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Genomic Cloning and Characterization of the Nonoccupied Allele Corresponding to the Integration Site of Human Papillomavirus Type 16 DNA in the Cervical Cancer Cell Line SiHa
VIROLOGY
217, 33–41 (1996) 0090
ARTICLE NO.
Genomic Cloning and Characterization of the Nonoccupied Allele Corresponding to the Integration Site of Human Papillomavirus Type 16 DNA in the Cervical Cancer Cell Line SiHa ¨ SL, and A. ALONSO1 R. BAUER-HOFMANN, C. BORGHOUTS, E. AUVINEN, E. BOURDA, F. RO Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, 69120 Heidelberg, Federal Republic of Germany Received May 15, 1995; accepted December 18, 1995 Human papillomavirus (HPV) type 16 DNA sequences have been found integrated into the host cell genome in a large number of cervical tumors and cell lines derived therefrom. In this study, we have cloned and analyzed the nonoccupied allele corresponding to the integration site of HPV-16 in the cervical cancer cell line SiHa. Our mapping analyses revealed an approximately 7.8-kb deletion of cellular DNA upon viral integration. Computer analysis of 2.3 kb of DNA sequences from the deleted genomic region as well as 1.0 kb of sequences upstream of the viral integration site showed no significant homology to any known human sequences. DNase I mapping experiments on native chromatin demonstrated the existence of two hypersensitive sites in both the HPV-16-containing and nonoccupied alleles located approximately 1.1 and 1.7 kb upstream of the viral integration site. This suggests that viral integration occurred close to putative regulatory sequences and that recombination with host cellular DNA was not followed by a reorganization of the chromatin structure upstream of the integration site. Nuclear run-on and RT-PCR experiments showed HPV-specific transcription spanning the E2, E4, E5, and L1/L2 open reading frames (ORFs) located upstream of the HPV-16 regulatory region (URR). Taken together, our data suggest that the cellular DNA region upstream of the HPV-16 integration site in the SiHa cell line contains regulatory elements affecting transcription of HPV-16 ORFs located upstream of the HPV-16 URR. q 1996 Academic Press, Inc.
INTRODUCTION
lished thereof showed preferential integration at or near fragile sites or protooncogenes (Du¨rst et al., 1987; Cannizzaro et al., 1988; Popescu and DiPaolo, 1989; Couturier et al., 1991; De Braekeleer et al., 1992). These findings suggest that integration may not only affect viral transcription but also could be accompanied by activation or inactivation of cellular genes involved in the control of cell proliferation and/or differentiation. The ORFs encoding the viral oncogenes E6 and E7, as well as the viral upstream regulatory region (URR), are consistently retained and transcribed in HPV integrates (Schwarz et al., 1985; Smotkin and Wettstein, 1986; Baker et al., 1987). Both viral gene products are involved in immortalization of human epithelial cells (Hawley-Nelson et al., 1989; Mu¨nger et al., 1989; Halbert et al., 1991), and their continuous transcription seems to be necessary for the maintenance of the malignant phenotype (von Knebel Doeberitz et al., 1988; Crook et al., 1989). Integration of HPV DNA frequently leads to an interruption of the viral E1 and E2 ORFs (Romanczuk and Howley, 1992). The E2 protein has been proposed to negatively interfere with transcription from the E6 and E7 ORFs through binding to its binding sites located within the URR (Thierry, 1993). Functional inactivation of the E2 ORF upon integration might therefore lead to an enhanced transcription from the E6 and E7 oncogenes which could
Human papillomavirus (HPV) DNA sequences have been identified in a variety of benign and malignant cervical tumors (zur Hausen and de Villiers, 1994). Whereas benign proliferations usually harbor low-risk HPVs, e.g., types 6 and 11, in malignant lesions high-risk HPV types, such as 16, 18, and 33, are predominant (zur Hausen and Schneider, 1987; zur Hausen, 1991a,b; de Villiers, 1994). The occurrence of particular HPV types in up to 90% of carcinomas (Meijer et al., 1992; Das et al., 1993) strongly suggests that viral infection is causally involved in the development of this type of cancer. Analysis of the physical state of HPV DNA has revealed that benign lesions mostly contain the viral sequences as episomes, whereas in malignant lesions the viral DNA is usually integrated into the host genome with subsequent disruption of one or more viral open reading frames (ORFs)(Cullen et al., 1991). Although there is no experimental evidence for a site-specific integration of HPV DNA, numerous cervical cancers and cell lines estabThe nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number 94741. 1 To whom correspondence and reprint requests should be addressed. Fax: /49-6221-424932. E-mail: [email protected].
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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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contribute to the malignization of the infected cells. Furthermore, it has recently been demonstrated that replacement upon integration of a mRNA instability element located within the 3* untranslated region of HPV16 between nt 4005 and nt 4213 results in increased steady-state E6/E7 mRNA levels (Jeon and Lambert, 1995; Jeon et al., 1995). Such increased mRNA levels may further contribute to the malignization process. Additionally, in some cases viral integration is accompanied by a deletion of E5 ORF sequences (Wilczynski et al., 1988), suggesting that an intact E5 gene product may be dispensable, at least for the maintenance of the malignant phenotype of HPV-infected keratinocytes. HPV-containing human cell lines have been widely used as a model to analyze the molecular mechanisms involved in HPV gene regulation and cervical tumor development. Whereas some of these cell lines, e.g., the CaSki cell line, harbor more than several hundred copies of integrated HPV genomes (Pater and Pater, 1985), the SiHa cell line, which was initially established from a squamous cervical carcinoma of a Japanese patient (Friedl et al., 1970), contains only one or two copies of HPV-16 DNA integrated at the chromosomal locus 13q2131 (Mincheva et al., 1987). As previously shown, viral integration into the host genome was accompanied by a deletion of cellular DNA (El Awady et al., 1987; Baker et al., 1987). Although approximately 0.3 and 0.8 kb of human DNA sequences from the left and right sites of the HPV integration locus have been sequenced, no information is available about the cellular sequences that have been deleted upon integration. Such information would be of importance for investigation of the viral integration mechanism and its effect on chromosomal sequence organization, especially the possible loss of important host regulatory sequences that might be implicated in growth control. In this report we present information about the nonoccupied allele corresponding to the HPV-16 integration site in the SiHa cell line and demonstrate that viral integration was not followed by a reorganization of the host cell genome. Furthermore, we show data indicating that cellular–viral fusion transcripts can be produced in SiHa cells.
the 5* and 3* cellular flanking regions of the integrated viral DNA present in the SiHa genome. These fragments are identical to the L and R probes described by El Awady et al. (1987) and were kindly provided by Dr. R. D. Burk. After three cycles of hybridization and dilution, a total of nine individual positive plaques were selected for DNA isolation. The isolated DNAs were digested with different restriction enzymes, blotted onto nylon membranes, and hybridized to the above-mentioned DNA probes. A partial restriction map was constructed and DNA fragments containing sequences upstream and downstream of the HPV-16 integration site as well as fragments representing the DNA region deleted upon viral integration were subcloned into the pBluescript II KS vector and further analyzed. DNase I and micrococcus nuclease (MNase) digestion experiments Chromatin structure was analyzed by digestion of isolated nuclei with increasing amounts of either DNase I or MNase as previously described (Ro¨sl et al., 1989). Following nuclease digestion, DNA was isolated, digested with appropriate restriction enzymes, separated in agarose gels, and blotted onto nylon membranes. Hybridization probes specific for both the deleted and nondeleted alleles are outlined in Fig. 3. Nuclear run-on experiments Run-on experiments were essentially carried out as described by Linial et al. (1985). For each reaction, 5 1 107 nuclei were incubated in 10 mM Tris–Cl (pH 7.4), 5 mM MgCl2 , 300 mM KCl in the presence of 100 units of RNasin, 0.5 mM rNTPs, 8 mM UTP, and 100 mCi of [32P]UTP for 30 min at 307. For hybridization of slot blot filters, 2–3 1 106 cpm/ml of labeled RNA was used per filter. Hybridization conditions were as described earlier (Ro¨sl et al., 1989). Reverse transcriptase (RT)-PCR
A genomic library from human lymphocyte DNA (D’Onofrio et al., 1985) was used to isolate DNA fragments representing the nonoccupied allele corresponding to the HPV-16 integration site in the SiHa cell line. A total of 2 1 106 plaques were screened using 32P-labeled 0.5-kb BglII–HindIII and 0.45-kb HindIII–EcoRI fragments (depicted as A and B, respectively, in Fig. 1A) covering
RT-PCR was essentially performed according to Kawasaki (1990). Briefly, after an initial denaturation step at 957 for 5 min, 1–2.5 mg of RNA was reverse transcribed at 427 for 30 min using primers 2, 3, and 5 diagnostic for transcription of the sense strand of the E2 ORF, the E5 ORF, and the E6 ORF, respectively, and 60 units of SuperScript II reverse transcriptase (GIBCO BRL). Thereafter, the samples were subjected to 30 cycles of PCR amplification using primers 1 (5*-TTGTTGCACAGAGACTCAGT-3*) and 2 (5*-AAGTGTAACAATTGCACTTT-3*) for identification of transcripts containing sequences corresponding to the E2 and E4 ORFs, primers 1 and 3 (5*GTATGTAGACACAGACAAAAG-3*) for identification of transcripts corresponding to sequences out of the E2, E4, and E5 ORFs, or primers 4 (5*-TGCAATGTTTCAGGA-
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MATERIALS AND METHODS Screening of the genomic library, DNA isolation, and Southern blot analysis
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FIG. 1. Partial restriction maps of the HPV-16-containing allele and the corresponding nonoccupied allele of the genome of SiHa cells. (A) Schematic representation of the HPV-16-containing allele. Solid line, cellular sequences. Boxed area, HPV-16 sequences. (B) Partial restriction maps of lambda clones l1 and l7 which were isolated from a human genomic library from lymphocytes after screening with probes A and B, depicted as hatched boxes in A. (C) Following digestion of these two clones with HindIII–EcoRI, DNA fragments corresponding to the region of interest were subcloned into the vector pBluescript II KS and further analyzed. (D) Partial restriction map of the nonoccupied allele derived from data obtained by restriction and sequence analysis of the cloned fragments illustrated in C. Abbreviations for restriction endonucleases: A, ApaI; B, BglII; E, EcoRI; H, HindIII; K, KpnI; N, NdeI; P, PstI; S, SphI.
