Klimek, Małgorzata

Kosz-Vnenchak, Magdalena

Szostek, Sława

Zawilińska, Barbara

Folia Biologica

English

Jagiellonian Univeristy Repository

Folia biologica (Kraków), vol. 56 (2008), No 3-4doi:10.3409/fb.56_3-4.269-271Physical State of Human Papillomavirus Type 16 in Cervical Cell DNA*S³awa SZOSTEK, Ma³gorzata KLIMEK, Barbara ZAWILIÑSKA, and Magdalena KOSZ-VNENCHAKAccepted April 22, 2008SZOSTEK S., KLIMEK M., ZAWILIÑSKA B., KOSZ-VNENCHAK M. 2008. Physical state ofhuman papillomavirus type 16 in cervical cell DNA. Folia biol. (Kraków) 56: 269-271.Multiplex PCR with specific primers for E2/E6 genes was used to assess the viral integrationstatus of HPV-16 in women with low and high grade squamous intraepithelial lesions (LSILand HSIL, respectively) in comparison to cervical cancer patients. Women with confirmedHPV-16 infection were examined: 30 with LSIL, 12 with HSIL and 23 with cervical cancer.The PCR products were separated electrophoretically in agarose gels and densitometricanalysis was performed using Bio-Rad Quantity One software. E2 and E6 sequences ofHPV-16 were detected in 91% of the women. The free episomal viral genome was notdetected in the cervical carcinoma group. Twenty six percent of the samples obtained fromthis group harboured the integrated form, whereas the remaining samples possessed amixture, i.e. episomal and integrated forms of viral DNA. The free episomal form dominatedin women with LSIL and HSIL. In 6 cases the episomal and integrated forms were detectedsimultaneously. HPV-16 integration occurred in a subset of LSILs and HSILs, not only in thecervical cancer patients and correlated with progression of cytological changes. Theassessment of the status of HPV-16 may be the molecular factor preceding the morphologicalfeatures leading to malignancy.Key words: Human papillomavirus genes, HPV-16, malignancy.S³awa SZOSTEK, Barbara ZAWILIÑSKA, Magdalena KOSZ-VNENCHAK Jagiellonian UniversityMedical College, Chair of Microbiology, Department of Virology, Czysta 18, 31-121 Kra-kow, Poland.E-mail: sszostek@cm-uj.krakow.plMa³gorzata KLIMEK, Centre of Oncology, M. Sk³odowska-Curie Memorial Institute, Depart-ment of Gynecol. Oncol., Garncarska 11, 31-115 Krakow, Poland.Magdalena KOSZ-VNENCHAK, Jagiellonian University, Faculty of Biochemistry, Biophysicsand Biotechnology, Laboratory of Molecular Genetics and Virology, Gronostajowa 7,30-387 Krakow, Poland.Cervical cancer is one of the most frequent fe-male malignant tumours and more than 500 000new cases are reported each year all over theworld. The etiological association between humanpapillomaviruses (HPV) and cervical cancer isnow well established. Among 100 types of HPVdiscovered to date, about 30 are involved in ano-genital infections. Epidemiological and biochemi-cal data support the division of HPVs into twogroups: high-risk, such as HPV-16, 18, 31, 33, 35,39, 45, 51, 52, 56, 58, 59, 68, 73 and 82, capable ofcausing a progression to cancer of the uterine cer-vix, and low-risk, such as HPV 6, 11, 40, 42, 43,44, 54, 61, 70, 72, and 81, rarely, if ever, leading tocancer (ZUR HAUSEN 2000; MUNOZ et al. 2003;BOSCH et al. 2002). HPV-16 is the most exten-sively studied HPV type and serves as an impor-tant model for studying viral carcinogenesis (ZURHAUSEN 2002). The E6 and E7 genes of high-riskHPV are known to play an important role in cervi-cal carcinogenesis because they are highly ex-pressed in cervical carcinoma cells and they havethe ability to transform and immortalize primarykeratinocytes (WOODWORTH et al. 1989; MUNGERet al. 1989). In the process of inactivation oftumour-suppressor gene products, the E6 proteinslead to the degradation of the p53 protein, and thebinding complex of E7 and pRB proteins disturbthe normal cell cycle and maintain abnormal cellu-lar proliferation (SCHEFFNER et al.1990; HECK etal. 1992).During a common infection and in most prema-lignant lesions, HPV is in an episomal state. How-ever, most cervical carcinomas and