose during passage in culture, although we did detect the duplicated region in DNA extracted from 140/7 cells as early as the third passage (data not shown). In addition, the duplication of the viral LCR was present in integrated HPV16 of the 140/5 cells (data not shown.) Rosen and Auborn (1991) reported that HPVll plasmid constructs that contained two copies of the HPVll LCR or an additional HPV16 LCR exhibited increased transforming activity in the baby rat kidney assay, most likely through a mechanism involving increased enhancer activity. Moreover, primary tumors associated with HPV6 or HPV11 frequently contain duplications in this region (Boshart and zur Hausen, 1986; Rando et al., 1986). The in vitro immortalization efficiency of HPV16 and HPV18 has been localized to the transcriptional regulatory region upstream of the

E6 and E 7 0 R F s (Romanczuk et al., 1991), but no previous studies have examined whether an additional copy of the HPV16 LCR confers enhanced transforming activity. When assayed in BRK cells, the transforming activity of the 10-kb full-length HPV16-140/7 was comparable to prototype HPV16 whereas the 8kb B a m H I fragment of HPV16-140/7 had very little transforming activity. The 8-kb fragment was nearly identical to prototype HPV16 by restriction analysis, yet it was only in combination with the partial duplication of the LCR (in the 2-kb B a m H I fragment) that the 8-kb B a m H I fragment showed significant transforming activity. Our findings suggest that a duplication of the HPV16 LCR is essential in the transforming activity of the HPV16-140/7 genome. However, since we did not sequence the entire 8-kb fragment, we cannot exclude the possibility that other mutations in the 8-kb fragment abrogate transforming activity. Possible mechanisms by which this duplication may alter the transforming activity are not known but may include the cellular and viral regulatory sites duplicated in the LCR* or unique base changes found in the LCR* that were not found in the full-length LCR of the 8-kb B a m H I fragment. Further studies, however, will be necessary to determine the significance of these sequence changes. In contrast to integrated HPV DNA, replication and transcription of episomal HPV16 is under viral control. The changes brought about by viral integration, including the disruption of the viral E1 and/or E2 proteins and the instability element in the Y-untranslated region of E6 and E7 mRNA, are absent. Therefore, additional alterations to the viral genome, which achieve the functional equivalence of integration, may be necessary for transformation by episomal viral DNA. The duplication of the LCR seen in HPV16-140/7 may represent one such molecular alteration. In addition to duplication of the LCR, there are several reports of other mutations in the viral genome in episomal HPV16 DNA in primary cervical tumors, including duplication of the entire E7 and part of the E 6 0 R F (Di Luca et al., 1989), deletion of the LCR (Kennedy et al., 1987), and deletion of three YY1 binding sites (May et

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al., 1994). In addition to the LCR duplication, we also noted a mutation in one of the four binding sites for the cellular transcriptional inhibitor YY1 in the LCR of HPV16-140/7. It has been reported that YY1 negatively regulates transcription of HPV16 DNA (May et al., 1994); therefore, the loss of this binding site may lead to deregulation of HPV gene expression. However, the YY1 mutation in HPV16-140/7 was in the 8-kb B a m H I fragment which did not have significant transforming activity on its own. Thus, the YY1 mutation does not appear to play a role in transformation by HPV16-140/7. Most of the lines described in the literature contain a mixture of integrated and extrachromosomal copies of HPV16, making it difficult to use these lines to determine whether the physical state of the viral genome influences the biological behavior of infected cells. We found no evidence of integration of HPV16 in the 140/7 line with passage in culture. Thus, this cell line, together with derivatives from the same primary tumor which contain exclusively integrated HPV16 DNA, represent an interesting model system to investigate the biological significance of the physical state of the HPV genome in cervical carcinogenesis and the mechanisms of transformation by episomal HPV16.

Acknowledgements We thank Dr Karen Auborn for helpful discussions regarding two-dimensional gel electrophoresis and Deborah S. Venturini for her help with DNA sequencing and for critical reading of the manuscript. This work was supported by Grant CA55892 from the National Cancer Institute.

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