CCCACAG-3*) and 5 (5*-TATAGTTGTTTGCAGCTCTGT3*) for identification of E6 transcripts. These primer combinations produced PCR fragments of 240 base pairs out of the E2/E4 ORFs (primer set 1/2), 446 base pairs out of the E2/E4/E5 ORFs (primer set 1/3), and 72 base pairs out of the E6 ORF (primer set 4/5), respectively. PCR products were analyzed by agarose gel electrophoresis. Other methods Isolation of total and cytoplasmic RNA and Southern and Northern hybridization experiments and DNA sequencing were performed according to standard protocols (Sambrook et al., 1989). RESULTS Genomic cloning and characterization of the nonoccupied allele To isolate and characterize the DNA region deleted upon HPV-16 integration into the cellular genome in SiHa cells, a human genomic library was screened with 32Plabeled probes A and B covering the 5* and 3* cellular flanking regions, respectively (see Fig. 1A). After three screening cycles, two lambda clones, depicted as l1 and l7 (Fig. 1B), were found to contain overlapping fragments covering approximately 40 kb of cellular DNA close to the viral integration site. Following digestion of these clones with HindIII– EcoRI, DNA fragments p7.1, p7.2, p1.1, and p1.2 (see Fig. 1C) corresponding to the region of interest were subcloned into the pBluescript II KS vector for further analysis. Figure 1 shows a partial restriction map of both the HPV-16-containing and the wild-type (wt) alleles. Comparison of both maps revealed that viral integration was followed by a deletion of 7.8 kb of wt allele se-
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quences. To obtain information about the deleted DNA sequences adjacent to the 5* and 3* integration sites, we sequenced subclone p7.2 and part of subclone p1.2 (see Fig. 1C). Figure 2A shows the DNA sequences of the wt-allele surrounding the 5* and 3* integration sites. A canonical full-length repetitive Alu sequence is located at the 5* integration site and was disrupted upon HPV-16 integration. In contrast, a unique sequence is present at the 3* integration site with no significant homology to any known human sequences available in the EMBL-GenBank. Interestingly, sequence homologies of four base pairs between HPV-16 and cellular sequences could be identified at both host–viral junctions (5*-TATT-3* and 5*ATGC-3* at the 5* and 3* integration sites, respectively; see Fig. 2B). Furthermore, small direct repeats are present at these junctions. A direct repeat of the sequence ATTTTT separated by two nucleotides was found at the 5* junction, and a TGC direct repeat without any spacer sequences is located at the 3* junction. These data suggest that insertion of the viral sequence into the host genome took place by a recombination event between two small direct repeats. In addition, no significant ORFs were found in both DNA strands of a total of about 2.3 kb of deleted DNA and 1.0 kb of sequences upstream of the 5* viral integration site. In addition, hybridization of the subcloned DNA fragments with radioactively labeled total genomic DNA resulted in strong hybridization signals, suggesting the presence of repetitive sequences in each of these fragments (data not shown). Analysis of the chromatin structure upstream of the 5* HPV integration site It has been shown that digestion of isolated nuclei or permeabilized cells with randomly cutting nucleases,
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FIG. 2. Human genomic DNA sequences at the 5* and 3* host–viral junctions in the genome of SiHa cells. (A) Clones p7.2 and p1.2 (see Fig. 1C) were partially sequenced by the dideoxy method. Sequences deleted upon HPV-16 integration are shown in bold italics. Small direct ATTTTT and TGC repeats found in the wt-allele at the 5* and 3* integration sites, respectively, are underlined. Asterisks above the sequence depict a canonical Alu repeat present at the 5* viral integration site. (B) Alignment of cellular and viral sequences at the 5* (top) and 3* (bottom) integration sites, showing the small regions of sequence homology at the 5* and 3* integration sites (TATT and ATGC, respectively; underlined), as well as the direct repeats (bold italics) present in the wt-allele.
such as DNase I, is a suitable method for analyzing chromatin structure. Such an analysis allows identification of hypersensitive sites that often coincide with regulatory sequences involved in transcription, replication, and recombination (Elgin, 1984; Hutchison and Weintraub, 1985). Molecular cloning of the wt-allele together with knowledge about the HPV-16-containing allele (El Awady et al., 1987; Baker et al., 1987) enabled us to analyze the chromatin organization of both alleles in independent mapping experiments. To address the question of whether HPV integration was accompanied by a change in cellular chromatin and/or took place in the vicinity of
regulatory elements, DNase I mapping analyses were performed. For this approach, nuclei were isolated from SiHa cells and treated with increasing amounts of either DNase I or MNase as described (Ro¨sl et al., 1989). Isolated genomic DNA was digested with either KpnI or ApaI, separated on agarose gels, and blotted onto nylon membranes. To analyze the chromatin structure of the HPV-16-containing allele, ApaI-digested DNA was hybridized to a 32P-labeled PstI–ApaI DNA fragment encompassing 836 bp of HPV-16 sequences (nucleotide positions 3693 to 4529, covering E5 and part of L2 ORFs). As shown in Fig. 3A,
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stream of the 5* HPV integration site seem to be present in both wt-and HPV-16-containing alleles. Similar hybridization patterns were observed when MNase was used for mapping analysis (data not shown). Taken together, these results demonstrate that integration of HPV-16 DNA into the genome of SiHa cells was not followed by reorganization of the chromatin structure upstream of the viral integration site. Transcription of HPV-16 sequences
FIG. 3. DNase I mapping analysis of the 5* cellular region of the HPV-16 integration site in the genome of SiHa cells. Isolated nuclei of SiHa cells were treated with 0.1, 0.2, 0.5, 1, and 4 units of DNase I (lanes 3 to 7, respectively). Following isolation, 10 mg of DNA was digested either with ApaI (A) or with KpnI (B), separated in 0.8% agarose gels, and transferred onto nylon membranes. For hybridization, a 32Plabeled PstI–ApaI DNA fragment covering approximately 0.8 kb of HPV16-specific DNA sequences (A) and a 526-bp SphI–NdeI fragment that had been deleted during viral integration (B; depicted as fragment C in Fig. 1D) were used. Lanes 1 and 2, controls without DNase I treatment. Taking into account the known positions for ApaI and KpnI cleavage, the radioactive signals at approximately 2.3 and 2.9 kb (A) and 4.0 and 4.6 kb (B) (middle and lower arrows) correspond to two DNase I hypersensitive sites (DHSS) located approximately 1.1 and 1.7 kb upstream of the HPV-16 integration site. The upper arrows indicate the positions of the parental restriction fragments of approximately 9 kb (A) and 18 kb (B). In the lower part of the figure, the HPV-16-containing and wt-alleles are schematically outlined showing the two DHSS (marked with arrows 1 and 2). Abbreviations for restriction endonucleases: H, HindIII; K, KpnI; N, NdeI; S, SphI.
As schematically outlined in Fig. 4, integration of HPV16 sequences into the host DNA in SiHa cells resulted in disruption of the viral URR-driven transcription unit and in location of both putative viral polyadenylation sites upstream of the URR transcription start point. Therefore, we were interested in whether the 5* DNA region, containing structurally intact E5, L2, and L1 ORFs, would be transcribed as fusion transcripts containing cellular and viral sequences. Northern hybridization experiments using SiHa cell RNA and a 32P-labeled E5 probe did not reveal any signals, even after long exposure times, suggesting that if transcripts exist, they must be present at a very low concentration. To further address this question, nuclear run-on experiments using different HPV-16-specific DNA fragments as probes were performed. Following immobilization of these fragments onto a nylon membrane, they were hybridized to [32P]UTP-labeled total RNA transcribed in vitro using nuclei isolated from SiHa cells. The results of these experiments are shown in Fig. 4. A 3.4-kb DNA fragment encompassing the E6/E7 ORFs located downstream of the HPV-16 URR gave a strong
DNA fragments of approximately 2.3 and 2.9 kb in length were detected, in addition to the signal for the parental restriction fragment of approximately 9 kb. Taking the known positions for ApaI cleavage (nt 4529) and HPV-16 integration (nt 3384) into account, these results suggest the presence of two hypersensitive sites located approximately 1.1 and 1.7 kb upstream of the 5* viral integration site. For analysis of the nonoccupied allele, KpnI-digested DNA was hybridized to a 526-bp SphI–NdeI restriction fragment that had been deleted upon HPV-16 integration (see Fig. 1D) and was found to be devoid of repetitive sequences. In addition to the signal for the parental restriction fragment of approximately 18 kb, two hybridizing fragments of approximately 4.0 and 4.6 kb in size were found, which also corresponds to hypersensitive sites located 1.1 and 1.7 kb upstream of the viral integration site (Fig. 3B). Thus, the same hypersensitive sites up-
FIG. 4. Analysis of HPV-16 transcription in SiHa cells by nuclear run-on analysis. 2.5 mg of different HPV-16-specific DNA fragments was heat-denatured and spotted onto nylon membranes. The membrane was hybridized to [32P]UTP-labeled RNA transcribed from SiHa nuclei. 1, 1.5 kb XcmI– KpnI fragment (HPV-16 nucleotides 3879– 5383); 2, 3.4 kb KpnI –KpnI fragment (nt 5383 –884); 3, pBluescript II KS fragment (negative control); 4, pUC 19 fragment (negative control); 5, cellular DNA (positive control). In the lower part of the figure, the HPV-16-containing allele is schematically outlined, showing the locations of the HPV-16-specific DNA probes used (hatched boxes, marked 1 and 2).