derived cell_______________________________________*Supported by grant N N401219034 from the Ministry of Science and Higher Education.lines maintain the HPV genome in an integratedform or in both integrated and episomal forms.Thus, integration into the chromosomes of the hostcell has been proposed as an activation mechanismfor progression from preinvasive lesions to cervi-cal cancer. The genome integration of HPV usu-ally disrupts or deletes E2 gene open readingframes, or rarely E1 open reading frames, whichresults in the loss of expression of the correspond-ing gene products. Disruption of these genes alsoleads to overexpression of the E6 and E7 oncopro-teins, since the E2 gene products can repress ac-tivities from the HPV promoters that direct theexpression of the E6 and E7 genes (ZUR HAUSEN2002). The disruption of the E2 gene will cause theabsence of the E2 gene sequences in the PCR prod-uct following integration.Our study was undertaken to determine thephysical status of HPV-16 by analyzing the ratiosof E2 and E6 genes by multiplex PCR and to assessthe viral integration of HPV-16 in cervical cellDNA from women with different grades of squa-mous intraepithelial lesions in comparison to cer-vical cancer patients.Material and MethodsSixty five HPV-16 positive cervical smears wereclassified by cytological examinations as lowgrade squamous intraepithelial lesions – LSIL (n=30),high grade squamous intraepithelial lesions – HSIL(n=12) and cervical carcinoma (n=23). Materialwas sampled from 20-75 year old women (averageage for LSIL woman 36 years, for HSIL – 43 yearsand for cervical carcinoma 48 years). Specimenswere collected in Digene Specimen Transport Me-dium (Digene Diagnostic, USA) and stored frozenat –70oC until processed.DNA isolationDNA was isolated using the Genomic DNA PrepPlus kit (A&A Biotechnology, Poland). Cell sampleswere lyzed in buffer with proteinase K (20mg/ml)and chaotropic salts. Addition of ethanol caused DNAto bind when the lysate was spun through a silicamembrane in a microcentrifuge tube. Followingwashing to remove contaminants, DNA was elutedin 10mM TRIS-HCl pH 8,5 (preheat to 75°C).PCRHPV-16 detection was performed using theSPF10 primers in order to amplify a 65 bp frag-ment from the L1 region of the HPV genome andwas followed by reverse hybridization (INNO-LiPA, Innogenetics, Belgium).Multiplex PCRThe E2 and E6 genes of HPV-16 were detectedby multiplex PCR using 2 pairs of primers synthe-sized according toYOSHINOUCHI (YOSHINOUCHIet al. 1999).The primers for each sequence were:5’-CTTGGGCACCGAAGAAACAC-3’,5’-TTGGTCACGTTGCCATTCAC-3’ for the E2gene and5’-AAGGGCGTAACCGAAATCGGT-3’,5’-CATATACCTCACGTCGCAG-3’ for the E6geneThe amplicon sizes were 352 bp and 208 bp forthe E2 and E6 sequences, respectively. PCR wasperformed in a final reaction volume of 25 Fl, con-taining PCR buffer (50 mM KCl, 10 mM Tris-HCl,pH 8.3), 1.5 mM MgCl2, 200 FM dNTPs, 0.4 FMof each primers, 2.5 U Ampli Taq Gold DNA po-lymerase (Perkin Elmer, USA) and template DNA(1Fg). The amount of isolated DNA was estimatedspectrophotometrically. DNA extracted from cer-vical carcinoma cell line CaSki was used as posi-tive control. The PCR conditions were as follows:initial cycle – 95°C for 5 min, 52°C for 1 min, 72°Cfor 1 min; the following 30 cycles: 95°C for 1 min,52°C for 1 min, 72°C for 1 min; the final cycle –95°C for 1 min, 52°C for 1 min, 72°C for 8 min.The PCR products were separated electropho-retically in 2% agarose gels. Transilluminated gelimages were digitalized and analyzed by the GelDoc 2000 documentation system (Bio-Rad, USA).Values corresponding to the intensity and densityof the gel bands of different PCR products wereobtained with the help of Quantity One 4.0 soft-ware (Bio-Rad, USA). The ratio of E2 to E6 copynumbers was calculated to determine the physicalstatus of the HPV-16 viral gene. HPV-16 in pureS. SZOSTEK et al.270Table 1Correlation between physical status ofHPV-16 DNA and cytological diagnosesCytologyPhysical