Cellular Transformation by a Unique Isolate of Human Papillomavirus Type 111 Ronald C

(CANCERRESEARCH52, 5872-5878, November1, 19921 Cellular Transformation by a Unique Isolate of Human Papillomavirus Type 111 Ronald C. McGlennen, Jyotsna Ghai, Ronald S. Ostrow,2 Kurt LaBresh, John F. Schneider, and AnthonyJ. Faras Departments ofLaboratory Medicine and Pathology fR. C. M.J, and Microbiology (I. G., R. S. 0., A. J. F.J, and the institute ofHuman Genetics (R. C. M., I. G., R. S. 0., K. L, I. F. S., A. I. F.], University ofMinnesota Medical School, Minneapolis, Minnesota 55455

ABSTRACT questions regarding the role of â€œlowoncogenic potentialâ€• vi ruses like HPV 11 to be involved in malignant transformation. Infection with human papillomvirus type 11 (HPV 11) is associated There are two aspects to the problem ofevaluating the role of with benign epithelial proliferations and rarely with malignant and HPVs in tumorigenesis in vivo. One consideration is the extent metastasizing tumors. Because of the biological diversity displayed in tissues InfectedwithHPV 11, wehaveexaminedthe capacityof various to which the infected cell or, in the larger context, the host and isolates of HPV 11 to transform cultured cells and compared their its environment play in promoting the progression of benign molecular differences by DNA sequence analysis. Five isolates of HPV HPV lesions on to malignant transformation. Included here are 11 were examinedfor their ability to transformprimaryneonatalrat factors such as environmental carcinogens in cigarette smoke, kidney epithellal cells and NIH 3T3 mouse flbroblasts in DNA trans and other chemicals which are tumor promotors (9, 10). fection experiments using calcium phosphate precipitation. Induded in Chronic immunosuppression, whether associated with preg these studies are the prototype isolate from a laryngeal papillonsa(HPV nancy or occurring with organ transplantation, has been shown liP); HPV 11VCfroma verrucouscarcinomaofthe penis HPV ilEpi to be an important predisposing factor to the development of from the viral episomes of a primary squamous cell carcinoma and two integrated genomes (HPV hInt 1 and HPV hInt 2) ofthe metastases. malignant cutaneous and mucosal HPV-associated tumors (11â€” Only HPV I1VC cotrausfectedwith the oncogeneHa-nsa transformed 13). Our laboratory has recently characterized a squamous cell neonatal rat kidney eplthelial cells with an efficiencycomparable to that carcinoma in a renal allograft recipient arising from the peria of HPV 16 DNA. HPV 1IVC DNA alone transformed NIH 3T3 cells. nal skin and containing HPV 11 (14). Analysis of the DNA sequence of HPV lip and 11VC revealed 16 A second aspect important in the evaluation of HPV-associ single nucleotide changes In the upstream regulatory region and open ated malignancy is to consider the oncogenic potential of the reading frames El, E2, E4, and ES, five resulting in amino acid substi virus itself. At the molecular level, human papillomaviruses tutions. This is the first demonstration of cellular transformation by a share a high degree of similarity in their genomic organization, natural Isolate HPV 11 DNA in ritro and illustrates that minimal changes In the DNA sequence of certain viruses confer oncogenicityto yet only a specific subset of HPV types are typically oncogenic what are normally nontransforming viruses. in vivo(1). Oneexplanationof the differencesin the oncogenic potential between these two groups of HPVs is thought to be due to simple base pair mutations in critical areas of the viral INTRODUCTION genome such as in the URR, which contains viral early promot HPVs3 are a group of small DNA viruses, of which there are ers and enhancers, or in the region of the early genes of the over 60 distinct types that cause a variety of epithelial lesions, virus, which encode for several putative oncoproteins (15). but are most notable because of their etiological association Transfection ofcloned viral DNAs into cultured mammalian with cervical cancer (1). Infection with HPV causes a wide cells has been used to measure the transforming potential of spectrum of mucosal and cutaneous lesions which include be certain viruses (15). Stable integration of the transfected viral nign epithelial proliferations and highly invasive and metasta DNA into cultured cells can induce morphological changes, sizing malignant tumors of the skin, respiratory tract, and gen with concomitant alterations in several growth parameters. ital mucosa (2, 3). Epidemiological data have demonstrated Among the human papillomaviruses, HPV 16 DNA has been that certain HPV types such as HPV 16, 18, 31, and 33 are shown to efficiently transform neonatal rat kidney epithelial more commonly associated with premalignant and malignant cells when cotransfected with the activated form of the onco lesions and thus have been designated as the oncogenic HPV gene Ha-ras and when in the presence of glucocorticoid hor types (4). By contrast, virus types with low oncogenic potential, mone (16, 17). Under identical conditions, however, the proto such as the closely related HPVs 6 and 11, frequently are as type HPV 11 DNA, molecularly cloned from a laryngeal sociated with condyloma acuminatum and orolaryngeal papil papilloma, failed to transform NRK cells (18). With the diver lomas, which except in cases where patients are immunocom sity of in vivo biological activities displayed in tumors contain promised or have received radiation therapy, have rarely been ing HPV 11 DNA from several patients, we have speculated shown to undergo malignant transformation (5, 6). In recent that different isolates of HPV 11 obtained from malignant tu years, several published reports have described patients with mors may have been altered in significant ways, rendering them primary and metastatic tumors of the lung, vulvar skin, and capable of cellular transformation in vitro, similar to HPV 16 anal mucosa found to harbor episomal and integrated forms of DNA. To examine this hypothesis, we have characterized five HPV 6 or 11 DNA (7, 8). These clinical examples have raised molecularly cloned, naturally occurring isolates of HPV 11 de rived from a spectrum of lesions and have performed in vitro Received9/23/91; accepted8/26/92. transfections to evaluate their respective potentials for trans The costs of publication of this article were defrayed in part by the payment of pagecharges.Thisarticle mustthereforebeherebymarkedadvertisementinaccord forming NRK and NIH 3T3 cells. The capacity of these viral ance with 18 U.S.C. Section 1734 solely to indicate this fact. isolates to transform cultured cells and several functional pa I Supported by NIH Grant CA25462 and the Leukemia Research Fund. rameters of the effects of these cloned viral DNAs on cellular 2 To whom requests for reprints should be addressed, at University of Minnesota Medical SChOOl,BOX206, Harvard Street at East River Road, Minneapolis, MN growth and differentiation were determined. Finally, by com 55455. paring the molecular structure of these viral isolates by DNA 3 The abbreviations usedare: HPV, human papillomavirus; NRK cells, neonatal rat kidneyepithelialcells URR, upstreamregulatoryregion;CM, completemedia, sequence analysis, we have attempted to explain their respective ORF,openreadingframe. biological differences. 5872

MATERIALS AND METHODS 20x standard saline citrate phosphate (lx is 0.12 M NaCl, 0.015 M sodium citrate, 0.013 Mpotassium phosphate, 0.001 MEDTA, pH 7.2) Molecular Cloning of Various HPV 11 Species. The prototype spe (24). Filters wereprobed with 5 nglml ofrandom-primed HPV 11DNA cies ofHPV (HPV 11P) was a generous gift from Dr. L. Gissmann and (specific activity, 4â€”8x 10@cpin/@ig)(25). Filters were washed under was derived from a benign laryngeal papilloma. The sample we received high stringency conditions (temperature midpoint â€”25C)and then had been molecularlycloned into a bacterialplasmid vector at the autoradiographed using Kodak XAR-5 X-ray film (Eastman Kodak, unique BamHI restriction endonuclease site that occurs in the struc Rochester, NY). tural gene Li (19). The full-length (8-kilobase) HPV 11P was excised Assay of Anchorage-independent Growth and Tumorigenesis in and recloned into the unique BamHI site of the vector pUC 19. HPV Athymic Nude Mice. The ability of transformed cells to display an 11VC is a full-length (8-kilobase) episomal viral genome originally chorage-independent growth was assayed by incubating various cell cloned in the BamHI site from the ACharon 27 genomic library derived lines in semisolid media. Tissue culture plates contained a support layer from a penile (Buschke-Lowenstein)verrucouscarcinomapreviously of 0.5%agarosein CM and a feederlayercomprising0.3%agarosein described (20). Three additional isolates of HPV 11 were cloned di CM seededwith 1.0, 2.0, or 4.0 x [email protected] were incubatedat rectly into pUC 19 from the cellular DNA extracts from tissues de 3TC in 7% CO2 and observed periodically for the appearance of cell scribed previously including a primary squamous cell carcinoma of the clusters. Tumorigenesis studies were performed by injecting 1.0 X l0@ perianalskinanda metastaticlivertumorofa renaltransplantrecipient cells s.c. from the various transformed cell lines and controls into the (14). Two-dimensional agarose gel electrophoresis ofuncleaved cellular hindquarter of 2â€”3-week-old BALB/c athymic nude mice. Animals DNA was used to separatecircularepisomalviralDNA fromlinearor were examined twice weekly for the appearance of palpable tumor integrated species (21). The isolate called HPV 1lEpi was obtained nodules, and when present the animals were sacrificed and the tumors from the region corresponding to nonintegrated monomers and multi were analyzed by histopathology and for the presence of viral DNA by mersofviral DNA fromtheprimaryanalcarcinomaandclonedintothe Southernblot hybridizations. BamHI site of pUC 19. Two independent clones of integrated forms of CharacterizationofHPV11IsolatebyDNASequenceAnalysis.All HPV II were isolated from BamHI digests of metastaticlung tumor determinations of nucleotide sequences were performed on double DNA containing only integratedDNA and are full-length8-kilobase stranded plasmids containing the HPV 11genome. Approximately 7 @ig genomic species (HPV 1lInt 1 and mt 2). All ofthe viral DNA isolates of plasmid DNA were denatured in 0.18 N sodium hydroxide and 0.18 werecharacterizedbyrestrictionendonucleasemappingand DNA hy mM EDTA for 5 mm at room temperature. The solution was neutral bridizationswith the known patternto the prototypeHPV 1lP. No ized with the addition of Tris-HC1 (jH 4.5) to 0.27 NI.The DNA was differences were observed after digestion with 22 common restriction precipitated with 0.27 M sodium acetate and two volumes of cold eth endonucleases. anol followed by an 80% ethanol wash and then dried in vacuo. Se DNA TransfectionwithCalciumPhosphatePrecipitationandEstab quencing reactions using â€œS-labeleddATP (New England Nuclear, lishment of Transformed Cell Lines in Vitro. DNA transfection was Wilmington, DE) were performed with a commercial kit (Sequenase; carriedout with a mammaliantransfectionkit (Stratagene,Inc., La United States Biochemicals, Cleveland, OH). In many cases, sets of Jolla, CA), which is a modification of the calcium phosphate precipi reactions containing dITP in place of dGTP were used as suggested by tation method previouslydescribed(22). Transfection experiments used the manufacturer to resolve DNA compressions. The sequences were either a low-passageSwiss-typeNIH 3T3 mouse fibroblasticcell line or determinedfollowingpolyacrylamide(6%w/v)gelelectrophoresis,fix neonatal rat kidney epithelial cells. NRK cells were prepared by minc ation in 5% (w/v) methanol:acetic acid, dehydration of the gel, and ing of 1â€”3-day-oldFischer rat kidneys under sterile conditions, fol autoradiography. Synthetic oligonucleotide primers 16 base pairs in lowed by enzymaticdissociationof cells using 0.83 mg/ml of type II size were generated at intervals of approximately 300 base pairs from collagenase and Dispase, a neutral protease (Boehringer-Mannheim nucleotide 7869 to 4231, beginning in the terminal portion of the Ll Biochemicals, Indianapolis, IN) and 0.4% trypsin in EDTA (Gibco open reading frame and coveringthe entire early region ofHPV liP, as Laboratories,Inc.,Grand Island,NY).The cellswereculturedin CM determinedfromthe publishedsequence(19). consisting ofmodified Eagle's medium (Gibco Laboratories), 10%fetal Expressionof HPV 11VC RNA in TransformedCells. NRK cells calfserum, penicillin (100 units/ml), streptomycin (100 units/ml), and transformed by HPV 11VC DNA were single cell cloned and expanded. Messenger RNA was extracted using a FastTrack mRNA kit (Invitro Fungizone (0.25 gig/ml) and plated at a density producing a 15â€”30% confluentculture.When the densityapproached60â€”80%confluency gen, Inc., San Diego, CA) according to the manufacturer's instructions. mRNA (100 ng) was incubated with two primers (5'-CCAGTTGTG transfections were performed using 10 @igofHPV 11 plasmid DNA, with or without 10 @tgHa-ras(pEJ6.6) and pSV2Neo, a plasmidcon CAAGACG-3', 5'-GAAGCGTGCCrTrCCC-3') for the E6 ORF of HPV 11 in the presence or absence of reverse transcriptase(BRL, taming the gene for the selective marker neomycin, to produce a cal cium phosphateDNA complex(17, 23). The precipitatewasincubated Gaithersburg, MD) according to the manufacturer's instructions. (Note thatthesecondprimermatchesthepublishedHPV 11sequence,but,as on the cells for 8â€”12hand then removed by thoroughly washing with indicated below, we found that the underlined nucleotide was actually a phosphate-buffered saline (pH 7.4), and the cells were reincubated in CMat37Â°Cat7%CO2.Theculturesgrewrapidlytofullconfluency,at T. Nonetheless, this primer can be used for sequencing or polymerase which time they were trypsinized and diluted 1:4and allowed to expand chain reaction amplification with the first primer to produce a 337- nucleotide product). Following deactivation of this enzyme and dena to confluency. Selection of neomycin-resistantfoci was achieved by turation of the nucleic acids by boiling for 5 mm, TaqI polymerase and incubation in media containing 1 @iMdexamethasone and the antibiotic buffer were added, and the reactions underwent 40 cyclesof polymerase Geneticin G4l8 (Gibco Laboratories) at 0.83 mg/ml, beginning 48 h chain reaction. An aliquot was analyzed by agarose gel electrophoresis followingtransfection.After18â€”2idaysfoci ofmorphologicallyaltered followed by hybridization to a HPV 11 E6 DNA probe. cells began to appear in samples treated with HPV 16 plus Ha-nix and adenovirus Ela plus Ha-ras plasmid DNAs and several days later with HPV i 1 plasmids.Transformedfoci of cells were isolated, dilutedto RESULTS produce single cell clones, and passed serially using routine culture The five isolates of HPV 11 examined in this study for their conditions. ability to transform primary and immortalized cultured cells DNA Analysis by SouthernBlot. Culturedcells wereextractedfor have unique origins and have been initially molecularly cloned total cellular DNA as describedpreviously(14). Total cellular DNA from each sample (8â€”10@@g)wasdigestedwith the restrictionenzyme from three separate clinical sources. The prototype HPV 11 BwnHI for 12â€”16hat 3TC. One-dimensional gel electrophoresis was viral DNA was originally cloned from a laryngeal papilloma, performed as previously described, and cellular DNA was denatured in and the genomic map and DNA sequence have been previously alkali, neutralized, and transferred to a nylon membrane (Zeta-bind; published (19). Four additional HPV 11 isolates were each BRL, Boston, MA) by the Southern procedure (23), using a solution of cloned into the bacterial vector pUC 19 either directly from 5873

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. UNIQUE HPV 11 ISOLATETRANSFORMSCELLS their tissue of origin or, in the case of HPV I IVC, subcloned Table2 ComparisonoffeaturesofcellulartransfectedHormone transformation cells withclonedviralpiasmidsin from an intermediate A phage genomic DNA library. All five isolates of HPV I 1 are 8 kilobases in length, which is the full SerumCloned agarTumorplasmidcellviral% ofclonal dependent depletionSoft length for most of the human papillomaviruses. Similar plas growthgrowthgrowthNRK lines4 growth mid constructs of cloned HPV 16 DNA have previously been cellsbNoaddedplasmid0 shown to transform NIH 3T3 by itself and, in cooperation with â€”â€”â€”Adenovirus â€” Ha-ras, primary NRK cells (16). An earlier study reports that a Ela100 No Yes++Yes 1â€”2wkHPV plasmid construct identical to our HPV 11P does not transform 1650 Yes Yes++++Yes NRK cells (18). 1â€”2wkHPV The ability of cultured cells to form morphologically altered 11VC45 Yes Yes+60% wkHPV11P0 5-6 cell colonies or foci is the first test of cell transformation assay â€”â€”â€”HPV1IEpi0 â€” and reflects two characteristics of a transformed cell: clonality â€”â€”â€”HPVllIntl0 â€” â€”â€”â€”HPV1IInt20 â€” and the loss of contact inhibition. The focus-forming capacity â€”â€”â€”NIH â€” was tested for the HPV 11 isolates in NRK cells transfected 3T3CNoaddedplasmid0 with 10 @igofeach ofthe respective plasmids, along with Ha-ras â€”â€”â€”Adenovirus â€” and pSV2Neo DNAs, and cultured for 4 weeks in selective Yes++++YesHPVEmiOO No media containing dexamethasone. Cells transfected with the Yes++++YesHPV16100 No HPV 11VC plus Ha-ras form large numbers offoci, comparable Yes++YesHPV11P011VC60 No Noâ€”â€”HPV11Epi No in appearance and number to cells transfected with HPV 16 No No plus Ha-ras (data not shown). By contrast, the plasmids con HPVllIntl0ND'@â€”â€”HPV11Int2ND taming HPV 1lP, HPV 1lEpi, HPV 1lInt 1 and HPV hInt 2, a Focus formation includes only those colonies of cells which show an altered the negative control Ha-ras, or pSV2Neo produced no foci of morphologyfromthe backgroundcells,and/orthosethat demonstratecellular morphologically altered cells, although occasional colonies of piling. nonmorphologically transformed cells appeared but could not b All transfections in NRK contained pSV2Neo and Ha-ms plasmids. C All transfections in NIH 3T3 cells contained the pSV2Neo plasmid. be expanded (data not shown). d ND, not done. The efficiency of focus formation compares the capacity of heterologous DNAs to transform primary and immortalized Similar experiments were performed using the immortalized cells. Table 1 lists the relative focus-forming efficiency for the cell line NIH 3T3 (Table 2), which has been shown to become HPV 11 isolates compared to HPV 16 and adenovirus Ela. As transformed when transfected with HPV 16 DNA alone and previously reported, the viral oncogene Ela, derived from the thus provides a second system in which to compare the onco type S adenovirus, morphologically transforms NRK cells in genicity of these respective viral DNAS. Transfection with ci combination with Ha-ras with a high level of efficiency (26). ther adenovirus Ela or HPV 16 DNAs produces more than 200 Relative to adenovirus Ela, transfection with HPV 16 plus Geneticin-resistant foci/l00-mm culture plate, with approxi Ha-ras DNA produces approximately 60% the number of trans mately 50â€”60%ofthese foci demonstrating the characteristic formed foci and is dependent on the addition of the glucocor refractile and piled-up appearance of transformed cells. Among ticoid hormone dexamethasone. The same quantity of HPV the HPV 11 isolates, only the HPV 11VC plasmid produced 11VC and Ha-ras DNA had a comparable efficiency of trans morphologically transformed foci. Isolation and expansion of formation of 45â€”50%in cells treated with dexamethasone in these foci (typically 15â€”20individual foci) for adenovirus Ela, three separate experiments. No transformed foci were observed HPV 16, and HPV 11VC revealed that only the foci with a in the absence ofhormone. Following the expansion of primary transformed appearance on the primary plate remained so in transformed foci and single cell cloning, HPV 11VC-transfect serial passages. ed cells were small, round in shape, and grew in dense piles The nature of cells transfected with HPV 11YC DNA was (data not shown). Furthermore, each of the foci on the HPV examined further by assaying whether morphologically trans I 1VC, HPV 16, and Ela could be subsequently expanded into formed-appearing foci of NRK cells could be maintained as cell continuous cell lines (Table 2). lines and could subsequently display anchorage-independent growth. Several foci from each plate were isolated by passing them 3â€”5timesand then diluting the cells to very low concen Table1 Efficiencyoffocusformationin NRKcelisfollowingtranswithFoci/gzg cloned viral piasmidsfection trations in an attempt to develop single cell clones. These mon oclonal cell lines were then expanded and passed again into DNAâ€• Cloned viral plasmid semisolid agar-containing CM. These plates were then incu DexHPV11VCtransfection of NRK cellsâ€• I MMDeXCNo bated under normal conditions and observed for the formation 113HPVI1P of cell clusters and spheres within the agar/media. Each HPV 00HPVI1Epi I0HPVllIntl 11VC, HPV 16, and adenovirus Ela cell line displaying a trans 00HPVlIInt2 formed morphology on the primary plates also grew in soft agar 00Adenovirus (Table 2). The rate of growth of HPV 11YC cells in soft agar 1615HPV16 Ela 151NoviraiDNA was about one-half of that observed for HPV 16- or adenovirus 00 Ela-transformed cells. The ability ofin vitro derived celllines to a All samples cotransfected with 10 @g pSV2Neo and Ha-ras and selected for form invasive tumors in animals is considered an essential and G4l8 resistance. b Results are the averages of 3 experiments (2 plates each) and represent both definitive test ofcellular transformation. The tumorigenicity of morphologicallytransformedand nontransformedfocinormalizedto the quantity HPV 11VC plus Ha-ms-derived NRK cell lines was assayed by ofDNA added.Noneofthe coloniesappearingonplatesotherthanHPV 16,Ela, injecting 1.0 X 10@cells s.c. into the hindquarter of 2â€”3-week andHPV llVCdemonstratedatransformedphenotypeorwereabletobepassaged into cell lines. old BALB/c athymic nude mice and found to produce large C Dex, dexamethasone. invasive tumors in 60% of the animals after about 6 weeks 5874

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. UNIQUE HPV I1 ISOLATETRANSFORMSCELLS (Table 2). The histological appearance of one such tumor is 1234 characteristic of a poorly differentiated carcinoma, with focal areas of glandular differentiation (data not shown). By compar 872 ison, adenovirus Ela- and HPV 16-transfected cell lines pro duced bulky invasive tumors within 9 days, also showing t'ea Size 03 tures of an undifferentiated carcinoma. Untransfected NRK (bp) 310 cells produced no tumors. .. The effects of serum deprivation on cellular growth in HPV 281/271 11VC cell lines was measured by counting the increase in cell 234 number over time for transformed and nontransfected NRK cells. HPV 11VC cell lines and other transformed cells were I 94 maintained for up to 96 h in media containing various amounts Fig. 2. Analysisof mRNA which was reverse-transcribedand amplified from variousHPV llVC DNA-containingcells.mRNAwasisolatedfrom independent of fetal calf serum (Table 2). The HPV 11VC grew slowly in cell lines 13(Lanes1 and 2) and 14(Lanes3 and 4), treated with (Lanes2 and 4) these conditions, while HPV 16 and adenovirus Ela DNA or without (Lanes I and 3) reversetranscriptase, subjectedto polymerasechain reactionamplificationfor 40 cyclesusingHPV 11E6 primers,electrophoresedin transformed cells or SiHa cells, a cervical carcinoma-derived 2% agarose, transferred to nylon membranes, and hybridized to a 32P-labeled cell line containing a single integrated copy of HPV 16, main probe made from HPV 11 DNA. No hybridization was observed in a buffer tamed high levels of proliferation even in markedly reduced control (data not shown).Left ordinate, some of the OX 174 HaeIII markers in base pairs. Arrow, position comigrating with a positive control band correspond serum concentrations (data not shown). By contrast, untrans ing to amplification from HPV 11 genomic DNA (337 base pairs; data not fected NRK cells were growth inhibited at serum levels of 2.5%, shown). whereas at 10% serum concentration, they had growth rates similar to those of the transformed cells. Although this combi treated extracts of total cellular DNA (Fig. 1). The results dem nation of findings concerning the growth characteristics of onstrate hybridization to an 8-kilobase band of high copy num HPV 11VC cells in vitro indeed confirms their transformed ber (20â€”50copies/cellequivalent). The hybridization of 2.6- nature, it appears that there are differences between the onco kilobase bands indicates the incorporation of the pUC-19 genic potentials of this and other viral DNAs. vector sequences in tandem with the HPV 11VC genomes. Molecular analysis of the HPV 11YC-transfected NRK and Other bands of varying intensity and size may be the results of NIH 3T3 cell lines demonstrated HPV 11 DNA in BamHI integration (Fig. 1, Lane 3). Two-dimensional gel analysis of uncut cell line DNA revealed hybridization of only the high molecular-weight curve of cellular DNA (data not shown) (14, 1234 23). These results indicate the stable integration ofHPV 11 and plasmid DNA into the cellular genome in multiple tandem repeats of the full-length viral genome. Messenger RNAs from these cells were examined by combined reverse transcriptase/ polymerase chain reaction followed by hybridization with an HPV 11 DNA probe. Two independent HPV 11YC-trans formed cell lines demonstrated active transcription of the ge nome (Fig. 2). In each case a HPV 11YC E6-specific band of 8Kb@ 337 base pairs was observed only in incubations which included reverse transcriptase, indicating that the bands did not result from DNA contamination of the samples. DNA sequence analysis of HPV 11YC and of HPV 11P was performed, and a comparison was made of the changes in the HPV 11VC genome from the published HPV 11 sequence (pro totype clone) (19). Because we were comparing the biological activity of the transforming HPV 11VC from this laboratory to the nontransforming prototype HPV 1lP, it was important to determine the nucleotide sequence of the early regions of both of these genomes in tandem in order to ensure that no bacteri ally derived mutations in the prototype clone might interfere @ â€”@â€” with our interpretation of the biological results. A total of 18 oligonucleotide primers were used to determine the sequence of both of these genomes, including the complete URR and early region on the coding strand. A total of 10 nucleotide changes were observed between the published sequence and the proto type HPV 1lP DNA (Table 3). These changes do not appear, however, to be the result of plasmid alteration during mainte Fig. I. Southern analysisand hybridizationof extracted transformed cell line nance, since each ofaltered nucleotides matches the sequence in DNA to an HPV 11 DNA probe.TotalcellularDNA waspreparedfromtrans HPV 11VC. In addition, a portion of the E6 region of HPV fectedcelllines,and 10 @igweretreated with the restrictionendonucleaseBamHI, 11Epi was sequenced as well, which included five of the total fractionated by electrophoresis on 0.8% agarose gels, transferred to nylon mem branes, and hybridized to a 32P-labeled random-primed HPV 11 DNA plasmid base pair changes, and primers were used that would read three probe(specificactivity> 3.0 X l0@cpm4ig) as describedin â€œMaterialsand of these sites on both strands. Here, too, the HPV 11p changes Methods.â€•Arrow,full.lengthlinearHPV 11 (8.0kilobases).pUC-19(2.6kilo bases) is visible at the bottom of each lane, and other bands are indicative of from the published sequence were identical to the HPV I lEpi integrationofthe plasmid. sequence. 5875

The comparison of the nucleotide sequence of HPV 11VC genome was subsequently lost upon passage (31). Also, using with the revised sequence of HPV 1lP revealed 16 single base rat embryo cells, full transformation was observed with the changes located in the URR and in the early region ORFs El, prototype of HPV 11 in cotransfections with activated Ha-ras E2, E4, E5a, and E5b of the genome (Table 4). No mutations (32), which are in contrast to results from various laboratories were identified in the E6 or E7 ORF, which have been shown to in NRK cells. We have utilized the prototype HPV 11, which be the major transforming regions of the HPV 16 genome (27). we have called HPV 11P, as well as four additional isolates of Seven of the changes are silent, i.e., the base substitutions do HPV 11, obtained from malignant tumors from two different not alter the codon for the amino acid, nor do they cause a shift patients, in transfection experiments to test their respective in the reading frame ofthe early gene open reading frames. Five capacities to transform NRK and NIH 3T3 cells. Only one of base changes would lead to amino acid substitutions; four these isolates, HPV I 1VC, derived from episomal viral DNA changes occur in the URR. Table 4 indicates the location and from a squamous cell carcinoma of the penis, was found to be the putative amino acid changes in the HPV 11VC genome. oncogenic at efficiencies oftransformation comparable to HPV 16 DNA. Cell lines containing stably integrated HPV 11VC DISCUSSION DNA were found to fulfill the prerequisite characteristics of transformed cells, namely that they have an altered morphology Cell transformation resulting from the transfection of the cloned DNA of HPV 16 and Ha-ras has become the paradigm in monolayer culture, have a diminished dependence on serum, of papillomavirus-induced oncogenesis in vitro (16). Subse display anchorage-independent growth, and are tumorigenic in quently, each of the â€œoncogenicâ€•HPVtypes have been shown animals. Based on the findings that only one of the five isolates to transform primary cells, when combined with Ha-ras, c-myc, of HPV 11 we have examined is oncogenic and the fact that or v-fos, with high efficiency (28, 29). Based on the genomic HPV 11-associated malignancies in patients are rare, we have organization and the in vivo induction of marked proliferation postulated that it is likely that features intrinsic to the HPV in infected cells, it might be expected that HPV 11 would im 11VC genome account for its ability to transform cells. mortalize or transform cells in vitro as well. Previous work has The analysis of the DNA sequence of HPV 11P and 11VC demonstrated that the prototype of HPV 11 DNA, cloned from revealed several interesting point mutations that could explain a laryngeal papilloma, when cotransfected with Ha-ras failed to the difference in biological activity of these two isolates. One transform NRK cells (18). Others have reported similar results important finding is that the sequence of the E6/E7 regions of in experiments using the highly related virus HPV 6 (30). Using each of the HPV 11 isolates are identical. It has been shown synthetic duplex URRs in an HPV 11 DNA construct, in previously that the major transforming region of HPV 16 and creased focus formation had been observed, but the HPV 11 18 genomes lie in the E6 and E7 ORFs, which encode for at least two oncoproteins that are translated from a series of either full-length or spliced mRNAs (15, 33). One hypothesis used to Table3 ObserveddsfferencesbetweenthepublishedHPV11 sequenceandthat explain the lack oftransforming function ofHPV 11 E6 and E7 determinedfor HPV lip relates to the fact that the HPV 1lP genome lacks the splice Nucleotide acid acceptor/donor sites within these ORFs and thus cannot pro locationPublishedâ€•sequenceHPVregion137 lipAmino changeAffected duce the full complement of mRNAs (18). The sequence of 186 A C E6 HPV 11YC also does not contain these putative RNA splice 380 C T E6 sites, and therefore, the differences between the transforming 431 G A E6 466 G A Gly-Glu E6 capacity of this and the other HPV 11 isolates cannot be due to 662 G T E7 the various E6 or E7 mRNAs. 1783 C G Thr-Ser El Alternatively, there may be qualitative differences in the func 1784 G C 3487 C T E2 tions of the E6 and E7 proteins between the â€œoncogenicâ€•and Ser-LeuE6 E4 low-oncogenic-potential viral types. The HPV 16 and 18 E7 URRaRef7718T CC CGCIle-Leu proteins have recently been shown to bind to the phosphory 19. lated form of the retinoblastoma gene product, thereby inhib iting its function, which relates to the suppression of cell pro Table4thePrototypenucleotideAmino Alterationsin the DNA ofcloned HPV iJVC corn to liferation (34). In a similar manner, the E6 protein appears to rototype HPV lippared psequence negate the function of p53, the product of another tumor sup pressor gene (35). The combination of these viral genes E6/E7 acidAffectedlocationHPV IVCchangeregion1312CGGIn-GluEl2170CT2580AG2884CT2888TC3436GAGly-GluE43626AC3645AGLys-ArgE23727AC3952ATIle-PheE5a3991GCVal-Leu11PHPV I of HPV 16 and 18, whether linked to its homologous URR or to a strong constitutive transcriptional promoter, can immor talize human keratinocytes (36, 37). Based on the amino acid sequence, one would also predict that the HPV 11 E6/E7 would also have a transforming function. However, the binding affin ity for HPV 11 E7 for the retinoblastoma protein has recently been shown to be markedly less than that of HPV 16 E7 and therefore may not efficiently inhibit the retinoblastoma protein function in the cell (38). Similarly, HPV 16 and 18 E6 are E5b4253GA7479CTURR7509TBase Cys-SerE5a known to avidly bind to p53 in F9 cell extracts, whereas the E6 of HPV 11 had no measurable binding (35). Functionally, how deletedURR7547TCURR7584CCCCURR ever, high levels ofexpression ofHPV 11 E6/E7, when cotrans fected with Ha-ras can indeed transform primary cells, but at a low efficiency (39). These data, in conjunction with our results, 5876

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. UNIQUE HPV I1 ISOLATETRANSFORMSCELLS could indicate that the E6 and E7 of HPV 11 may be qualita isolate with the capacity to efficiently transform cells in culture. tively less active at inhibiting the function of their respective It may be that no single mutation in the HPV 11VC genome cellular substrates, but a high level of expression of HPV 11 accounts for the difference in transforming function of this proteins could overcome this deficiency and lead to cell trans virus; rather, it may be due to a combination of changes occur formation. ring in the URR, E2, El and ES. Our laboratory is currently Regulation of papillomavirus gene expression is controlled investigating the functional effects of these changes in experi by combinations ofproteins interacting on the URR, a region of ments designed to exchange the respective regions of the HPV the viral genome containing numerous enhancers and binding 11VCand HPV 1iP, measure levelsofexpression ofthe URRs, sites for cellular and virus-derived transcription factors (40). and determine the effect of ES and El mutations on viral and There appears to be a relationship between the dose effect of the cellular biological functions. HPV 11 URR and cellular transformation. This fact is exem plified in a recent report which characterized an isolate of HPV REFERENCES 11presentina hepaticmetastasisofa lungcarcinomafroma 1. Lorincz, A., Temple, G., Kurman, R., Jensen, A., and Lancaster, W. Onco patient with laryngotracheobronchial papillomatosis contain genic association of specific human papillomavirus types with cervical neo ing a duplication of the URR (7). A similar duplication in the plasia. J. Natl. Cancer Inst., 79:671â€”677,1987. 2. Lancaster, W., and Jensen, A. Natural history of human papillomavirus URR ofan isolate ofHPV 6 was characterized from a Buschke infection of the anogenital tract. Cancer Metastasis Rev., 6: 653â€”664,1987. LÃ¶wenstein tumor (41). Multiple small repeats of a transcrip 3. Ostrow, R., and Farm, A. The molecular biology ofhuman papillomaviruses tional enhancer sequence has also been identified in a HPV and the pathogenesis of genital papillomas and neoplasms. Cancer Metasta sisRev.,6: 383â€”395,1987. 6-containing verrucous carcinoma (42). 4. Pecoraro, G., Morgan, D., and Defendi, V. Differential effects of human Analysis of the DNA sequence of the HPV 11VC URR re papillomavirus type 6, 16, and 18 DNAs on immortalization and transfor mation of human cervical epithelial cells. Proc. Natl. Acad. Sci. USA, 86: vealed several novel mutations that may ultimately contribute 563â€”567,1989. to its observed effects in cells. One change is the insertion of 5. Rabbett, W. Juvenile laryngealpapillomatosis:the relation of irradiation to two additional nucleotides in the HPV 11VC clone just at the 3' malignant degeneration in this disease. Ann. Otol. Rhinol. Laryngol., 74: 1149â€”1163,1965. end of the putative binding site for the transcriptional factor 6. Ostrow, R., Zachow, K., Thompson, 0., and Farm, A. Molecularcloning and AP2 (7575â€”7589).Whether this mutation affects the binding of characterizationof a uniquetype of human papillomavirusfrom an immune any cellular transcription factors to the putative AP2 site re deficientpatient. J. Invest. Dermatol., 82: 362â€”366,1984. 7. Byrne, J., Tsao, M., Fraser, R., and Howley, P. Human papillomavirus-ll mains to be determined. This site is also within 5 base pairs of DNA in a patient with chronic laryngotracheobronchialpapillomatosisand the most 5' E2-responsive element and could possibly alter the metastatic squamous-cellcarcinoma of the lung. N. Engl. J. Med., 317: 873â€”878,1988. binding of E2 at this locus. The E2 protein as it acts upon this 8. Schneider, A., de Villiers, E., and Schneider, V. Multifocal squamous neo domain has both positive and negative effects on viral gene plasia of the femalegenital tract: significanceof human papillomavirusin transcription, dependent on the balance of E2 with other cell fection of the vagina after hysterectomy. Obstet. Gynecol., 70: 294â€”298, 1987. ular transcription factors (43â€”45).In addition to changes oh 9. Greenberg, E., Vessey, M., McPherson, K., and Yeates, D. Cigarette smoking served in the URR of HPV 11YC, the conservative amino acid and cancer of the uterine cervix. Br. J. Cancer, Si: 139â€”141,1985. change occurring at 3645 in the sequence of E2 may in some 10. WHO. WHO CollaborativeStudy of Neoplasiaand Steroid Contraceptives: invasive cervical cancer and combined oral contraceptives. Br. J. Med., 290: way affect how this protein interacts with URR. The signifi 961â€”965,1985. cance of the other mutations occurring in the URR of HPV 11. Purtillo, D. Opportunistic cancers in patients with immunodeficiency syn 11VC are currently unknown, but none appear to affect the dromes. Arch. Pathol. Lab. Med., iii: 1123â€”1129,1987. 12. Penn, I. Tumors arising in organ transplant recipients. Adv. Cancer Res., 28: binding domains of other known transcriptional factors or of 31â€”58,1978. the glucocorticoid-responsive element (46). 13. Schneider, A., Hotz, M., and Gissmann, L. Increased prevalence of human papillomaviruses in the lower genital tract ofpregnant women. Int. J. Cancer, Mutations in several of the other early-region ORFs El, E4, 40:198â€”204,1987. and E5a and E5b of the HPV 11VC genome may also contrib 14. Manias, D., Ostrow, R., McGlennen, R., Estensen, R., and Farm, A. Human ute to the overall ability of this isolate to transform cells. Some papillomavirus type 11 DNA is integrated in primary and metastatic tumors from a renal transplant patient. Cancer Res., 49: 2514â€”2519,1989. nucleotide base changes were found to encode conservative 15. Smotkin, D., Prokoph, H., and Wettstein, F. Oncogenic and nonocogenic amino acid substitutions. However, the mutation at 3952, re human genital papillomavirusesgenerate the E7 mRNA by different mech sulting in a change from isoleucine to phenylalanine, may be anisms. J. Virol., 63: 1441â€”1447,1989. 16. Matlashewski,G., Schneider,J., Bank,L., Jones, N., Murray, A., and Craw significant to the hydrophobic anchoring domain of the E5 ford, L. Human papillomavirus type 16 DNA cooperates with activated ras in protein, a region shown to be critical to the function of E5 in transforming primary cells. EMBO J., 6: 1741â€”1746,1987. BPV 1-transformed cells (47, 48). Similarly, the alteration at 17. Shib, C., and Weinberg, R. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell, 29: 161â€”169,1982. 3991, cysteine to serine, may alter the hydrophilic domain of 18. Pater, M., Hughes, G., Hyslop, D., Nakshatri, H., and Pater, A. Glucocor ES, a region critical to that portion of the protein that stimu ticoid-dependent oncogenic transformation by type 16 but not type I I human papilloma virus DNA. Nature (Lond.), 335: 832â€”835,1989. lates DNA synthesis (49). The E5 ORF of CRPV has also been 19. Dartmann, K., Schwarz, E., Gissmann, L, and zur Hausen, H. The nude shown to be an oncogene (50). The function of E5 ofthe human otide sequence and genome organization of human papillomavirus type I 1. papillomaviruses is currently unknown, but the structures of the Virology, 151: 124-130, 1986. 20. Zachow, K., Ostrow, R., Bender, M., Watts, S., Okagaki, T., Pass, F., and HPV 11 and 16 ES proteins are similar to that of BPV-1 E5, Faras, A. Detection of human papillomavirus DNA in anogenital neoplasias. and one investigator has recently demonstrated that ES of HPV Nature (Lond.), 300: 771â€”773,1982. 6 can transform NIH 3T3 cells (Si). 21. Wettstein, F., and Stevens, J. Variable-sized free episomes of Shope papil loma virus DNA are present in all non-virus-producing neoplasms and inte Malignant transformation appears to be a rare consequence grated episomes are detected in some. Proc. Natl. Acad. Sci. USA, 79: 790â€” of an HPV 11 infection. The detection of HPV 11 and other 794, 1982. 22. Gorman, C. High efficiency gene transfer into mammalian cells. In. D. nononcogenic viral DNAs in a significant number of tumors Glover (ed.), DNA Cloning, VoL 2, pp. 143â€”165.Oxford:IRL Press, 1986. from immunosuppressed patients suggests that host factors 23. Southern, P., and Berg, P. Transformation of mammalian cells to antibiotic strongly influence the process of carcinogenesis. Despite this, resistance with a bacterial gene under control of the SV4O early region pro moter. J. Mol. Appl. Genet., 1: 327â€”34,1983. however, characteristics inherent to the virus are also impor 24. Southern, E. Detection of specific sequences among DNA fragments sepa tant, as evidenced by our demonstration of a unique HPV 11 rated by gel electrophoresis. J. MoI. Biol., 98: 503â€”517,1975. 5877

25. Feinberg, A., and Vogeistein, B. A technique for radiolabeling DNA restric in retinoblastoma protein binding and other properties. J. ViroL, 64: 723â€” lion endonuclease fragments to high specific activity. Ana. Biochem., 137: 730, 1990. 6â€”13,1983 39. Storey, A., Osborn, K., and Crawford,L Co-transformationby human pap 26. Schneider, J., Fisher, F., Goding, R., and Jones, N. Mutational analysisof illomavirustypes 6 and 11.J. Gen. ViraL, 71: 165â€”171,1990. the adenovirus Ela gene: the role of transcriptional regulation in transfor 40. Gloss, B., Chong, T., and Bernard, H. Numerous nuclear proteins bind the mation. EMBO J., 6: 2053â€”2060,1987. long control region of human papillomavirus type 16: a subset of 6 of 23 27. Phelps, W., Yee, C., Munger, K., and Howley, P. The human papillomavirus DNase I-protectedsegmentscoincideswith the location of cell-type-specific type 16E7 geneencodestransactivatingand transformationfunctionssimilar enhancer. J. Virol., 63: 1142â€”1152,1989. to those of adenovirus Ela. Cell, 53: 539â€”547,1988. 41. Boshart, M., and zur Hausen, H. Human papillomavirusesin Buschke-LÃ¶ 28. Crook, T., Almond, N., Murray, A., Stanley, M., and Crawford, L. Consti wenstein tumors: physical state of the DNA and identification of a tandem tutive expression of c-rnyc confers hormone independence and enhanced duplicationin the noncodingregionofa human papillomavirus6 subtype.J. growth factor responsiveness on cells transformed by human papillomavirus Virol., 58: 963â€”966,1986. type 16. Proc. NatI. Acad. Sci. USA, 86: 5713â€”5717,1989. 42. Rando, R., Groff, D., Chiridjian, J., and Lancaster, W. Isolation and char 29. Crook, T., Storey, A., Almond, N., Osborn, K., and Crawford, L Human acterization of a novel human papillomavirus type 6 DNA from an invasive papillomavirustype 16cooperateswithactivatedms and fosoncogenesin the vulvarcarcinoma.J. Virol., 57: 353â€”356,1986. hormone-dependent transformation of primary mouse cells. Proc. Natl. 43. Hirochika, H., Hirochika, R., Broker, T., and Chow, L Functional mapping Acad. Sci. USA, 85: 8820â€”8824,1988. of the human papillomavirustype 11 transcriptional enhancer and its inter 30. Kasher, M., and Roman, A. Characterization of human papillomavirus type action with the trans-acting E2 proteins. Genes Dev., 2: 54â€”67,1988. 6b DNA isolated from an invasivesquamouscarcinomaof the vulva.Virol 44. Chin, M., Broker, T., and Chow, L. Identification of a novel constitutive ogy, 165: 225â€”233,1988. enhancer element and an associated binding protein: implications for human 31. Rosen, R., and Auborn, K. Duplication of the upstream regulatory sequences papillomavirus type 11 enhancer regulation. J. Virol., 63: 2967â€”2976,1989. increases the transformation potential of human papillomavirus type I I. 45. Auborn, K., Galli, R., Dilorenzo, T., and Steinberg, B. Identification of Virology, 185: 484â€”487,1991. DNA-proteininteraction and enhancer activityat the 5' end of the upstream 32. Cerni, C., Patocka, K., and Meneguzzi, G. Immortalization of primary rat regulatory region in human papillomavirus type I 1. Virology, 170: 123â€”130, embryo cells by human papillomavirus type 11 DNA is enhanced upon 1989. cotransfer of ras. Virology, 177: 427â€”436,1990. 46. Gloss, B., Bernard, H., Seedorf, K., and Klock, G. The upstream regulatory 33. Munger, K., Phelps, W., Bubb, V., Howley, P., and Schlegel, R. The E6 and regionofthe human papillomavirus-16contains an E2 protein-independent E7 genes of the human papillomavirus type 16 together are necessary and enhancer which is specificfor cervicalcarcinoma cells and regulated by glu sufficientfor transformation of primary human keratinoctyes.J. Virol., 63: cocorticoid hormones. EMBO J., 6: 3735â€”3743,1987. 4417â€”4421,1988. 47. Schiller, J., Vast, W., Vousden, K., and Lowy, D. ES open reading frame of 34. Dyson, N., Howley,P., Munger, K., and Harlow, E. The human papilloma bovine papillomavirus type I encodes a transforming gene. J. Virol., 57: 1â€”6, virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. 1986. Science (Washington DC), 243: 934â€”936,1989. 48. Burkhardt, A., DiMaio, D., and Schlegel, R. Genetic and biochemical defi 35. Werness, B., Levine, A., and Howley, P. Association of human papillomavi nition of the bovine papillomavirusES transforming protein. EMBO J., 6: rus type 16 and 18 E6 proteins with p53. Science (Washington DC), 248: 2381â€”2385,1987. 76â€”79,1990. 49. Rawls,J., Loewenstein,P.,and Green,M. Mutationalanalysisof bovine 36. Barbosa, M., and Schlegel, R. The E6 and E7 genes ofHPV 18 are sufficient papillomavirustype I ES peptide domains involvedin induction of cellular for inducing two-stage in vitro transformation of human keratinocytes. On DNA synthesis.J. ViroL,63: 4962â€”4964,1989. cogene,4:1529â€”1532,1989. So. Meyers,C., Harry,J., Lin, Y., and Wettstein,F. Identificationofthree 37. Kaur, P., McDougall, J., and Cone, R. Immortalization ofprimary epithelial transformingproteins encodedbycottontail rabbit papillomavirus.J. ViroL, cells by cloned cervical carcinoma DNA containing human papillomavirus 66:1655â€”1664,1992. type 16 E6/7 open reading frames.J. Gen. Virol., 70: 1261â€”1266,1989. 51. Chen, S., and Mounts, P. Transforming activity of E5a protein of human 38. Gage, J., Meyers, C., and Wettstein, F. The E7 proteins ofthe nononcogenic papillomavirus type 6 in NIH 3T3 and Cl27 cells. J. Virol., 64: 3226â€”3233, human papillomavirus type 6b(HPV 6b)and ofthe oncogenic HPV 16 differ 1990.

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Ronald C. McGlennen, Jyotsna Ghai, Ronald S. Ostrow, et al.

Cancer Res 1992;52:5872-5878.

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