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hybridization signal (slot 2). Interestingly, a hybridization signal, although with weaker intensity, was also obtained when using a fragment containing the E5 and part of the L2 ORFs, located 5* of the viral URR (slot 1). Total cellular DNA (slot 5) used as positive control showed a strong hybridization signal, whereas no signals could be observed with pBluescript II KS and pUC 19 vector DNA (slots 3 and 4). To confirm these results, we performed RT-PCR using total SiHa RNA and primers specific for either the E2/E4 or E6 ORFs. After agarose gel electrophoresis, the PCR products were transferred onto a nylon membrane and hybridized to the entire HPV-16 genome. As can be seen in Fig. 5A, a strong hybridization signal was observed when using the E6-specific primers (lane 1). A weaker signal was found using the E2/E4-specific primers (lane 3). The signals were not the result of contaminating DNA since there was no signal when reverse transcriptase was omitted. To further strengthen these data that suggested transcription of HPV-16 ORFs located 5* of the HPV-16 URR, we performed RT-PCR on cytoplasmic SiHa RNA using two primers spanning a DNA fragment of 446 bp in size of the upstream E2, E4, and E5 ORFs. As illustrated in Fig. 5B, a fragment of expected length was obtained that hybridized to a 32P-labeled E5-specific
FIG. 6. Southern blot analysis of cervical carcinomas. A total of 10 mg of genomic DNA from four cervical carcinomas (lanes 1 to 3, 5 to 8, 10) and from SiHa cells (lanes 4 and 9) was digested with HindIII, separated in an 0.8% agarose gel, transferred onto a nylon membrane, and hybridized to 32P-labeled probes A (compare Fig. 1A, lanes 1 to 5) and B (compare Fig. 1A, lanes 6 to 10). Note the absence of hybridization signals in tumor sample #1 (lanes 1 and 6).
probe. These results, together with the data obtained by nuclear run-on analysis described above, suggest that transcription of HPV sequences is initiated from two different promoter elements: a weak cellular one located 5* of the viral integration site and a second, stronger one, probably corresponding to the P97 promoter within the HPV-16 URR. Thus, SiHa cells seem to express host– viral fusion transcripts initiated from a weak promoter located within the cellular host sequences upstream of the 5* HPV-16 integration site. Genomic organization of the wt-allele in human cervical tumors
FIG. 5. Analysis of HPV-16 transcription in SiHa cells by RT-PCR. (A) 2.5 mg of total cellular RNA was used for RT-PCR with either E6-specific primers 4 and 5 (lanes 1 and 2) or E2/E4-specific primers 1 and 2 (lanes 3 and 4), spanning DNA fragments of 72 bp (E6 ORF) or 240 bp (E2/E4 ORF). Reactions were performed in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of reverse transcriptase. An aliquot of 15 ml out of a total of 50 ml of each PCR mixture was separated in a 2% agarose gel, transferred onto a nylon membrane, and hybridized to a 32P-labeled probe covering the entire HPV-16 genome. (B) One microgram of cytoplasmic RNA was used for RT-PCR with primers 1 and 3, specific for amplification of a 446-bp DNA fragment out of the E2, E4, and E5 ORFs (lane 1). Lane 2, RT-PCR after RNase A digestion. Lane 3, template-free negative control. Ten microliters (out of a total of 50 ml) was separated in a 2% agarose gel, transferred onto a nylon membrane, and hybridized to a 32P-labeled E5-specific probe. In the lower part of the figure, the HPV-16-harboring allele is schematically outlined, showing the locations of PCR oligonucleotides 1 to 5 used for RT-PCR.
Although no specific integration sites have been described for papillomavirus DNA, several reports have shown that some chromosomal loci are preferred to others (Popescu and DiPaolo, 1989). Therefore we analyzed the structure of the wt-allele described in this report in human tumors by Southern hybridization. Genomic DNA was isolated from four HPV-16-containing cervical carcinomas and from SiHa cells, digested with HindIII, blotted onto a nylon membrane, and hybridized to probes A and B (compare Fig. 1A). Signals at approximately 4.6 kb (probe A, lanes 2–5) and 4.7 kb (probe B, lanes 7–10) could be observed in three of the four tumors analyzed. Interestingly, no signal was observed in tumor sample #1 (Fig. 6, lanes 1 and 6), suggesting that the corresponding DNA region was deleted in this particular tumor. As expected, two hybridizing bands were found with SiHa DNA, corresponding to the wild-type and the occupied alleles. These results indicate that within one of four tumors analyzed, genomic reorganization took place at the same locus which was altered in the genome of SiHa cells.
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Genomic analysis carried out on human cervical cancers containing high-risk HPVs, such as HPV-16 or HPV18, has shown that the viral sequences are frequently integrated into the host genome (Cullen et al., 1991). In some studies, evidence has been provided that integration of HPV genomes occurs at or near fragile sites (Cannizzaro et al., 1988; De Braekeleer et al., 1992), DNA regions that have been considered as regions of genomic instability (Popescu and DiPaolo, 1989; Zimonjic et al., 1994), and in some cases at sites proximal to genes relevant for control of growth and differentiation (Du¨rst et al., 1987; Couturier et al., 1991). Furthermore, some chromosomes, such as chromosomes 3 and 8, seem to be more accessible to integration than others (Popescu and DiPaolo, 1989). In some cell lines derived from HPVcontaining cervical cancers, integration near protooncogenes, such as c-myc (Du¨rst et al., 1987; Couturier et al., 1991), has been described, suggesting that viral integration may cause alterations in the expression of these genes. Integration of HPVs into the host genome is frequently followed by deletion of host cellular sequences. In M50 cells, for example, a deletion of approximately 1.1 kb occurred at the integration site of HPV-16 (Choo et al., 1990). In the Sk-v cell line, which was established from a Bowen’s carcinoma of the vulva, integration of HPV-16 at a single site resulted in a deletion of approximately 5.5 kb of cellular DNA (Schneider-Maunoury et al., 1987). In agreement with data presented by El Awady et al. (1987) and Baker et al. (1987), our results indicate that integration of HPV-16 DNA into the genome of SiHa cells was followed by a deletion of host sequences. Restriction mapping of the wt-allele cloned in this study allowed us to state the size of the deleted region more precisely as 7.8 kb. We sequenced a total of 3.3 kb of DNA, comprising 2.3 kb of sequences from the deleted region and 1.0 kb of cellular sequences upstream of the 5* viral integration site. Computer analysis of these sequences with GenBank did not reveal significant homologies to known human sequences nor were any significant ORFs found. In addition, Northern analyses of RNA isolated from different epithelial cell lines (HeLa, CaSki, C4-I, and SiHa) using fragments p1.1 and p1.2 (see Fig. 1C) as probes did not reveal any appreciable signals, suggesting that these sequences are either not transcribed or transcribed at a very low rate. Sequence analysis of the deleted fragment showed that integration of viral DNA took place in the middle of an Alu repeat at the 5* host–viral junction, whereas a unique sequence was present at the 3* junction. Comparison of the DNA sequences of the wt- and the HPVcontaining alleles revealed a homology of four base pairs
at both host–viral junctions (see Fig. 2). Similarly, when analyzing the integration locus of HPV-16 DNA in a cervical cancer biopsy, Du¨rst et al. (1986) observed a fourbase pair homology between viral and host sequences. Moreover, small direct repeats that overlap both junctions were identified in the wt-allele, suggesting their involvement in the recombination process between host and viral DNA sequences. A similar kind of integration mechanism has been suggested by Choo et al. (1990) for integration of HPV-16 DNA into the host genome in a primary cervical carcinoma. Interestingly, short patches of sequence homology at the host–virus junctions are also a common finding for adeno- and polyomaviruses (Gahlmann et al., 1982; Ruley and Fried, 1983). Thus, it is tempting to speculate that the short sequence homologies observed at both host–viral junctions in SiHa cells may play a role in the integration process, similar to that found for adeno- and polyomaviruses. Chromatin analysis of the viral integration locus in HeLa cells revealed that HPV-18 DNA is integrated in the vicinity of a cellular promoter which cooperates with HPV-18 URR in early transcription of viral genes (Ro¨sl et al., 1989). In the present study, we were interested in whether integration of HPV-16 DNA into the host genome in SiHa cells impairs transcription of viral ORFs due to chromosomal position effects. It is well known that such effects frequently affect transcriptional activation of, e.g., transgenes (Wilson et al., 1990). It has recently been demonstrated for the chicken lysozyme gene that suppression of gene expression in transgenic mice due to chromosomal position effects correlates with alterations in DNase I hypersensitive site (DHSS) formation. Moreover, DHSS formation on cis-regulatory elements seems to correlate directly with transcriptional activation of chicken lysozyme transgenes, suggesting that positionindependent gene expression in transgenic mice is dependent on regeneration of the original DHSS pattern (Huber et al., 1994). In our study, we provide evidence that two DNase I hypersensitive sites are present, approximately 1.1 and 1.7 kb upstream of the 5* host–viral junction, in both the wt- and the HPV-16-containing alleles. Thus, integration of HPV-16 DNA occurred near a hypersensitive region in SiHa cells. However, this integration event did not lead to major chromatin changes. Transcriptional analysis of HPV-16 ORFs in SiHa cells demonstrated the presence of viral transcripts from the E6 and E7 ORFs, mapping downstream of the HPV-16 URR, as well as from the E2, E4, E5, and the L2/L1 ORFs, located upstream of the viral URR, close to the 5* viral integration site (see Figs. 4 and 5). The strong quantitative differences in the signal intensities between upstream and downstream ORFs suggest that two different promoters are used for transcription of HPV-16 ORFs: one upstream of the 5* host–viral junction and another one, probably the P97 promoter, within the viral URR. The
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Baker, C. C., Phelps, W. C., Lindgren, V., Braun, M. J., Gonda, M. A., and Howley, P. M. (1987). Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J. Virol. 61, 962–971. Cannizzaro, L. A., Du¨rst, M., Mendez, M. J., Hecht, B. K., and Hecht, F. (1988). Regional chromosome localization of human papillomavirus integration sites near fragile sites, oncogenes, and cancer chromosome breakpoints. Cancer Genet. Cytogenet. 33, 93–98. Choo, K. B., Lee, H. H., Liew, L. N., Chong, K. Y., and Chou, H. F. (1990).
Analysis of the unoccupied site of an integrated human papillomavirus 16 sequence in a cervical carcinoma. Virology 178, 621–625. Couturier, J., Sastre-Garau, X., Schneider-Maunoury, S., Labib, A., and Orth, G. (1991). Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for proto-oncogene expression. J. Virol. 65, 4534–4538. Crook, T., Morgenstern, J. P., Crawford, L., and Banks, L. (1989). Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV-16 plus EJ-ras. EMBO J. 8, 513–519. Cullen, A. P., Reid, R., Campion, M., and Lorincz, A. T. (1991). Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. J. Virol. 65, 606–612. Das, B. C., Gopalkrishna, V., Das, D. K., Sharma, J. K., Singh, V., and Luthra, U. K. (1993). Human papillomavirus DNA sequences in adenocarcinoma of the uterine cervix in Indian women. Cancer 72, 147– 153. De Braekeleer, M., Sreekantaiah, C., and Haas, O. (1992). Herpes simplex virus and human papillomavirus sites correlate with chromosomal breakpoints in human cervical carcinoma. Cancer Genet. Cytogenet. 59, 135–137. de Villiers, E.-M. (1994). Human pathogenic papillomavirus types: An update. Curr. Top. Microbiol. Immunol. 186, 1–12. D’Onofrio, C., Colantuoni, V., and Cortese, R. (1985). Structure and cellspecific expression of a cloned human retinol binding protein gene: The 5*-flanking region contains hepatoma specific transcriptional signals. EMBO J. 4, 1981–1989. Du¨rst, M., Schwarz, E., and Gissmann, L. (1986). Integration and persistence of human papillomavirus DNA in genital tumors. In ‘‘Banury Report 21: Viral Etiology of Cervical Cancer’’ (R. Peto and H. zur Hausen, Eds.), pp. 273–280. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Du¨rst, M., Croce, C. M., Gissmann, L., Schwarz, E., and Huebner, K. (1987). Papillomavirus sequences integrate near cellular oncogenes in some cervical carcinomas. Proc. Natl. Acad. Sci. USA 84, 1070– 1074. El Awady, M. K., Kaplan, J. B., O’Brien, S. J., and Burk, R. D. (1987). Molecular analysis of integrated human papillomavirus 16 sequences in the cervical cancer cell line SiHa. Virology 159, 389– 398. Elgin, S. C. R. (1984). Anatomy of hypersensitive sites. Nature 309, 213– 214. Friedl, F., Kimura, I., Osato, T., and Ito, Y. (1970). Studies on a new human cell line (SiHa) derived from carcinoma of uterus. I. Its establishment and morphology. Proc. Soc. Exp. Biol. Med. 135, 543–545. Gahlmann, R., Leisten, R., Vardimon, L., and Doerfler, W. (1982). Patch homologies and the integration of adenovirus DNA in mammalian cells. EMBO J. 1, 1101–1104. Halbert, C. L., Demers, G. W., and Galloway, D. A. (1991). The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65, 473–478. Hawley-Nelson, P., Vousden, K. H., Hubbert, N. L., Lowy., D. R., and Schiller, J. T. (1989). HPV-16 E6 and E7 proteins cooperate to immortalize primary human foreskin keratinocytes. EMBO J. 8, 3905–3910. Huber, M. C., Bosch, F. X., Sippel, A. E., and Bonifer, C. (1994). Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation. Nucleic Acids Res. 22, 4195–4201. Hutchison, N., and Weintraub, H. (1985). Localization of DNase I-sensitive sequences to specific regions of interphase nuclei. Cell 43, 471– 482. Jeon, S., and Lambert, P. F. (1995). Integration of human papillomavirus type 16 DNA into the human genome leads to increased stability of E6 and E7 mRNAs: Implications for cervical carcinogenesis. Proc. Natl. Acad. Sci. USA 92, 1654–1658. Jeon, S., Allen-Hoffmann, B. L., and Lambert, P. F. (1995). Integration
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proximity of the primers used for RT-PCR of E2/E4/E5 ORF sequences to the 5* host–viral junction together with the presence of two DHSS immediately upstream of the 5* viral integration site suggests the presence of host–viral fusion transcripts. Viral–cellular fusion transcripts have been found to be more stable than intact viral copies (Kennedy et al., 1991). Very recently, it was demonstrated that replacement of an A / U-rich mRNA instability element, which is present within the viral 3* untranslated region of HPV-16 between nt 4005 and nt 4213, by cellular sequences upon integration of HPV-16 DNA into the human genome can result in increased steady-state levels of E6- and E7-specific mRNAs through altered mRNA stability (Jeon and Lambert, 1995; Jeon et al., 1995). Integration of HPV-16 DNA into the genome in SiHa cells took place at HPV-16 nt 3132 accompanied by a small deletion of 251 bp of viral sequences (nt 3133–3384) and did therefore not lead to a disruption of the viral 3* UTR. Nevertheless, it is not known whether this region contains cis-elements that might affect URR-driven viral transcription and should be further investigated. Alterations in the genomic organization at the locus corresponding to the HPV-16 integration site in the SiHa cell line were found in one of four human cervical carcinomas analyzed. Although the number of tumors analyzed is too small to allow an unambiguous conclusion to be drawn concerning the biological significance of these genomic alterations, our results suggest the presence of a genetically instable chromosomal locus at the HPV-16 integration site. Cloning and characterization of such regions from a greater number of human tumors would be necessary to assess the possible importance of the integration sites for the biological activity of integrated papillomavirus sequences in infected human cells. ACKNOWLEDGMENTS We thank Dr. R. D. Burk for providing us with the L and R probes of the HPV-16-containing allele of SiHa cells and Dr. M. Du¨rst for the cloned HPV-16 fragment from SiHa cells as well as for the cervical tumor samples analyzed in this study. We also thank Dr. M. Du¨rst for critical reading of the manuscript and J. Fischer for excellent technical assistance.
REFERENCES
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of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells. J. Virol. 69, 2989–2997. Kawasaki, E. S. (1990). Amplification of RNA. In ‘‘PCR Protocols: A Guide to Methods and Applications’’ (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, Eds.), pp. 21–27. Academic Press, San Diego. Kennedy, I. M., Haddow, J. K., and Clements, J. B. (1991). A negative regulatory element in the human papillomavirus type 16 genome acts at the level of late mRNA stability. J. Virol. 65, 2093–2097. Linial, M., Gunderson, N., and Groudine, M. (1985). Enhanced transcription of c-myc in bursal lymphoma cells requires continuous protein synthesis. Science 230, 1126–1132. Meijer, C. J. L. M., van den Brule, A. J. C., Snijders, P. J. F., Helmerhorst, T., Kenemans, P., and Walboomers, J. M. M. (1992). Detection of human papillomavirus in cervical scrapes by the polymerase chain reaction in relation to cytology: Possible implications for cervical cancer screening. In ‘‘The Epidemiology of Cervical Cancer and Human Papillomavirus’’ (N. Munoz, F. X. Bosch, K. V. Shah, and A. Meheus, Eds.), pp. 271–281. Int. Agency Res. Cancer, Lyon. Mincheva, A., Gissmann, L., and zur Hausen, H. (1987). Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridization. Med. Microbiol. Immunol. 176, 245–256. Mu¨nger, K., Phelps, W. C., Bubb, V., Howley, P. M., and Schlegel, R. (1989). The E6 and E7 genes of the human papilllomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol. 63, 4417–4421. Pater, M. M., and Pater, A. (1985). Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix. Virology 145, 313–318. Popescu, N. C., and DiPaolo, J. A. (1989). Preferential sites for viral integration on mammalian genome. Cancer Genet. Cytogenet. 42, 157–171. Romanczuk, H., and Howley, P. M. (1992). Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity. Proc. Natl. Acad. Sci. USA 89, 3159– 3163. Ro¨sl, F, Westphal, E.-M., and zur Hausen, H. (1989). Chromatin structure and transcriptional regulation of human papillomavirus type 18 DNA in HeLa cells. Mol. Carcinog. 2, 72–80. Ruley, H. E., and Fried, M. (1983). Clustered illegitimate recombination
events in mammalian cells involving very short sequence homologies. Nature 304, 181–184. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). ‘‘Molecular Cloning: A Laboratory Manual,’’ 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schneider-Maunoury, S., Croissant, O., and Orth, G. (1987). Integration of human papillomavirus type 16 DNA sequences: A possible early event in the progression of genital tumors. J. Virol. 61, 3295–3298. Schwarz, E., Freese, U. K., Gissmann, L., Mayer, W., Roggenbuck, B., Stremlau, A., and zur Hausen, H. (1985). Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314, 111–114. Smotkin, D., and Wettstein, F. O. (1986). Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancerderived cell line and identification of the E7 protein. Proc. Natl. Acad. Sci. USA 83, 4680–4684. Thierry, F. (1993). Proteins involved in the control of HPV transcription. Papillomavirus Rep. 4, 27–32. von Knebel Doeberitz, M., Oltersdorf, T., Schwarz, E., and Gissmann L. (1988). Correlation of modified human papilloma virus early gene expression with altered growth properties in C4-1 cervical carcinoma cells. Cancer Res. 48, 3780–3786. Wilczynski, S. P., Pearlman, L., and Walker, J. (1988). Identification of HPV-16 early genes retained in cervical carcinomas. Virology 166, 624–627. Wilson, C., Bellen, H. J., and Gehring, W. J. (1990). Position effects on eukaryotic gene expression. Annu. Rev. Cell Biol. 6, 679–714. Zimonjic, D. B., Popescu, N. D., and DiPaolo, J. A. (1994). Chromosomal organization of viral integration sites in human papillomavirus-immortalized human keratinocyte cell lines. Cancer Genet. Cytogenet. 72, 39–43. zur Hausen, H. (1991a). Human papillomaviruses in the pathogenesis of anogenital cancer. Virology 184, 9–13. zur Hausen H. (1991b). Papillomavirus–host cell interactions in the pathogenesis of anogenital cancer. In ‘‘Origins of Human Cancer: A Comprehensive Review’’ (J. Brugge, T. Curran, E. Harlow, and F. McCormick, Eds.), pp. 685–705. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. zur Hausen, H., and de Villiers, E.-M. (1994). Human papillomaviruses. Annu. Rev. Microbiol. 48, 427–447. zur Hausen H., and Schneider. A. (1987). The role of papillomaviruses in human anogenital cancers. In ‘‘The Papovaviridae’’ (N. P. Salzman and P. M. Howley, Eds.), pp. 245–263. Plenum Press, NY.
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Genomic Cloning and Characterization of the Nonoccupied Allele Corresponding to the Integration Site of Human Papillomavirus Type 16 DNA in the Cervical Cancer Cell Line SiHa ¨ SL, and A. ALONSO1 R. BAUER-HOFMANN, C. BORGHOUTS, E. AUVINEN, E. BOURDA, F. RO Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, 69120 Heidelberg, Federal Republic of Germany Received May 15, 1995; accepted December 18, 1995 Human papillomavirus (HPV) type 16 DNA sequences have been found integrated into the host cell genome in a large number of cervical tumors and cell lines derived therefrom. In this study, we have cloned and analyzed the nonoccupied allele corresponding to the integration site of HPV-16 in the cervical cancer cell line SiHa. Our mapping analyses revealed an approximately 7.8-kb deletion of cellular DNA upon viral integration. Computer analysis of 2.3 kb of DNA sequences from the deleted genomic region as well as 1.0 kb of sequences upstream of the viral integration site showed no significant homology to any known human sequences. DNase I mapping experiments on native chromatin demonstrated the existence of two hypersensitive sites in both the HPV-16-containing and nonoccupied alleles located approximately 1.1 and 1.7 kb upstream of the viral integration site. This suggests that viral integration occurred close to putative regulatory sequences and that recombination with host cellular DNA was not followed by a reorganization of the chromatin structure upstream of the integration site. Nuclear run-on and RT-PCR experiments showed HPV-specific transcription spanning the E2, E4, E5, and L1/L2 open reading frames (ORFs) located upstream of the HPV-16 regulatory region (URR). Taken together, our data suggest that the cellular DNA region upstream of the HPV-16 integration site in the SiHa cell line contains regulatory elements affecting transcription of HPV-16 ORFs located upstream of the HPV-16 URR. q 1996 Academic Press, Inc.
INTRODUCTION
lished thereof showed preferential integration at or near fragile sites or protooncogenes (Du¨rst et al., 1987; Cannizzaro et al., 1988; Popescu and DiPaolo, 1989; Couturier et al., 1991; De Braekeleer et al., 1992). These findings suggest that integration may not only affect viral transcription but also could be accompanied by activation or inactivation of cellular genes involved in the control of cell proliferation and/or differentiation. The ORFs encoding the viral oncogenes E6 and E7, as well as the viral upstream regulatory region (URR), are consistently retained and transcribed in HPV integrates (Schwarz et al., 1985; Smotkin and Wettstein, 1986; Baker et al., 1987). Both viral gene products are involved in immortalization of human epithelial cells (Hawley-Nelson et al., 1989; Mu¨nger et al., 1989; Halbert et al., 1991), and their continuous transcription seems to be necessary for the maintenance of the malignant phenotype (von Knebel Doeberitz et al., 1988; Crook et al., 1989). Integration of HPV DNA frequently leads to an interruption of the viral E1 and E2 ORFs (Romanczuk and Howley, 1992). The E2 protein has been proposed to negatively interfere with transcription from the E6 and E7 ORFs through binding to its binding sites located within the URR (Thierry, 1993). Functional inactivation of the E2 ORF upon integration might therefore lead to an enhanced transcription from the E6 and E7 oncogenes which could
Human papillomavirus (HPV) DNA sequences have been identified in a variety of benign and malignant cervical tumors (zur Hausen and de Villiers, 1994). Whereas benign proliferations usually harbor low-risk HPVs, e.g., types 6 and 11, in malignant lesions high-risk HPV types, such as 16, 18, and 33, are predominant (zur Hausen and Schneider, 1987; zur Hausen, 1991a,b; de Villiers, 1994). The occurrence of particular HPV types in up to 90% of carcinomas (Meijer et al., 1992; Das et al., 1993) strongly suggests that viral infection is causally involved in the development of this type of cancer. Analysis of the physical state of HPV DNA has revealed that benign lesions mostly contain the viral sequences as episomes, whereas in malignant lesions the viral DNA is usually integrated into the host genome with subsequent disruption of one or more viral open reading frames (ORFs)(Cullen et al., 1991). Although there is no experimental evidence for a site-specific integration of HPV DNA, numerous cervical cancers and cell lines estabThe nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number 94741. 1 To whom correspondence and reprint requests should be addressed. Fax: /49-6221-424932. E-mail: [email protected].
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contribute to the malignization of the infected cells. Furthermore, it has recently been demonstrated that replacement upon integration of a mRNA instability element located within the 3* untranslated region of HPV16 between nt 4005 and nt 4213 results in increased steady-state E6/E7 mRNA levels (Jeon and Lambert, 1995; Jeon et al., 1995). Such increased mRNA levels may further contribute to the malignization process. Additionally, in some cases viral integration is accompanied by a deletion of E5 ORF sequences (Wilczynski et al., 1988), suggesting that an intact E5 gene product may be dispensable, at least for the maintenance of the malignant phenotype of HPV-infected keratinocytes. HPV-containing human cell lines have been widely used as a model to analyze the molecular mechanisms involved in HPV gene regulation and cervical tumor development. Whereas some of these cell lines, e.g., the CaSki cell line, harbor more than several hundred copies of integrated HPV genomes (Pater and Pater, 1985), the SiHa cell line, which was initially established from a squamous cervical carcinoma of a Japanese patient (Friedl et al., 1970), contains only one or two copies of HPV-16 DNA integrated at the chromosomal locus 13q2131 (Mincheva et al., 1987). As previously shown, viral integration into the host genome was accompanied by a deletion of cellular DNA (El Awady et al., 1987; Baker et al., 1987). Although approximately 0.3 and 0.8 kb of human DNA sequences from the left and right sites of the HPV integration locus have been sequenced, no information is available about the cellular sequences that have been deleted upon integration. Such information would be of importance for investigation of the viral integration mechanism and its effect on chromosomal sequence organization, especially the possible loss of important host regulatory sequences that might be implicated in growth control. In this report we present information about the nonoccupied allele corresponding to the HPV-16 integration site in the SiHa cell line and demonstrate that viral integration was not followed by a reorganization of the host cell genome. Furthermore, we show data indicating that cellular–viral fusion transcripts can be produced in SiHa cells.
the 5* and 3* cellular flanking regions of the integrated viral DNA present in the SiHa genome. These fragments are identical to the L and R probes described by El Awady et al. (1987) and were kindly provided by Dr. R. D. Burk. After three cycles of hybridization and dilution, a total of nine individual positive plaques were selected for DNA isolation. The isolated DNAs were digested with different restriction enzymes, blotted onto nylon membranes, and hybridized to the above-mentioned DNA probes. A partial restriction map was constructed and DNA fragments containing sequences upstream and downstream of the HPV-16 integration site as well as fragments representing the DNA region deleted upon viral integration were subcloned into the pBluescript II KS vector and further analyzed. DNase I and micrococcus nuclease (MNase) digestion experiments Chromatin structure was analyzed by digestion of isolated nuclei with increasing amounts of either DNase I or MNase as previously described (Ro¨sl et al., 1989). Following nuclease digestion, DNA was isolated, digested with appropriate restriction enzymes, separated in agarose gels, and blotted onto nylon membranes. Hybridization probes specific for both the deleted and nondeleted alleles are outlined in Fig. 3. Nuclear run-on experiments Run-on experiments were essentially carried out as described by Linial et al. (1985). For each reaction, 5 1 107 nuclei were incubated in 10 mM Tris–Cl (pH 7.4), 5 mM MgCl2 , 300 mM KCl in the presence of 100 units of RNasin, 0.5 mM rNTPs, 8 mM UTP, and 100 mCi of [32P]UTP for 30 min at 307. For hybridization of slot blot filters, 2–3 1 106 cpm/ml of labeled RNA was used per filter. Hybridization conditions were as described earlier (Ro¨sl et al., 1989). Reverse transcriptase (RT)-PCR
A genomic library from human lymphocyte DNA (D’Onofrio et al., 1985) was used to isolate DNA fragments representing the nonoccupied allele corresponding to the HPV-16 integration site in the SiHa cell line. A total of 2 1 106 plaques were screened using 32P-labeled 0.5-kb BglII–HindIII and 0.45-kb HindIII–EcoRI fragments (depicted as A and B, respectively, in Fig. 1A) covering
RT-PCR was essentially performed according to Kawasaki (1990). Briefly, after an initial denaturation step at 957 for 5 min, 1–2.5 mg of RNA was reverse transcribed at 427 for 30 min using primers 2, 3, and 5 diagnostic for transcription of the sense strand of the E2 ORF, the E5 ORF, and the E6 ORF, respectively, and 60 units of SuperScript II reverse transcriptase (GIBCO BRL). Thereafter, the samples were subjected to 30 cycles of PCR amplification using primers 1 (5*-TTGTTGCACAGAGACTCAGT-3*) and 2 (5*-AAGTGTAACAATTGCACTTT-3*) for identification of transcripts containing sequences corresponding to the E2 and E4 ORFs, primers 1 and 3 (5*GTATGTAGACACAGACAAAAG-3*) for identification of transcripts corresponding to sequences out of the E2, E4, and E5 ORFs, or primers 4 (5*-TGCAATGTTTCAGGA-
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MATERIALS AND METHODS Screening of the genomic library, DNA isolation, and Southern blot analysis
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FIG. 1. Partial restriction maps of the HPV-16-containing allele and the corresponding nonoccupied allele of the genome of SiHa cells. (A) Schematic representation of the HPV-16-containing allele. Solid line, cellular sequences. Boxed area, HPV-16 sequences. (B) Partial restriction maps of lambda clones l1 and l7 which were isolated from a human genomic library from lymphocytes after screening with probes A and B, depicted as hatched boxes in A. (C) Following digestion of these two clones with HindIII–EcoRI, DNA fragments corresponding to the region of interest were subcloned into the vector pBluescript II KS and further analyzed. (D) Partial restriction map of the nonoccupied allele derived from data obtained by restriction and sequence analysis of the cloned fragments illustrated in C. Abbreviations for restriction endonucleases: A, ApaI; B, BglII; E, EcoRI; H, HindIII; K, KpnI; N, NdeI; P, PstI; S, SphI.