status of HPV-16 DNAEpisomalE2/E6$1Episomaland integrated1>E2/E6>0IntegratedE2/E6=0LSILn=3028 2 0HSILn=128 4 0Ca*n=230 17 6Ca* – cervical carcinomaepisomal form was expected to have equivalentcopy numbers of E2 and E6 genes (E2/E6 ratio> 1or =1), whereas preferential disruption of E2 uponviral integration should result in less E2 gene cop-ies than E6. This means that an E2/E6 ratio of lessthan 1 would indicate the presence of both inte-grated and episomal forms, while a ratio of 0would indicate the presence of integrated formonly.Results and DiscussionA total of 65 samples were examined in whichHPV-16 was previously confirmed by the SPF-10primer set and reverse hybridization. Several tech-niques have been described for the elucidation ofHPV-16 status in infected cells, such as Southernblot, real-time PCR and two dimensional gel elec-trophoresis (PARK et al. 1997; NAGAO et al. 2002;HUDELIST et al. 2004). In this study we used multi-plex PCR with primers specific for E2 and E6genes and analyzed the quantitative ratio of PCRproducts.HPV-16 E6 was found in all specimens. The E2sequence of HPV-16 was detected in 59 (91%)samples, suggesting that in the remaining negativesamples the virus may have been in integratedform only. The physical status of HPV-16 in sam-ples with different cytological results are shown inTable 1. In women with LSIL and HSIL, the episo-mal form dominated (93% and 67%, respectively,with E2/E6 ratio>1 or =1). In 6 cases the episomaland integrated forms of HPV-16 were detected si-multaneously. In material obtained from patientswith cervical cancer the E2 region was amplifiedin 17 (74%) of cases. In this group 26% of patientsexclusively harboured the integrated form (E2/E6ratio = 0). In women with cervical carcinoma nofree episomal HPV-16 was found but only mixed(1>E2/E6 ratio>0) or integrated forms of the viralgenome. The examinations showed a tendency ofincreasing frequency of HPV-16 integration withprogression to malignancy. We confirmed alsothat HPV-16 integration occurred in a subset ofLSILs and HSILs, not only in the cervical cancerspecimens. This may suggest that viral integrationmay occur earlier than the onset of morphologicalchanges, which could indicate a high grade lesion.The assessment of HPV-16 status may be a helpfulcomplementary tool for cytological study in cervi-cal screening to identify women at risk for devel-oping high grade squamous intraepithelial lesionsor carcinoma.ReferencesBOSCH F. X., LORINCZA., MUNOZN., MEIJERC. J., SHAHK.V. 2002. The causal relation between human papillomavirusand cervical cancer. J.Clin.Pathol. 55: 244-265.HECKD. V., YEEC. L., HOWLEY P. M., MUNGERK. 1992.Ef-ficiency of binding the retinoblastoma protein correlateswith the transforming capacity of the E7 oncoproteins of thehuman papillomavirus. Proc. Natl. Acad. Sci. USA 89:4442-4446.HUDELIST G., MANAVI M., PISCHINGER K.,WATKINS-RIEDEL T., SINGER C. F., KUBISTA E.,CZERWENKA K. F. 2004. Physical state and expression ofHPV DNA in benign and dysplastic cervical tissue: differentlevels of viral integration are correlated with lesion grade.Gynecol. Oncol. 92: 873-880.MUNGER K., PHELPS W. C., BUBB V., HOWLEY P. M.,SCHLEGEL R. 1989. The E6 and E7 genes of the human pa-pillomavirus type 16 together are necessary and sufficientfor transformation primary human keratinocytes. J. Virol.63: 4417-4421.MUÒOZ N., BOSCH F. X, DE SANJOSE S., HERRERO R.,CASTELLSAGUE X., SHAH K. V., SNIJDERS P. J. F., MEIJERC.J. L. M. 2003. Epidemiologic classification of human pa-pillomavirus types associated with cervical cancer. N. Engl.J. Med. 348: 518-527.NAGAO S., YOSHINOUCHI M., MIYAGI Y., HONGO A.,KODAMA J., ITOH S., KUDOT. 2002. Rapid and sensitive de-tection of physical status of human papillomavirus type 16DNA by quantitative real-time PCR. J. Clin. Microbiol. 40:863-867.PARK J., HWANG E., PARK S., AHN H., UM S., KIM C., KIMS., NAMKOONG S. 1997. Physical status and expression ofHPV genes in cervical cancers. