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Previous Article | Next Article 
Journal of Virology, October 2004, p. 11172-11186, Vol. 78, No. 20
0022-538X/04/$08.00+0 DOI: 10.1128/JVI.78.20.11172-11186.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Lilin Lai,3 Renske D. M. Steenbergen,6 Chris L. J. M. Meijer,6 Peter J. F. Snijders,6 Pamela Chatis,7,8 Thomas R. Broker,2 Phillip T. Moen Jr.,7,
and Louise T. Chow2*
Department of Pathology,1 Department of Biochemistry and Molecular Genetics,2 Department of Medicine,3 Department of Microbiology,4 Laboratory of Medical Genetics University of Alabama at Birmingham, Birmingham, Alabama,5 Department of Pathology, Vrije Universitat Medical Center, Boelelaan, Amsterdam, The Netherlands,6 New Technologies Research and Development Group, Perkin-Elmer Life Sciences, Inc.,7 Division of Infectious Disease, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, Massachusetts8
Received 30 April 2004/ Accepted 16 June 2004
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ABSTRACT |
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INTRODUCTION |
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The molecular basis for HPV oncogenesis is predicated on the mechanisms by which these normally benign viruses reproduce themselves. In productive infections, the 8-kb, double-stranded, circular viral DNAs amplify as extrachromosomal nuclear plasmids, and this amplification is restricted to postmitotic differentiated cells of the squamous epithelium. DNA synthesis requires the E2 origin recognition protein and the E1 viral helicase protein, as well as the reactivated host replication machinery. Thus, HPVs encode E6 and E7 oncoproteins that bind to and inactivate the tumor suppressor proteins p53 and the retinoblastoma family of tumor suppressor proteins, respectively, to bypass cell cycle controls (37, 38). Typically, there is little or no viral E6 or E7 oncoprotein gene expression in basal cells due to negative controls exerted on the enhancer-promoter by cellular factors and possibly by the viral E2 protein, which is also a transcription factor (10). As with cellular genes (27), chromatin remodeling by posttranslational modifications of histones has also been implicated in viral gene regulation (15, 72).
In contrast to the noncycling, differentiated keratinocytes comprising the suprabasal strata of benign productive HPV lesions, much or all of the epithelium in high-grade dysplasias and carcinomas consists of basal cell-like proliferating cells in which viral DNA is often integrated and viral E6 and E7 oncogenes are invariably highly expressed (60, 73). These neoplastic changes are probably brought about by inopportune and prolonged expression of the oncogenic HPV E6 and E7 proteins in the normally rather quiescent reserve or stem cells of the epithelia during repeated wounding and healing (10). Consequently, excessive cell proliferation, accumulation of mutations in key host regulatory genes, and chromosome instability occur in these cells (37, 38). Cell lines such as SiHa, CaSki, and HeLa were established from HPV-induced cervical cancers, and they contain integrated HPV DNA from which the continuous expression of the viral oncogenes is critical for the maintenance of cell growth and the transformed phenotypes (3, 14, 33, 41, 48, 50, 67, 70).
Viral DNA integration invariably occurs downstream of the E6/E7 genes, often in the E1 or E2 gene region accompanied by a deletion of downstream sequences, resulting in a loss of feedback control of oncogene expression by the E2 protein. Moreover, transcripts of the E6/E7 genes capture 3' cellular untranslated sequences and polyadenylation signals (26, 48, 53, 68), and these fusion transcripts are more stable than those derived from extrachromosomal viral DNA. This increase in viral mRNA stability and the resulting elevated levels of viral oncoproteins are considered to be important factors in neoplastic progression (23).
HPV-16 and HPV-18 oncogenes can immortalize primary human keratinocytes in vitro (73). The immortal cells contain multiple integrated HPV DNA, express telomerase and are often hyperdiploid, with a gain or loss of partial or entire chromosomes (36, 54, 57, 59). Early-passage immortal cells are typically not tumorigenic in nude mice, whereas cells at higher passages exhibit more dysplastic histology when grown as organotypic raft cultures, simulating the histopathological characteristics of low-, moderate-, and high-grade dysplasias (21, 58). Eventually, tumorigenic clones may emerge after long-term culturing, presumably due to additional mutations in cellular genes incurred during passage (73). Along with cervical carcinoma cell lines, these immortalized cells serve as experimental models with which to investigate chromosomal changes that occur in patient lesions during the progression to dysplasias and cancers. However, the regulation of viral transcription in these immortalized cells or cancers had not been examined in detail.
In the present study, we used in situ hybridization in conjunction with tyramide-fluorescence enhanced signal deposition (T-FISH) (1, 4, 65) to visualize viral RNA transcription foci in HPV DNA-immortalized human keratinocytes. We also identified chromosomes that harbored active centers in CaSki and SiHa cells by simultaneous detection of nascent viral RNA, integrated viral DNA, or chromosomal territories. Both CaSki and SiHa cells are hyperdiploid. CaSki cells harbor ca. 600 copies of HPV-16 DNA distributed over 11 or more chromosomal sites, mainly in tandem arrays. The complex chromosomal rearrangements and the large number of sites of integration have up till now prevented cytogenetic mapping of the integrated viral DNAs. SiHa cells, in contrast, possess only two copies of HPV DNA, one each in the two copies of chromosome 13 at locus 13q21 (1, 3, 17, 33). Our results demonstrate that, regardless of the copy number of integrated viral DNA or number of distinct integration sites, natural selection favors cells containing only one or at most two transcriptionally active viral DNA center(s), which was preferentially localized to the perinucleolar region. All other integrated viral DNA copies become transcriptionally inactive. This perinucleolar domain, designated the papillomavirus transcriptional domain (PVTD), is distinct from other major nuclear domains commonly studied. Some of the silent copies can be reactivated by growth for one to two cell divisions in medium containing 5-azacytidine, implicating DNA methylation in the silencing. Conversely, the active copy can be silenced under challenge for cell survival. Of particular medical relevance, examination of HPV-associated primary cancers also revealed one nascent viral transcription center per cell. Collectively, these observations indicate that the selection for the fittest cells via transcriptional silencing is a dynamic and reversible process. The implications of this process of transcriptional modulation during oncogenesis are discussed.
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MATERIALS AND METHODS |
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Nucleic acid hybridization probes and probe labeling.
HPV genomic DNA probes for in situ hybridization were prepared by nick translation of genomic plasmid DNAs in the presence of biotinylated dUTP. The probes were precipitated in the presence of 0.4 µg of Cot-1 DNA, 0.20 µg of yeast tRNA, and 0.6 µg of salmon sperm DNA/µl. The probe cocktail was prepared in Hybridsol VII (Ventana Medical Systems, Tucson, Ariz.) as described previously (65). HPV genomic DNA probes for Southern and Northern blot hybridization were prepared by nick translation in the presence of [
-32P]dCTP (Perkin-Elmer Life Sciences, Boston, Mass.).
In situ detection of nascent viral transcripts, viral DNA, chromosomal DNA, or protein. Simultaneous detection of nascent viral RNAs and viral DNA or chromosomal domains was performed essentially as described previously (24, 35, 69), with modifications pertaining to the use of tyramide signal amplification (4). Hybridization to nascent RNA transcripts was performed without prior denaturation of nucleic acids. Briefly, cells were permeabilized in CSK solution (19) containing 0.5% Triton X-100 detergent for 3 min at 4°C, fixed with 4% paraformaldehyde-1 mM MgCl2 (pH 7.2) for 10 min, dehydrated in ethanol, and hybridized with probes. After overnight incubation with probes in Hybrizol VII at 37°C, slides were washed extensively and then incubated with a 1:100 dilution of streptavidin-horseradish peroxidase (HRP) conjugate in 4x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 37°C for 1 h. After a washing step, signals were revealed with a tyramide-fluorophor (1:100 dilution in Amplification Diluent; Perkin-Elmer). First-round HRPs were inactivated by 3% hydrogen peroxide in 4x SSC at room temperature prior to further probing.
To detect viral DNA or for interphase chromosome paints, slides were treated with 100 µg of RNase A (Sigma)/ml in 2x SSC at 37°C, dehydrated in ethanol, denatured in 70% formamide-2x SSC (pH 7.0) at 72°C, dehydrated in ethanol, and hybridized with probes. After overnight incubation at 37°C, slides were treated as described for nascent RNA detection. For simultaneous detection of viral DNA and chromosome paints, viral DNA was probed first. Direct fluorescein isothiocyanate (FITC)-conjugated chromosomal paints from Vysis (Downers Grove, Ill.) were detected by using the manufacturer's protocol. Digoxigenin paint probes from Oncor (Gaithersburg, Md.) were detected by using either an anti-digoxigenin (DIG)-FITC or an anti-DIG-Texas red antibody (Roche, Indianapolis, Ind.). Cells were stained with DAPI (4',6'-diamidino-2-phenylindole; Sigma) at 0.05 mg/ml and mounted in Antifade.