CCCACAG-3*) and 5 (5*-TATAGTTGTTTGCAGCTCTGT3*) for identification of E6 transcripts. These primer combinations produced PCR fragments of 240 base pairs out of the E2/E4 ORFs (primer set 1/2), 446 base pairs out of the E2/E4/E5 ORFs (primer set 1/3), and 72 base pairs out of the E6 ORF (primer set 4/5), respectively. PCR products were analyzed by agarose gel electrophoresis. Other methods Isolation of total and cytoplasmic RNA and Southern and Northern hybridization experiments and DNA sequencing were performed according to standard protocols (Sambrook et al., 1989). RESULTS Genomic cloning and characterization of the nonoccupied allele To isolate and characterize the DNA region deleted upon HPV-16 integration into the cellular genome in SiHa cells, a human genomic library was screened with 32Plabeled probes A and B covering the 5* and 3* cellular flanking regions, respectively (see Fig. 1A). After three screening cycles, two lambda clones, depicted as l1 and l7 (Fig. 1B), were found to contain overlapping fragments covering approximately 40 kb of cellular DNA close to the viral integration site. Following digestion of these clones with HindIII– EcoRI, DNA fragments p7.1, p7.2, p1.1, and p1.2 (see Fig. 1C) corresponding to the region of interest were subcloned into the pBluescript II KS vector for further analysis. Figure 1 shows a partial restriction map of both the HPV-16-containing and the wild-type (wt) alleles. Comparison of both maps revealed that viral integration was followed by a deletion of 7.8 kb of wt allele se-
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quences. To obtain information about the deleted DNA sequences adjacent to the 5* and 3* integration sites, we sequenced subclone p7.2 and part of subclone p1.2 (see Fig. 1C). Figure 2A shows the DNA sequences of the wt-allele surrounding the 5* and 3* integration sites. A canonical full-length repetitive Alu sequence is located at the 5* integration site and was disrupted upon HPV-16 integration. In contrast, a unique sequence is present at the 3* integration site with no significant homology to any known human sequences available in the EMBL-GenBank. Interestingly, sequence homologies of four base pairs between HPV-16 and cellular sequences could be identified at both host–viral junctions (5*-TATT-3* and 5*ATGC-3* at the 5* and 3* integration sites, respectively; see Fig. 2B). Furthermore, small direct repeats are present at these junctions. A direct repeat of the sequence ATTTTT separated by two nucleotides was found at the 5* junction, and a TGC direct repeat without any spacer sequences is located at the 3* junction. These data suggest that insertion of the viral sequence into the host genome took place by a recombination event between two small direct repeats. In addition, no significant ORFs were found in both DNA strands of a total of about 2.3 kb of deleted DNA and 1.0 kb of sequences upstream of the 5* viral integration site. In addition, hybridization of the subcloned DNA fragments with radioactively labeled total genomic DNA resulted in strong hybridization signals, suggesting the presence of repetitive sequences in each of these fragments (data not shown). Analysis of the chromatin structure upstream of the 5* HPV integration site It has been shown that digestion of isolated nuclei or permeabilized cells with randomly cutting nucleases,
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FIG. 2. Human genomic DNA sequences at the 5* and 3* host–viral junctions in the genome of SiHa cells. (A) Clones p7.2 and p1.2 (see Fig. 1C) were partially sequenced by the dideoxy method. Sequences deleted upon HPV-16 integration are shown in bold italics. Small direct ATTTTT and TGC repeats found in the wt-allele at the 5* and 3* integration sites, respectively, are underlined. Asterisks above the sequence depict a canonical Alu repeat present at the 5* viral integration site. (B) Alignment of cellular and viral sequences at the 5* (top) and 3* (bottom) integration sites, showing the small regions of sequence homology at the 5* and 3* integration sites (TATT and ATGC, respectively; underlined), as well as the direct repeats (bold italics) present in the wt-allele.
such as DNase I, is a suitable method for analyzing chromatin structure. Such an analysis allows identification of hypersensitive sites that often coincide with regulatory sequences involved in transcription, replication, and recombination (Elgin, 1984; Hutchison and Weintraub, 1985). Molecular cloning of the wt-allele together with knowledge about the HPV-16-containing allele (El Awady et al., 1987; Baker et al., 1987) enabled us to analyze the chromatin organization of both alleles in independent mapping experiments. To address the question of whether HPV integration was accompanied by a change in cellular chromatin and/or took place in the vicinity of
regulatory elements, DNase I mapping analyses were performed. For this approach, nuclei were isolated from SiHa cells and treated with increasing amounts of either DNase I or MNase as described (Ro¨sl et al., 1989). Isolated genomic DNA was digested with either KpnI or ApaI, separated on agarose gels, and blotted onto nylon membranes. To analyze the chromatin structure of the HPV-16-containing allele, ApaI-digested DNA was hybridized to a 32P-labeled PstI–ApaI DNA fragment encompassing 836 bp of HPV-16 sequences (nucleotide positions 3693 to 4529, covering E5 and part of L2 ORFs). As shown in Fig. 3A,
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stream of the 5* HPV integration site seem to be present in both wt-and HPV-16-containing alleles. Similar hybridization patterns were observed when MNase was used for mapping analysis (data not shown). Taken together, these results demonstrate that integration of HPV-16 DNA into the genome of SiHa cells was not followed by reorganization of the chromatin structure upstream of the viral integration site. Transcription of HPV-16 sequences
FIG. 3. DNase I mapping analysis of the 5* cellular region of the HPV-16 integration site in the genome of SiHa cells. Isolated nuclei of SiHa cells were treated with 0.1, 0.2, 0.5, 1, and 4 units of DNase I (lanes 3 to 7, respectively). Following isolation, 10 mg of DNA was digested either with ApaI (A) or with KpnI (B), separated in 0.8% agarose gels, and transferred onto nylon membranes. For hybridization, a 32Plabeled PstI–ApaI DNA fragment covering approximately 0.8 kb of HPV16-specific DNA sequences (A) and a 526-bp SphI–NdeI fragment that had been deleted during viral integration (B; depicted as fragment C in Fig. 1D) were used. Lanes 1 and 2, controls without DNase I treatment. Taking into account the known positions for ApaI and KpnI cleavage, the radioactive signals at approximately 2.3 and 2.9 kb (A) and 4.0 and 4.6 kb (B) (middle and lower arrows) correspond to two DNase I hypersensitive sites (DHSS) located approximately 1.1 and 1.7 kb upstream of the HPV-16 integration site. The upper arrows indicate the positions of the parental restriction fragments of approximately 9 kb (A) and 18 kb (B). In the lower part of the figure, the HPV-16-containing and wt-alleles are schematically outlined showing the two DHSS (marked with arrows 1 and 2). Abbreviations for restriction endonucleases: H, HindIII; K, KpnI; N, NdeI; S, SphI.
As schematically outlined in Fig. 4, integration of HPV16 sequences into the host DNA in SiHa cells resulted in disruption of the viral URR-driven transcription unit and in location of both putative viral polyadenylation sites upstream of the URR transcription start point. Therefore, we were interested in whether the 5* DNA region, containing structurally intact E5, L2, and L1 ORFs, would be transcribed as fusion transcripts containing cellular and viral sequences. Northern hybridization experiments using SiHa cell RNA and a 32P-labeled E5 probe did not reveal any signals, even after long exposure times, suggesting that if transcripts exist, they must be present at a very low concentration. To further address this question, nuclear run-on experiments using different HPV-16-specific DNA fragments as probes were performed. Following immobilization of these fragments onto a nylon membrane, they were hybridized to [32P]UTP-labeled total RNA transcribed in vitro using nuclei isolated from SiHa cells. The results of these experiments are shown in Fig. 4. A 3.4-kb DNA fragment encompassing the E6/E7 ORFs located downstream of the HPV-16 URR gave a strong
DNA fragments of approximately 2.3 and 2.9 kb in length were detected, in addition to the signal for the parental restriction fragment of approximately 9 kb. Taking the known positions for ApaI cleavage (nt 4529) and HPV-16 integration (nt 3384) into account, these results suggest the presence of two hypersensitive sites located approximately 1.1 and 1.7 kb upstream of the 5* viral integration site. For analysis of the nonoccupied allele, KpnI-digested DNA was hybridized to a 526-bp SphI–NdeI restriction fragment that had been deleted upon HPV-16 integration (see Fig. 1D) and was found to be devoid of repetitive sequences. In addition to the signal for the parental restriction fragment of approximately 18 kb, two hybridizing fragments of approximately 4.0 and 4.6 kb in size were found, which also corresponds to hypersensitive sites located 1.1 and 1.7 kb upstream of the viral integration site (Fig. 3B). Thus, the same hypersensitive sites up-
FIG. 4. Analysis of HPV-16 transcription in SiHa cells by nuclear run-on analysis. 2.5 mg of different HPV-16-specific DNA fragments was heat-denatured and spotted onto nylon membranes. The membrane was hybridized to [32P]UTP-labeled RNA transcribed from SiHa nuclei. 1, 1.5 kb XcmI– KpnI fragment (HPV-16 nucleotides 3879– 5383); 2, 3.4 kb KpnI –KpnI fragment (nt 5383 –884); 3, pBluescript II KS fragment (negative control); 4, pUC 19 fragment (negative control); 5, cellular DNA (positive control). In the lower part of the figure, the HPV-16-containing allele is schematically outlined, showing the locations of the HPV-16-specific DNA probes used (hatched boxes, marked 1 and 2).
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hybridization signal (slot 2). Interestingly, a hybridization signal, although with weaker intensity, was also obtained when using a fragment containing the E5 and part of the L2 ORFs, located 5* of the viral URR (slot 1). Total cellular DNA (slot 5) used as positive control showed a strong hybridization signal, whereas no signals could be observed with pBluescript II KS and pUC 19 vector DNA (slots 3 and 4). To confirm these results, we performed RT-PCR using total SiHa RNA and primers specific for either the E2/E4 or E6 ORFs. After agarose gel electrophoresis, the PCR products were transferred onto a nylon membrane and hybridized to the entire HPV-16 genome. As can be seen in Fig. 5A, a strong hybridization signal was observed when using the E6-specific primers (lane 1). A weaker signal was found using the E2/E4-specific primers (lane 3). The signals were not the result of contaminating DNA since there was no signal when reverse transcriptase was omitted. To further strengthen these data that suggested transcription of HPV-16 ORFs located 5* of the HPV-16 URR, we performed RT-PCR on cytoplasmic SiHa RNA using two primers spanning a DNA fragment of 446 bp in size of the upstream E2, E4, and E5 ORFs. As illustrated in Fig. 5B, a fragment of expected length was obtained that hybridized to a 32P-labeled E5-specific
FIG. 6. Southern blot analysis of cervical carcinomas. A total of 10 mg of genomic DNA from four cervical carcinomas (lanes 1 to 3, 5 to 8, 10) and from SiHa cells (lanes 4 and 9) was digested with HindIII, separated in an 0.8% agarose gel, transferred onto a nylon membrane, and hybridized to 32P-labeled probes A (compare Fig. 1A, lanes 1 to 5) and B (compare Fig. 1A, lanes 6 to 10). Note the absence of hybridization signals in tumor sample #1 (lanes 1 and 6).
probe. These results, together with the data obtained by nuclear run-on analysis described above, suggest that transcription of HPV sequences is initiated from two different promoter elements: a weak cellular one located 5* of the viral integration site and a second, stronger one, probably corresponding to the P97 promoter within the HPV-16 URR. Thus, SiHa cells seem to express host– viral fusion transcripts initiated from a weak promoter located within the cellular host sequences upstream of the 5* HPV-16 integration site. Genomic organization of the wt-allele in human cervical tumors
FIG. 5. Analysis of HPV-16 transcription in SiHa cells by RT-PCR. (A) 2.5 mg of total cellular RNA was used for RT-PCR with either E6-specific primers 4 and 5 (lanes 1 and 2) or E2/E4-specific primers 1 and 2 (lanes 3 and 4), spanning DNA fragments of 72 bp (E6 ORF) or 240 bp (E2/E4 ORF). Reactions were performed in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of reverse transcriptase. An aliquot of 15 ml out of a total of 50 ml of each PCR mixture was separated in a 2% agarose gel, transferred onto a nylon membrane, and hybridized to a 32P-labeled probe covering the entire HPV-16 genome. (B) One microgram of cytoplasmic RNA was used for RT-PCR with primers 1 and 3, specific for amplification of a 446-bp DNA fragment out of the E2, E4, and E5 ORFs (lane 1). Lane 2, RT-PCR after RNase A digestion. Lane 3, template-free negative control. Ten microliters (out of a total of 50 ml) was separated in a 2% agarose gel, transferred onto a nylon membrane, and hybridized to a 32P-labeled E5-specific probe. In the lower part of the figure, the HPV-16-harboring allele is schematically outlined, showing the locations of PCR oligonucleotides 1 to 5 used for RT-PCR.
Although no specific integration sites have been described for papillomavirus DNA, several reports have shown that some chromosomal loci are preferred to others (Popescu and DiPaolo, 1989). Therefore we analyzed the structure of the wt-allele described in this report in human tumors by Southern hybridization. Genomic DNA was isolated from four HPV-16-containing cervical carcinomas and from SiHa cells, digested with HindIII, blotted onto a nylon membrane, and hybridized to probes A and B (compare Fig. 1A). Signals at approximately 4.6 kb (probe A, lanes 2–5) and 4.7 kb (probe B, lanes 7–10) could be observed in three of the four tumors analyzed. Interestingly, no signal was observed in tumor sample #1 (Fig. 6, lanes 1 and 6), suggesting that the corresponding DNA region was deleted in this particular tumor. As expected, two hybridizing bands were found with SiHa DNA, corresponding to the wild-type and the occupied alleles. These results indicate that within one of four tumors analyzed, genomic reorganization took place at the same locus which was altered in the genome of SiHa cells.
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Genomic analysis carried out on human cervical cancers containing high-risk HPVs, such as HPV-16 or HPV18, has shown that the viral sequences are frequently integrated into the host genome (Cullen et al., 1991). In some studies, evidence has been provided that integration of HPV genomes occurs at or near fragile sites (Cannizzaro et al., 1988; De Braekeleer et al., 1992), DNA regions that have been considered as regions of genomic instability (Popescu and DiPaolo, 1989; Zimonjic et al., 1994), and in some cases at sites proximal to genes relevant for control of growth and differentiation (Du¨rst et al., 1987; Couturier et al., 1991). Furthermore, some chromosomes, such as chromosomes 3 and 8, seem to be more accessible to integration than others (Popescu and DiPaolo, 1989). In some cell lines derived from HPVcontaining cervical cancers, integration near protooncogenes, such as c-myc (Du¨rst et al., 1987; Couturier et al., 1991), has been described, suggesting that viral integration may cause alterations in the expression of these genes. Integration of HPVs into the host genome is frequently followed by deletion of host cellular sequences. In M50 cells, for example, a deletion of approximately 1.1 kb occurred at the integration site of HPV-16 (Choo et al., 1990). In the Sk-v cell line, which was established from a Bowen’s carcinoma of the vulva, integration of HPV-16 at a single site resulted in a deletion of approximately 5.5 kb of cellular DNA (Schneider-Maunoury et al., 1987). In agreement with data presented by El Awady et al. (1987) and Baker et al. (1987), our results indicate that integration of HPV-16 DNA into the genome of SiHa cells was followed by a deletion of host sequences. Restriction mapping of the wt-allele cloned in this study allowed us to state the size of the deleted region more precisely as 7.8 kb. We sequenced a total of 3.3 kb of DNA, comprising 2.3 kb of sequences from the deleted region and 1.0 kb of cellular sequences upstream of the 5* viral integration site. Computer analysis of these sequences with GenBank did not reveal significant homologies to known human sequences nor were any significant ORFs found. In addition, Northern analyses of RNA isolated from different epithelial cell lines (HeLa, CaSki, C4-I, and SiHa) using fragments p1.1 and p1.2 (see Fig. 1C) as probes did not reveal any appreciable signals, suggesting that these sequences are either not transcribed or transcribed at a very low rate. Sequence analysis of the deleted fragment showed that integration of viral DNA took place in the middle of an Alu repeat at the 5* host–viral junction, whereas a unique sequence was present at the 3* junction. Comparison of the DNA sequences of the wt- and the HPVcontaining alleles revealed a homology of four base pairs
at both host–viral junctions (see Fig. 2). Similarly, when analyzing the integration locus of HPV-16 DNA in a cervical cancer biopsy, Du¨rst et al. (1986) observed a fourbase pair homology between viral and host sequences. Moreover, small direct repeats that overlap both junctions were identified in the wt-allele, suggesting their involvement in the recombination process between host and viral DNA sequences. A similar kind of integration mechanism has been suggested by Choo et al. (1990) for integration of HPV-16 DNA into the host genome in a primary cervical carcinoma. Interestingly, short patches of sequence homology at the host–virus junctions are also a common finding for adeno- and polyomaviruses (Gahlmann et al., 1982; Ruley and Fried, 1983). Thus, it is tempting to speculate that the short sequence homologies observed at both host–viral junctions in SiHa cells may play a role in the integration process, similar to that found for adeno- and polyomaviruses. Chromatin analysis of the viral integration locus in HeLa cells revealed that HPV-18 DNA is integrated in the vicinity of a cellular promoter which cooperates with HPV-18 URR in early transcription of viral genes (Ro¨sl et al., 1989). In the present study, we were interested in whether integration of HPV-16 DNA into the host genome in SiHa cells impairs transcription of viral ORFs due to chromosomal position effects. It is well known that such effects frequently affect transcriptional activation of, e.g., transgenes (Wilson et al., 1990). It has recently been demonstrated for the chicken lysozyme gene that suppression of gene expression in transgenic mice due to chromosomal position effects correlates with alterations in DNase I hypersensitive site (DHSS) formation. Moreover, DHSS formation on cis-regulatory elements seems to correlate directly with transcriptional activation of chicken lysozyme transgenes, suggesting that positionindependent gene expression in transgenic mice is dependent on regeneration of the original DHSS pattern (Huber et al., 1994). In our study, we provide evidence that two DNase I hypersensitive sites are present, approximately 1.1 and 1.7 kb upstream of the 5* host–viral junction, in both the wt- and the HPV-16-containing alleles. Thus, integration of HPV-16 DNA occurred near a hypersensitive region in SiHa cells. However, this integration event did not lead to major chromatin changes. Transcriptional analysis of HPV-16 ORFs in SiHa cells demonstrated the presence of viral transcripts from the E6 and E7 ORFs, mapping downstream of the HPV-16 URR, as well as from the E2, E4, E5, and the L2/L1 ORFs, located upstream of the viral URR, close to the 5* viral integration site (see Figs. 4 and 5). The strong quantitative differences in the signal intensities between upstream and downstream ORFs suggest that two different promoters are used for transcription of HPV-16 ORFs: one upstream of the 5* host–viral junction and another one, probably the P97 promoter, within the viral URR. The
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Baker, C. C., Phelps, W. C., Lindgren, V., Braun, M. J., Gonda, M. A., and Howley, P. M. (1987). Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J. Virol. 61, 962–971. Cannizzaro, L. A., Du¨rst, M., Mendez, M. J., Hecht, B. K., and Hecht, F. (1988). Regional chromosome localization of human papillomavirus integration sites near fragile sites, oncogenes, and cancer chromosome breakpoints. Cancer Genet. Cytogenet. 33, 93–98. Choo, K. B., Lee, H. H., Liew, L. N., Chong, K. Y., and Chou, H. F. (1990).
Analysis of the unoccupied site of an integrated human papillomavirus 16 sequence in a cervical carcinoma. Virology 178, 621–625. Couturier, J., Sastre-Garau, X., Schneider-Maunoury, S., Labib, A., and Orth, G. (1991). Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for proto-oncogene expression. J. Virol. 65, 4534–4538. Crook, T., Morgenstern, J. P., Crawford, L., and Banks, L. (1989). Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV-16 plus EJ-ras. EMBO J. 8, 513–519. Cullen, A. P., Reid, R., Campion, M., and Lorincz, A. T. (1991). Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. J. Virol. 65, 606–612. Das, B. C., Gopalkrishna, V., Das, D. K., Sharma, J. K., Singh, V., and Luthra, U. K. (1993). Human papillomavirus DNA sequences in adenocarcinoma of the uterine cervix in Indian women. Cancer 72, 147– 153. De Braekeleer, M., Sreekantaiah, C., and Haas, O. (1992). Herpes simplex virus and human papillomavirus sites correlate with chromosomal breakpoints in human cervical carcinoma. Cancer Genet. Cytogenet. 59, 135–137. de Villiers, E.-M. (1994). Human pathogenic papillomavirus types: An update. Curr. Top. Microbiol. Immunol. 186, 1–12. D’Onofrio, C., Colantuoni, V., and Cortese, R. (1985). Structure and cellspecific expression of a cloned human retinol binding protein gene: The 5*-flanking region contains hepatoma specific transcriptional signals. EMBO J. 4, 1981–1989. Du¨rst, M., Schwarz, E., and Gissmann, L. (1986). Integration and persistence of human papillomavirus DNA in genital tumors. In ‘‘Banury Report 21: Viral Etiology of Cervical Cancer’’ (R. Peto and H. zur Hausen, Eds.), pp. 273–280. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Du¨rst, M., Croce, C. M., Gissmann, L., Schwarz, E., and Huebner, K. (1987). Papillomavirus sequences integrate near cellular oncogenes in some cervical carcinomas. Proc. Natl. Acad. Sci. USA 84, 1070– 1074. El Awady, M. K., Kaplan, J. B., O’Brien, S. J., and Burk, R. D. (1987). Molecular analysis of integrated human papillomavirus 16 sequences in the cervical cancer cell line SiHa. Virology 159, 389– 398. Elgin, S. C. R. (1984). Anatomy of hypersensitive sites. Nature 309, 213– 214. Friedl, F., Kimura, I., Osato, T., and Ito, Y. (1970). Studies on a new human cell line (SiHa) derived from carcinoma of uterus. I. Its establishment and morphology. Proc. Soc. Exp. Biol. Med. 135, 543–545. Gahlmann, R., Leisten, R., Vardimon, L., and Doerfler, W. (1982). Patch homologies and the integration of adenovirus DNA in mammalian cells. EMBO J. 1, 1101–1104. Halbert, C. L., Demers, G. W., and Galloway, D. A. (1991). The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65, 473–478. Hawley-Nelson, P., Vousden, K. H., Hubbert, N. L., Lowy., D. R., and Schiller, J. T. (1989). HPV-16 E6 and E7 proteins cooperate to immortalize primary human foreskin keratinocytes. EMBO J. 8, 3905–3910. Huber, M. C., Bosch, F. X., Sippel, A. E., and Bonifer, C. (1994). Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation. Nucleic Acids Res. 22, 4195–4201. Hutchison, N., and Weintraub, H. (1985). Localization of DNase I-sensitive sequences to specific regions of interphase nuclei. Cell 43, 471– 482. Jeon, S., and Lambert, P. F. (1995). Integration of human papillomavirus type 16 DNA into the human genome leads to increased stability of E6 and E7 mRNAs: Implications for cervical carcinogenesis. Proc. Natl. Acad. Sci. USA 92, 1654–1658. Jeon, S., Allen-Hoffmann, B. L., and Lambert, P. F. (1995). Integration
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proximity of the primers used for RT-PCR of E2/E4/E5 ORF sequences to the 5* host–viral junction together with the presence of two DHSS immediately upstream of the 5* viral integration site suggests the presence of host–viral fusion transcripts. Viral–cellular fusion transcripts have been found to be more stable than intact viral copies (Kennedy et al., 1991). Very recently, it was demonstrated that replacement of an A / U-rich mRNA instability element, which is present within the viral 3* untranslated region of HPV-16 between nt 4005 and nt 4213, by cellular sequences upon integration of HPV-16 DNA into the human genome can result in increased steady-state levels of E6- and E7-specific mRNAs through altered mRNA stability (Jeon and Lambert, 1995; Jeon et al., 1995). Integration of HPV-16 DNA into the genome in SiHa cells took place at HPV-16 nt 3132 accompanied by a small deletion of 251 bp of viral sequences (nt 3133–3384) and did therefore not lead to a disruption of the viral 3* UTR. Nevertheless, it is not known whether this region contains cis-elements that might affect URR-driven viral transcription and should be further investigated. Alterations in the genomic organization at the locus corresponding to the HPV-16 integration site in the SiHa cell line were found in one of four human cervical carcinomas analyzed. Although the number of tumors analyzed is too small to allow an unambiguous conclusion to be drawn concerning the biological significance of these genomic alterations, our results suggest the presence of a genetically instable chromosomal locus at the HPV-16 integration site. Cloning and characterization of such regions from a greater number of human tumors would be necessary to assess the possible importance of the integration sites for the biological activity of integrated papillomavirus sequences in infected human cells. ACKNOWLEDGMENTS We thank Dr. R. D. Burk for providing us with the L and R probes of the HPV-16-containing allele of SiHa cells and Dr. M. Du¨rst for the cloned HPV-16 fragment from SiHa cells as well as for the cervical tumor samples analyzed in this study. We also thank Dr. M. Du¨rst for critical reading of the manuscript and J. Fischer for excellent technical assistance.
REFERENCES
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of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells. J. Virol. 69, 2989–2997. Kawasaki, E. S. (1990). Amplification of RNA. In ‘‘PCR Protocols: A Guide to Methods and Applications’’ (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, Eds.), pp. 21–27. Academic Press, San Diego. Kennedy, I. M., Haddow, J. K., and Clements, J. B. (1991). A negative regulatory element in the human papillomavirus type 16 genome acts at the level of late mRNA stability. J. Virol. 65, 2093–2097. Linial, M., Gunderson, N., and Groudine, M. (1985). Enhanced transcription of c-myc in bursal lymphoma cells requires continuous protein synthesis. Science 230, 1126–1132. Meijer, C. J. L. M., van den Brule, A. J. C., Snijders, P. J. F., Helmerhorst, T., Kenemans, P., and Walboomers, J. M. M. (1992). Detection of human papillomavirus in cervical scrapes by the polymerase chain reaction in relation to cytology: Possible implications for cervical cancer screening. In ‘‘The Epidemiology of Cervical Cancer and Human Papillomavirus’’ (N. Munoz, F. X. Bosch, K. V. Shah, and A. Meheus, Eds.), pp. 271–281. Int. Agency Res. Cancer, Lyon. Mincheva, A., Gissmann, L., and zur Hausen, H. (1987). Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridization. Med. Microbiol. Immunol. 176, 245–256. Mu¨nger, K., Phelps, W. C., Bubb, V., Howley, P. M., and Schlegel, R. (1989). The E6 and E7 genes of the human papilllomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol. 63, 4417–4421. Pater, M. M., and Pater, A. (1985). Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix. Virology 145, 313–318. Popescu, N. C., and DiPaolo, J. A. (1989). Preferential sites for viral integration on mammalian genome. Cancer Genet. Cytogenet. 42, 157–171. Romanczuk, H., and Howley, P. M. (1992). Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity. Proc. Natl. Acad. Sci. USA 89, 3159– 3163. Ro¨sl, F, Westphal, E.-M., and zur Hausen, H. (1989). Chromatin structure and transcriptional regulation of human papillomavirus type 18 DNA in HeLa cells. Mol. Carcinog. 2, 72–80. Ruley, H. E., and Fried, M. (1983). Clustered illegitimate recombination
events in mammalian cells involving very short sequence homologies. Nature 304, 181–184. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). ‘‘Molecular Cloning: A Laboratory Manual,’’ 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schneider-Maunoury, S., Croissant, O., and Orth, G. (1987). Integration of human papillomavirus type 16 DNA sequences: A possible early event in the progression of genital tumors. J. Virol. 61, 3295–3298. Schwarz, E., Freese, U. K., Gissmann, L., Mayer, W., Roggenbuck, B., Stremlau, A., and zur Hausen, H. (1985). Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314, 111–114. Smotkin, D., and Wettstein, F. O. (1986). Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancerderived cell line and identification of the E7 protein. Proc. Natl. Acad. Sci. USA 83, 4680–4684. Thierry, F. (1993). Proteins involved in the control of HPV transcription. Papillomavirus Rep. 4, 27–32. von Knebel Doeberitz, M., Oltersdorf, T., Schwarz, E., and Gissmann L. (1988). Correlation of modified human papilloma virus early gene expression with altered growth properties in C4-1 cervical carcinoma cells. Cancer Res. 48, 3780–3786. Wilczynski, S. P., Pearlman, L., and Walker, J. (1988). Identification of HPV-16 early genes retained in cervical carcinomas. Virology 166, 624–627. Wilson, C., Bellen, H. J., and Gehring, W. J. (1990). Position effects on eukaryotic gene expression. Annu. Rev. Cell Biol. 6, 679–714. Zimonjic, D. B., Popescu, N. D., and DiPaolo, J. A. (1994). Chromosomal organization of viral integration sites in human papillomavirus-immortalized human keratinocyte cell lines. Cancer Genet. Cytogenet. 72, 39–43. zur Hausen, H. (1991a). Human papillomaviruses in the pathogenesis of anogenital cancer. Virology 184, 9–13. zur Hausen H. (1991b). Papillomavirus–host cell interactions in the pathogenesis of anogenital cancer. In ‘‘Origins of Human Cancer: A Comprehensive Review’’ (J. Brugge, T. Curran, E. Harlow, and F. McCormick, Eds.), pp. 685–705. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. zur Hausen, H., and de Villiers, E.-M. (1994). Human papillomaviruses. Annu. Rev. Microbiol. 48, 427–447. zur Hausen H., and Schneider. A. (1987). The role of papillomaviruses in human anogenital cancers. In ‘‘The Papovaviridae’’ (N. P. Salzman and P. M. Howley, Eds.), pp. 245–263. Plenum Press, NY.
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