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Physical status of the E2 human papilloma virus 16 viral gene in cervical preneoplastic and neoplastic lesions

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Physical status of the E2 human papilloma virus 16 viral gene in cervical preneoplastic and neoplastic lesions
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  Journal of Clinical Virology 21 (2001) 129–134 Physical status of the E2 human papilloma virus 16 viralgene in cervical preneoplastic and neoplastic lesions S.A. Tonon  a, *, M.A. Picconi  b , P.D. Bos  a , J.B. Zinovich  a , J. Galuppo  a ,L.V. Alonio  b , A.R. Teyssie  b a Laboratorio de Biologia Molecular Aplicada ,  Facultad de Ciencias Exactas ,  Quimicas y Naturales , Uni   ersidad Nacional de Misiones ,  Felix de Azara  1552  ,  Posadas ,  Misiones  3300  ,  Argentina b Ser  icio   irus oncogenicos ,  INEI  - ANLIS   ‘‘  Dr .  Carlos G  .  Malbran ’’  .  A  .  Velez Sarsfield   570  ,  Buenos Aires ,  Argentina Received 10 July 2000; received in revised form 26 December 2000; accepted 19 January 2001 Abstract Background  :   Integration of human papilloma virus (HPV) 16 DNA is considered an important genetic change incervical lesion progression towards ICC. The viral E2 gene is often disrupted by this process, releasing suppressionof viral E6 / E7 oncogenes, a key factor for oncogenic progression.  Objecti   es :   To evaluate the physical status of HPV16 E2 gene in cervical preneoplastic and neoplastic lesions and its relation with lesion severity.  Study design :   Asensitive PCR approach for the detection of an intact E2 HPV 16 gene in infected epithelial cells from the cervix withlow grade squamous intraepithelial lesion (LGSIL), high grade squamous intraepithelial lesion (HGSIL) and invasivecervical carcinoma (ICC) diagnosis was applied. The correlation between gene disruption and lesion stage wasexamined.  Results :   Sixty-two LGSIL, 39 HGSIL and 24 ICC samples were analyzed. Fifty-seven LGSIL [92%], 13HGSIL [33%] and 4 ICC [17%] showed results compatible with an intact E2 gene, while 5 LGSIL [8%], 26 HGSIL[67%] and 20 ICC [83%] samples gave no signal.  Conclusions :   HPV 16 E2 gene disruption showed a positivecorrelation with cervical lesion progression, particularly from LGSIL to HGSIL. Although additional genetic eventsare very likely to be needed for HGSIL to ICC progression, the E2 gene disruption is a putative early marker toconsider in the prognostic analysis of HPV 16 chronically infected women. © 2001 Elsevier Science B.V. All rightsreserved. Keywords :   Human papilloma virus 16; E2; Integration; Cervical neoplasiawww.elsevier.com / locate /  jcv 1. Introduction Cervical cancer is a significant public healthproblem, approximately 500000 new cases arediagnosed worldwide each year. The etiologic roleof specific human papilloma virus (HPV) types incervical neoplasia has been demonstrated and it is Abbre  iations :   HGSIL, high grade squamous intraepitheliallesion; HPV, human papilloma virus; ICC, invasive cervicalcarcinoma; LGSIL, low grade squamous intraepithelial lesion.* Corresponding author. Tel.:  + 54-3752-427687; fax:  + 54-3752-425414. E  - mail address :   tonon@arnet.com.ar (S.A. Tonon).1386-6532 / 01 / $ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S1386-6532(01)00155-X  S  . A .  Tonon et al  .  /   Journal of Clinical Virology  21 (2001) 129  –  134  130 already well established, although the precisemechanisms of transformation have yet to beelucidated (zur Hausen, 1996).Epidemiological studies strongly support thehypothesis that high risk HPV types (16, 18, 31,33) may act as solitary cervical carcinogens (zurHausen, 1991, 1996). Certain regions of theworld present high incidences of cervical car-cinomas, directly related to an elevated preva-lence of high risk HPV infection (Mun ˜ oz et al.,1992; Kjaer et al., 1993; Tonon et al., 1999).There is a substantial time lag between pri-mary cervical high risk HPV infection and thedevelopment of lesions. Cervical cancer has itsgreater incidence in age groups between 55 and65 years, meanwhile low grade squamous in-traepithelial lesions (LGSIL) are common in agegroups of 25  –  35 years; so malignant progressionseems to require 20  –  30 years (zur Hausen, 1994).The gradual accumulation of mutations withinlatently infected host cells resulting from thefunction of viral oncoproteins would lead to aselection of clones with speci fi c cellular DNAmodi fi cations. It has been reported that the ex-pression in human cells of HPV type 16 E6 andE7 gene products results in an accumulation of genetic alterations within those infected cells;however, the expression of HPV type 16 E6 orE7 genes did not lead to detectable modi fi cationsof host cell DNA (Hashida and Yasumoto, 1991;White et al., 1994)Infections by high risk HPV types have beenfound in about 97% of all cervical carcinomasamples tested throughout the world, HPV 16being by far the most frequently virus type de-tected, accounting for approximately 60% of allpositive data (Bosch et al., 1995). The biologicalproperties of this viral type include the capabilityof immortalizing human keratinocytes and to in-duce chromosomal aberrations by overriding cellcycle control mechanisms via viral oncoproteinscellular signalling interference (IARC, 1995).An incremental upregulation of viral E6 / E7genes from HPV 16 has been implied as a pre-requisite for continued cellular growth stimula-tion, existing a correlation between quantity of viral oncogene product and severity of the lesion(zur Hausen, 1996). Mutational analysis showedthat both E6 and E7 were required for ef  fi cientimmortalization of primary human genital tractkeratinocytes (Hudson et al., 1990). Continuousexpression of these two viral oncogenes appearsto be necessary for the induction as well as themaintenance of the transformed cellular state(von Knebel-Doeberitz et al., 1994).Analyses of promoter activity in human ep-ithelial cells have shown that the HPV 16 majorpromoter possesses a basal activity which can berepressed by full-length E2 gene products. Theseare DNA-binding proteins that can repress tran-scription from the HPV promoters that governexpression of the E6 and E7 oncogenes (Thierryand Howley, 1991). The E2 suppressive actioncan decrease the ef  fi ciency of HPV 16 E6 / E7dependent immortalization and even haltinggrowth of HPV-positive cervical cancer lines(Hwang et al., 1993). The detection of altered E2DNA sequences is more common in carcinomasthan in cervical intraepithelial neoplasia, and isfrequently associated with malignant progression(Cullen et al., 1991).These  fi ndings have led to the speculation thatthe loss of a functional E2 gene is one of themajor genetic changes that facilitates transforma-tion of human cells and the transition into amalignant state (Hoppe-Seyler and Butz, 1994).In the proposed stepwise progression of highrisk HPV induced lesions, partial deregulation of E6 / E7 oncoprotein expression lies at the border-line of LGSIL to high grade squamous intraep-ithelial lesion (HGSIL) clinical change. Thisgenetic modi fi cation is generally caused by inte-gration of viral DNA into the host cell chromo-some causing disruption of the viral E2 gene(McBride et al., 1991). Therefore, the capabilityof detecting the integrity of this viral gene maybe useful as an early marker for cervical lesionfollow-up in HPV 16 chronically infected pa-tients.We have applied a sensitive PCR approach forthe detection of an intact E2 HPV 16 gene ininfected epithelial cells from the cervix withLGSIL, HGSIL and invasive cervical carcinoma(ICC) diagnosis, examining the correlation be-tween gene disruption and lesion stage.  S  . A .  Tonon et al  .  /   Journal of Clinical Virology  21 (2001) 129  –  134   131 2. Materials and methods One hundred and twenty  fi ve clinical sampleswere analyzed, corresponding to 62 LGSIL, 39HGSIL and 24 ICCs of the squamous type. Freshcytological specimens were obtained from theecto- and endocervix using cytobrush and pro-cessed the same day of collection; neoplastic tissuewas obtained fresh at the time of surgicaloperation.Patient age ranged from 30 to 69 years with amean of 46. All gave their written informed con-sent for participation in this study in accord withthe International Ethical Normative for Biomedi-cal Research presented by the PanAmericanHealth Organization.Cytobrushes were washed with sterile PBS solu-tion, pH 7.4, and tissue samples minced in thesame buffer. Collected cells were lysed adding 200  g / ml of proteinase K in presence of 0.5% SDS ina total reaction volume of 500   l with an incuba-tion of 4 h at 55 ° C, and nucleic acid puri fi ed(Astori et al., 1995). Isolated DNAs were PCRtested for the beta-globin gene to con fi rm tem-plate suitability (Bauer et al., 1992).All specimens were tested for HPV infection bygeneric PCR with the GP5 +/ GP6 +  primer pair(de Roda Husman et al., 1995). The PCR prod-ucts were dot-blotted onto nylon membranes andhybridized with a speci fi c HPV type 16 biotini-lated oligonucleotide probe for 1 h at 55 ° C, mem-branes were washed twice for 10 min at 58 ° C anddeveloped by chemiluminescence. Hybridationpositive samples were further con fi rmed for HPV16 infection by type-speci fi c PCR, targeting theE6 gene (Das et al., 1992). The ampli fi cation of a260 bp speci fi c fragment was considered a positivereaction. DNAs from the CaSki and SiHa celllines harboring HPV 16 were used as positive andwater as negative controls.A pair of HPV 16 E2 type-speci fi c primers weremade from the 2700  –  2717 (E2 forward) and the3859  –  3878 (E2 reverse) positions of the HPV 16Rviral genome. This pair of primers  fl ank the entireHPV 16 E2 ORF (2726  –  3853).The ampli fi cation of a 1,178 bp speci fi c frag-ment was considered positive for the presence of an intact E2 gene. The identity of E2 PCRproduct was con fi rmed by restriction digestionwith  Rsa I and  Pst I generating three (124, 196,858 bp) and two (181, 997 bp) products, respec-tively (data not shown). CaSki DNA was used asa positive control and SiHa DNA as negativecontrol (Meissner, 1999).The PCR reactions were carried out in a 25   lsolution, containing 5   l of template DNA, 10mM Tris  –  HCl (pH 8.3), 50 mM KCl, 3 mMMgCl2, 100   M dATP, dCTP, dTTP and dGTP,10 pmol of each primer and 1 U of AmpliTaq (5U /  l; Perkin Elmer, NJ, USA ™ ). A hot starttechnique was used, and the thermal cycling con-ditions were: for GP HPV generic PCR: 94 ° C for3 min and then 40 cycles at 92 ° C for 30 s, 50 ° Cfor 30 s and 72 ° C for 30 s; for HPV 16 typing:94 ° C for 3 min and then 35 cycles at 92 ° C for 30s, 65 ° C for 30 s and 72 ° C for 30 s; for E2 geneintegrity testing: 94 ° C for 5 min and then 40cycles at 92 ° C for 45 s, 55 ° C for 45 s and 72 ° C for1 min. The ampli fi cation products were analysedon 1.5% agarose gels and photographed underUV light. 3. Results and discussion The disruption of the E2 gene of high risk HPVtypes, particularly HPV 16, is considered an im-portant genetic change in the process of cervicalcarcinogenesis. This alteration, in conjunctionwith gene ampli fi cation, has been proposed as anactivation mechanism for progression from prein-vasive lesions to cervical cancer (Berumen et al.,1994; Vernon et al., 1996; Casas et al., 1999).It is known that a number of HPV 16 chroni-cally infected patients older than 35 years, inaverage, will eventually develop HGSIL, whileothers will spontaneously regress (Ho et al., 1998).It is in this age group that lesion progressionmarkers are needed in order to gauge relative riskand help in the correct selection of therapeuticprotocols.To analyze the integrity of the E2 viral gene inHPV 16 infected cells of the cervix, total isolatedDNAs from cervical lesions of different gradeswere examined by PCR using primers that encom-pass the entire E2 ORF.  S  . A .  Tonon et al  .  /   Journal of Clinical Virology  21 (2001) 129  –  134  132Fig. 1. Integrity of the E2 gene ORF in HPV 16 infected cervical cells with preneoplastic and neoplastic lesions. Agarose gelelectrophoresis of PCR products. Lanes 1  –  2: CaSki DNA showing intact HPV 16 E2 (1178 bp) and E6 (260 bp) genes; 3: SiHaDNA disrupted E2; 4: ICC sample, disrupted E2; 5  –  6: HGSIL samples, disrupted E2; 7:100 bp ladder; 8  –  11: LGSIL samples, intactE2; 12: negative PCR control (water). One hundred and twenty  fi ve clinical samplesfrom HPV 16 infected patients older than 30,corresponding to 62 LGSIL, 39 HGSIL and 24ICCs of the squamous type were processed. Allsamples were con fi rmed as HPV 16 infected bytwo methods: (a) generic HPV PCR and positivehybridization with a type 16 speci fi c olignucle-otide probe, and (b) positive type-speci fi c PCR bythe E6 gene fragment speci fi c ampli fi cation. Theseresults were correlated with the presence or ab-sence of an E2 viral gene signal (Scheffner et al.,1990). Table 1Physical status of the HPV 16 E2 gene in preneoplastic and neoplastic lesionsLGSILDiagnosis ICCHGSIL13 (33%) a 4 (17%) a Intact E2 gene 57 (92%)Disrupted E2 gene 26 (67%)5 (8%) a 20 (83%)39 24Total 62 a P  0.001.  S  . A .  Tonon et al  .  /   Journal of Clinical Virology  21 (2001) 129  –  134   133 DNA from the CaSki cell line was used as apositive E2 control. This cell line carries 60  –  600copies of the HPV 16 genome integrated as fulllength, head-to-tail tandem repeats with intact E2ORFs (Meissner, 1999). Meanwhile, DNA fromthe SiHa cell line served as negative reactioncontrol. This cell line has an estimated copy num-ber of 1  –  2 HPV 16 genomes integrated as singlesubgenomic fragments with disruption of the E2ORF (positions 3134  –  3384) (Bauer-Hoffman etal., 1996).From the 125 clinical samples analyzed, 92% of LGSIL, 33% of HGSIL and 17% of ICCs showedresults compatible with an intact E2 gene; while8% of LGSIL, 67% of HGSIL and 83% of ICCsamples gave no signal (Table 1).A representative gel electrophoresis of ampli fi -cation products is shown in Fig. 1. The absence of E2 speci fi c product ampli fi cation in the presenceof an ampli fi cation with E6 primers correlatedvery well with higher grade cervical lesions.The preferential disruption of the E2 gene willcause an absence of the speci fi c PCR product,suggesting a pure chromosomal integration event.In the remaining cases with an intact E2 gene,there are two possibilities: a pure episomal formor the concomitant presence of episomal and inte-grated forms. The applied experimental approachcan not distinguish between these two, for thepresence of any intact E2 ORF will produce adetectable signal (Cullen et al., 1991; Park et al.,1997).Recently, the determination of the E2 / E6 copynumber relative ratio by multiplex or real timePCR has been proposed for discerning pure episo-mal from the mixed form of viral infection(Yoshinouchi et al., 1999). Furthermore, a novelapproach targeting the ampli fi cation of papillo-mavirus oncogene transcripts, so-called APOT, isable to distinguish episome- from integrate-derived HPV mRNAs (Klaes et al., 1999). It islikely that the simultaneous presence of episomaland integrated forms in LGSIL and HGSIL be-comes more frequent, further analysis of our E2positive population by these new proposed meth-ods will allow a sub-classi fi cation.Our results support previous observations thatdisruption of the E2 ORF correlates with cervicallesion progression, particularly from LGSIL toHGSIL (Das et al., 1992). Although additionalgenetic events or altered responsiveness to ligandsare very likely to be needed for HGSIL to tumorprogression (Daniel et al., 1997), the E2 genedisruption is a putative early marker to considerin the prognostic analysis of HPV 16 chronicallyinfected older women. References Astori G, Pipan C, Muffato G, Botta G. Detection of HPV-DNA in semen, urine, urethral samples by dot blot andPCR. Microbiologica 1995;18:143  –  9.Bauer H, Greer C, Manos M. Determination of genital humanpapillomavirus infection by consensus polymerase chainreaction ampli fi cation. In: Herrington C, McGee J, editors.Diagnostic molecular pathology, a practical approach. Ox-ford: IRL Press, 1992:131  –  52.Bauer-Hoffman R, Bofghouts C, Auvinen E, Bourda E, RosiF, Alonso A. Genomic cloning and characterization of thenonoccupied allele corresponding to the integration site of HPV 16 DNA in the cervical cancer cell line SiHa. Virol1996;217:33  –  41.Berumen J, Casas L, Segura E, Amezcua JL, Garcia-CarrancaA. Genome ampli fi cation of human papillomavirus types16 and 18 in cervical carcinomas is related to the retentionof E1 / E2 genes. Int J Cancer 1994;56(5):640  –  5.Bosch FX, Manos MM, Mun ˜ oz N, Sherman M, Jansen AM,Peto J, et al. Prevalence of human papillomavirus incervical cancers, a Worldwide perspective. J Nat CancerInst 1995;87:796  –  802.Casas L, Galvan SC, Ordonez RM, Lopez N, Guido M,Berumen J. Asian-American variants of human papillo-mavirus type 16 have extensive mutations in the E2 geneand are highly ampli fi ed in cervical carcinomas. Int JCancer 1999;83(4):449  –  55.Cullen A, Reid R, Campion M, Lorincz A. Analysis of thephysical state of different HPV DNAs in intraepithelialand invasive cervical neoplasms. J Virol 1991;65:606  –  12.Daniel B, Rangarajan A, Mukherjee G, Vallikad E, Krishna S.The link between integration and expression of HPV 16genomes and cellular changes in the evolution of CINlesions. J Gen Virol 1997;78:1095  –  101.Das B, Sharma J, Gopalakrishna V, Luthra U. Analysis byPCR of the physical state of HPV type 16 DNA in cervicalpreneoplastic and neoplastic lesions. J Gen Virol1992;73:2327  –  36.de Roda Husman A, Walboomers J, van den Brule A, MeijerC, Snijders P. The use of general primers GP5 and GP6elongated at their 3   ends with adjacent highly conservedsequences improves HPV detection by PCR. J Gen Virol1995;76:1057  –  62.Hashida T, Yasumoto S. Induction of chromosomal abnor-
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