Antibodies used in the present study were: SC35 (Sigma, St. Louis, Mo.); PML (PG-M3), C23 (MS-3; Santa Cruz Biotechnology, Santa Cruz, Calif.), and p220npat (a gift from J. Wade Harper) (31). For C23 antigen detection (Sigma), slides were incubated with a 1:100 dilution of primary antibody in 50% goat serum, 1x phosphate-buffered saline at 37°C, washed, and then incubated again with a 1:100 dilution of a goat anti-mouse-TRITC (tetramethyl rhodamine isothiocyanate) antibody. For the other antigens, Antigen Retrieval (Dako, Carpinteria, Calif.) was performed prior to probing with 1:100 dilution of primary antibodies. After a washing step, a secondary goat anti-rabbit or anti-mouse-HRP antibody was then applied for 1 h at 37°C (Roche). The slides were washed, developed by using a 1:100 dilution of fluorescence-tyramide (Perkin-Elmer), stained with DAPI, and mounted with Antifade.
HPV typing of paraffin-embedded patient biopsy specimens was conducted on DNA recovered from tissue sections by using nested PCR amplification, followed by diagnostic restriction enzyme digestions. After deparaffinization, 4-µm sections were treated with a proteinase K solution (Dako) for 6 min, ethanol dehydrated, and probed for viral RNA and then reprobed for viral DNA with nick-translated probes.
Southern blot analysis, Northern blot analysis, and 3' RACE. A total of 7 µg of total cellular DNA from FK16A and FK18B cells at different passages were digested with PstI and separated in a 0.8% agarose gel. Southern blot hybridization was performed with 32P-labeled HPV-16 genomic DNA probes. For Northern blot analysis, total RNA was isolated by using TRIzol reagent (Invitrogen) from cell lines. Total RNA were also isolated from CaSki cells with or without prior treatment with 10 µM 5-azacytidine (Sigma) at 37°C for 40 to 48 h. RNA was digested with RNase-free DNase I before being separated in a 1% formaldehyde gel, transferred to nitrocellulose membranes, and probed. The blots were analyzed with a Molecular Dynamics PhosphorImager. 3' RACE (rapid amplification of cDNA ends) analysis of HPV RNAs from CaSki cells was performed as described previously (68). Five cDNA clones were randomly selected and sequenced by the UAB Sequencing Core Facility.
Lentiviral vectors and transduction. An human immunodeficiency virus (HIV)-based vector was used to introduce genes expressing either enhanced green fluorescent protein (GFP)-internal ribosome entry site (IRES)-puro or HPV-18 E6/E7-IRES-puro into the FK18B cell line. The vector was similar to that described (7, 66). The eGFP or E6/E7 coding sequence was inserted 5' of the IRES, and each was under control of the early cytomegalovirus promoter. Infectious stocks of vectors were prepared by transfection of 293T cells as described previously (39) and used at a multiplicity of infection of 1 or 10 to transduce FK18B cells at passage 109.
Light microscopy. Slides were viewed with an Olympus AX70 microscope equipped with a Speicher Filter Set (ChromaTechnologies, Brattleboro, Vt.). Photographs were taken by using a Zeiss Axiocam and Zeiss software. Quantification was conducted on at least 200 cells per event.
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RESULTS |
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One nascent HPV transcription center in CaSki cells. DNA probes were used to detect transcriptional centers in CaSki cells. Unexpectedly, of the several hundred copies of integrated viral DNA, only a single nuclear RNA hybridization signal was typically observed (Fig. 1F, green). This center consistently colocalized with a low-intensity viral DNA focus (Fig. 1G, red) but not with the much larger tandem arrays of viral DNA. A merged image is shown in Fig. 1H. This nascent RNA center was RNase sensitive and was not observed with HIV probes (data not shown).
Identification of small viral integration centers in the CaSki cells. To determine whether the single transcriptionally active, small DNA center in CaSki cells was clonal, or one that differed among individual cells, we proceeded to identify chromosomes that harbor low copy numbers of HPV DNA by using T-FISH on metaphase chromosomes. We found that CaSki cells contained between 75 and 78 chromosomes, and we detected between 12 and 16 distinct integration sites (Fig. 2A, red). On the basis of their fluorescence probe signals, most loci contained multiple copies of viral DNA, and only three chromosomes contained a low copy number of viral DNA.
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Localization of a clonal transcription center to a derivative chromosome 14 in CaSki cells. To identify the chromosome that harbored the transcriptionally active HPV DNA in CaSki cells, we simultaneously probed for nascent HPV-16 RNA (in red) and for one of several individual chromosome territory domains (in green) in interphase cells. Visual inspection of the spatial localization of viral RNA center revealed a 70% association with chromosome 14 (Fig. 2E) and a statistically insignificant (<15%) association with chromosomes 3 or 21 (data not shown). Because our 3' RACE analysis suggested a link to chromosome 6 or 22 (see below), we also examined these chromosomal territory domains. Analysis of interphase cells showed a 91% association with chromosome 6 (Fig. 2H) but no significant association with chromosome 22 (Fig. 2I). Indeed, by metaphase chromosome painting, one derivative chromosome 14 was observed to harbor a low copy of HPV integrant (Fig. 2D, in green). The chromosome 14 paint (Fig. 2D, in red) did not hybridize to a small region that was proximal to the HPV DNA but distal to the centromere. Collectively, these results suggest that a clonal selection occurred for a transcriptionally active HPV DNA located on a derivative chromosome 14, which may contain some translocated chromosome 6 materials. However, metaphase paint analysis failed to demonstrate chromosome 6 sequences proximal to the HPV genome (data not shown). Thus, this locus may have a complex chromosomal rearrangement. As controls for specificity, nascent HPV RNA did not associate with large tandem HPV DNA repeats on chromosome 2, nor with chromosomes such as X or 13 (<15% association) that had no integrated HPV (Fig. 2F and G).
Clonal RNA transcription in CaSki cells by 3' RACE.
To substantiate a clonal selection for the viral transcription center, we performed a 3' RACE experiment to determine the virus-host RNA junction sequences. Sequence analysis of five independent cDNA clones placed the 3' viral-cellular junction at HPV-16 nucleotide position 3728 in the overlapping E2/E4 open reading frames. All five cDNAs had the same cellular sequence of 98 or 99 bases, followed by the posttranscriptionally added poly(A), in agreement with previous reports (3, 52, 68). Thus, we are indeed dealing with the same CaSki cells as in previous reports. Interestingly, our cDNAs contained alternative E1
E4 mRNA splices that differ in the acceptor site at either nucleotide 3357 (two cDNAs) or, one codon later, nucleotide 3360 (three cDNAs). Alternative splicing of this message was not reported previously but was predicted based on comparative sequence analysis (11). BLASTN analysis of the host sequence revealed a homology or identity with sequences that are associated with L2 line elements and a possible match with sequences on chromosomes 6 and 22. The former, but not the latter, is consistent with the chromosome paint analyses. Taken together, these results support the conclusion that a single active HPV center is in direct association with DNA from chromosome 6 located on a derivative chromosome 14.
Selection for a single transcriptional center during passage of HPV-immortalized keratinocytes. To test whether a single transcription center observed in CaSki cells is a unique case or is a common and reproducible event, we examined four previously well characterized primary human foreskin keratinocyte lines immortalized by either HPV-16 (FK16A, FK16B) or HPV-18 (FK18A, FK18B) (57, 58, 59). The FK16A cell line contains ca. 5 to 10 integrated copies of HPV DNA, whereas FK16B contains more than 100 integrated copies. The FK18A and FK18B lines both contain more than 100 copies of viral DNA, but the two lines might be of clonal origin based on Southern blot hybridization (59). These cell lines went from a normal karyotype to a tetraploid state accompanied by various chromosomal changes. In organotypic cultures, they exhibited progressively more dysplastic phenotypes with increasing passage numbers (58).
At least 200 interphase cells each of FK16A, FK16B, FK18A, and FK18B at different passages were analyzed for their nascent viral RNA centers. The FK16A cell line demonstrated up to five centers at passage 23, but at passages 64 and 82 there was only a single transcription center (Fig. 3A and B, in green, and data not shown). The FK16B cell line at passage 27 had an average of 3.1 RNA centers per cell, but some cells had as many as 7 centers. At passage 64, the average number of transcription centers per cell had dropped to 2.3. At passage 92, the percentage of cells containing one center increased to 70%, whereas most of the remaining population contained two centers (Fig. 3C and D, in green, and data not shown). Thus, there appears to be a distinct trend toward selection for one transcription center during passage of HPV-16 immortalized cells. In FK18A and FK18B cells, only a single HPV transcription center was observed, even at early passage, and this pattern was maintained at later passages (Fig. 3E to F, in green, and data not shown). Interphase viral DNA analysis of FK18A and FK18B showed that both possessed a single viral DNA integration focus. In these cell lines, the focus of viral RNA transcripts (green) was always positioned at one end of the larger DNA center (red) (Fig. 3G). Enlarged images of two of the three cells in Fig. 3G are shown in Fig. 3H and I. We suggest that that this pattern is consistent with transcription of a DNA template located near or at the 3' end of the viral DNA array, allowing the use of a downstream host cleavage site and poly(A) signal, as in the cases of the CaSki and SiHa cell lines. Had the entire array been actively transcribed, the RNA signals would have colocalized with the larger DNA signal (see Fig. 7D and E). Collectively, our results strongly suggest that cells with a single transcription center may have a distinct growth advantage over cells with multiple RNA centers.
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Single transcription centers in human cancer tissues. To ascertain the biological relevance of the results obtained from transformed and immortalized cell lines to HPV cancers, we examined viral RNA transcription centers in formalin-fixed, paraffin-embedded tissue biopsies of HPV-associated lesions. To determine the feasibility of using these methods with paraffin-embedded specimens, we first simultaneously detected HPV RNA and DNA in 4-µm sections of comparably fixed and paraffin-embedded organotypic raft cultures of FK16 and FK18 lines. Nascent transcription centers were detected, and the number of bright RNA foci in the nuclei correlated with those present in cells grown in submerged cultures at similar passage numbers. An example of a single DNA and RNA center per cell in the FK18B cells at passage 118 is shown in Fig. 6A. Some nuclei did not contain any RNA center, probably because the relevant portion of the nucleus was sectioned off. In contrast, in productively infected laryngeal papillomas that harbor a large number of extrachromosomal viral DNA, abundant RNA was detected throughout the cells, as previously described (12, 61), masking all nascent transcription centers (Fig. 6B). Thus, a low transcriptional center number is a prerequisite for center detection. These results also suggest a high concentration of nascent viral RNA at the site of transcription relative to low concentrations of completed and dispersed viral transcripts.
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Activation of transcription from silent viral DNA centers by growth in 5-azacytidine. To begin to identify mechanisms by which the majority of the integrated HPV DNA copies are transcriptionally inactivated, we treated growing CaSki cells for 40 to 48 h with 10 µM 5-azacytidine, an inhibitor of DNA methylation. Northern blot analysis of RNA revealed an increase in the intensity of the existing messages and the appearance of new transcripts in treated versus untreated cells (Fig. 7A). In situ hybridization revealed a number of large transcription centers in treated cells (Fig. 7B, in red). However, activation was not observed in the majority of cells in these experiments. This incomplete induction might be due to a cell cycle effect, to an incomplete demethylation of the HPV DNA, or to a toxic or lethal effect of the compound (25, 47). Indeed, we observed a decreased cell number in treated cultures compared to the untreated cultures, and cells with activated centers were frequently rounded up. In contrast, treatment of cells with trichostatin A, a potent inhibitor of histone deacetylases, failed to activate the silenced centers (data not shown).
We also tested late passages of HPV-immortalized cell lines that had only one nascent RNA center. In treated FK16A passage 82 cells, multiple centers were observed (Fig. 7C, in green). In treated FK18A passage 113 and FK18B passage 115 cells (Fig. 7D and E), the nascent HPV RNA signals (in green) either colocalized with or were larger in size than the corresponding DNA center (in red), a finding distinctively different from the pattern in untreated cells (Fig. 3G to I). However, as with CaSki cells, some treated cells contained only the original, active 3' boundary copy (Fig. 7D). These observations suggest that the long-term silencing of HPV DNA centers is mediated at least in part by methylation on cytosine.
Perinucleolar localization of viral transcription. The nucleus is a functionally organized environment, with many gene expression and regulatory processes occurring in distinct subnuclear domains (34, 35, 55). We investigated whether the PVTD is spatially associated with previously described nuclear domains that are known to be involved in transcription regulation, RNA splicing, or cell cycle regulation, such as the nuclear domain 10 (ND10) or PML bodies (revealed by PML) and Cajal bodies (20, 22, 46). Extrachromosomal HPV DNA replication may occur in the neighborhood of ND10 (62), as is the case with a number of other DNA viruses (18). Cajal bodies (20) are nuclear domains implicated in the control of S-phase-associated histone gene transcription mediated by p220npat (31, 71).
Colocalization studies were performed first with CaSki cells by probing for nascent HPV-16 transcripts (in red) and then for PML (Fig. 8A, in green) or for p220npat (Fig. 8B, in green), with or without an antigen retrieval process. We observed no associations. Additional domains involved with gene transcription and RNA metabolism are the interchromatin granule clusters that are visualized as immunofluorescent "speckles" with antibodies to spliceosome assembly factor SC-35 (56). Simultaneous visualization of SC-35 antibody (in green) and HPV-16 DNA in CaSki cells (in red) (Fig. 8C) showed only stochastic colocalization with the larger tandem viral DNA arrays. Although most of the transcripts of oncogenic HPVs spanning the E6 region are spliced, nascent viral RNA (Fig. 8D, in green) did not spatially associate with SC-35 domains (red) (Fig. 8D). This lack of association has also been reported for transcripts of some cellular genes as well (51).
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DISCUSSION |
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Using T-FISH to probe for nascent viral RNA simultaneously with integrated viral DNA, metaphase or interphase chromosomes, or nuclear domains with T-IF, we demonstrate here for the first time directly that there is a strong selection for cells containing only a single transcriptionally active viral DNA integrant and that this locus is perinucleolar in the majority of the cells examined, regardless of the number of centers (Fig. 1, 3, 5, and 8). We have named this the PVTD. Additional viral copies do not have associated nascent viral RNA transcripts. The SiHa cell line appears to be an exception, since both copies of HPV-16, one each at genetically identical loci on the pair of chromosomes 13 (1, 3, 17, 33), are transcriptionally active (Fig. 1). We speculate that this double RNA center might even have arisen as a result of positive selection for a chromosome duplication or gene conversion event. Oncoprotein production in their progenitor cells might have been less than optimal and became outcompeted by cells that underwent the duplication during the development of the cancer or the establishment of the cell line. Similarly, cells that no longer express viral oncogenes will not be maintained, as clearly demonstrated by in vitro experimentation (14, 67).
Our data also indicate that, within any one cell line, the single RNA center is clonal rather than stochastically variable among individual cells, implicating a regulated expression. First, our systematic in situ mapping data revealed that the transcriptionally active template in CaSki cells is located at a single-copy or low-copy-number site on a derivative chromosome 14 (Fig. 2), among the dozen loci that together harbor up to 600 copies of viral DNA. These mapping data are supported by 3' RACE in which five of five cDNAs have the same virus-host junction sequence. Second, in FK18 cells harboring multiple copies of the HPV-18 DNA in a single large tandem array, only the virus-host junction copy appears to be transcribed (Fig. 3). Of greatest relevance, each of 13 primary HPV-associated cancers examined also exhibited one viral transcription center per cell, when observable (Fig. 6).
Our data show that the evolving viral transcription patterns are attributed to epigenetic rather than genetic alterations, in that transcription from the inactive integrants can be reactivated by 5-azacytidine, implicating DNA methylation as at least being partially responsible for silencing transcription (Fig. 7). Reactivation was not achieved by exposing the cells to trichostatin A, an inhibitor of histone deacetylases (data not shown). These results agree with the current view that DNA methylation confers long-term DNA silencing, whereas deacetylases shut off transcription on a short-term basis (13, 43). Our observations are in excellent agreement with a recent report (2) that HPV-16 DNA is targeted for CpG methylation in vivo and that changing methylation patterns were observed with different lesion grades. The data are also consistent with reports that transfected and integrated HPV DNA is heavily methylated and transcriptionally silent (5, 29, 45). However, reactivation was not demonstrated in those studies.
Our findings explain why one predominant viral transcript was identified in cancers and carcinoma cell lines (26, 53, 68) and also why little or no E2 protein is expressed. We speculate that transcription from the internal copies may have contributed relatively little to the expression of viral oncoproteins relative to the junction copy due to low stability of mRNA utilizing the viral poly(A) site (23). Furthermore, there is also a selection against cells that express high levels of E2 which would downregulate the promoter for the viral oncogenes (9, 14, 16, 44, 63, 67). An additional impetus for silencing comes from our recent observation that the HPV E2 protein associates with the mitotic spindles to enable extrachromosomal HPV DNA plasmids to segregate as minichromosomes and to establish persistence (64). However, upon viral DNA integration, E2 might contribute to genomic instability if E2 bound to the viral origin in tandemly integrated HPV DNA creates a viro-centromere, causing chromosome breakage when it is pulled to the opposite centrosome relative to the kinetochore. Mitotic catastrophe associated with the breakage-fusion bridge cycle (32) can be avoided only when the internal tandem viral copies capable of encoding E2 are silenced. Selection for the fittest cells for progression can then begin.
The selection for a single dominant expression locus appears to be a dynamic and plastic process. Various immortalized cell lineages reached this state after different numbers of cell division during passage in vitro, perhaps depending on the number of viral integration sites, the transcriptional activities from these various loci, and the relative mRNA stabilities conferred by the captured 3' host untranslated regions and poly(A) sequences. The selection may ultimately be predicated on the absolute level of viral oncoprotein expression, as was suggested by our oncogene challenge experiment (Fig. 5). By introducing an extra copy of the HPV oncogenes into a cell line which had already arrived at a single expression center, we presented a striking example in which another round of transcription center selection was attained within a matter of weeks when the cells experienced a new and strong selection pressure for the newly introduced viral oncogenes that are transcriptionally and translationally coupled to a drug resistance gene. The surviving cells that emerged from an apparent "crisis period" turned off the original viral RNA expression center and replaced it with the newly introduced copy, gaining puromycin resistance while maintaining E6/E7 production. Thus, the crisis may have been caused by too much of the viral oncoproteins. Had the higher level of viral oncogene gene expression from both the resident and the newly introduced copies been of no consequence or even advantageous to the cells, we would not have observed the silencing of the original viral RNA center. We presume that the silencing of the original RNA center might be attributed to chromatin remodeling by DNA methylation, as was observed in other cell lines. However, extensive cell death upon exposure to 5-azacytidine prevented us from testing this hypothesis.
Although the selection for cells with more stable transcripts and higher levels of viral oncoproteins in proliferating cells is important at the early stage of viral oncogenesis, in the long run, what seems to be important is an optimal level of oncogene expression from a locus most suitable for the particular environment in which the cells find themselves. Neither too little nor too much of the viral oncoproteins will do. Cells that fail to cycle rapidly are overgrown; those that divide too quickly may not be able to replenish their cellular components and are lost during passage, and others may undergo senescence. Our results with primary HPV cancers are consistent with a clonal selection for the fittest cells, which is in agreement with the notion that cancer is monoclonal in origin (6, 28). We suggest that the ability to detect a single viral RNA center might provide for a novel, highly relevant biomarker of neoplastic progression. Additional experiments will be required to test this hypothesis. Cellular oncogenes are amplified in a variety of cancers, e.g., the N-myc locus in neuroblastomas (49), ErbB2 in breast cancer, and EGRF in other cancers (42). It will be most interesting to determine how many of the amplified copies are actively transcribed. Finally, the ability to conduct multiplex assays with tyramide-enhanced reagents and the gene regulatory concepts described here will have considerable applicability to attempts at cancer control, analysis of diseases, production of transgenic stem cells, and gene replacement therapy.
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ACKNOWLEDGMENTS |
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We thank Ge Jin, Martha Hayes, Heather Benedict-Hamilton, Brian Streib, and Susie Godbey for technical assistance; Albert Tousson, Julie Decker, and George M. Shaw for support during the initial phase of this project; Mark Bobrow, Kim Fitzpatrick, and Yoshika Sherring from Perkin-Elmer for some of the reagents and advice; and Wade Harper for the p220npat antibody. We also gratefully acknowledge the 1998 Advanced In Situ and Immunohistochemistry Class at Cold Spring Harbor Laboratory for being instrumental to the conduct of this research.
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FOOTNOTES |
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Present address: Genzyme Genetics, Santa Fe, NM 87505.
Present address: One Cell Systems, Inc., Cambridge, MA 02139.
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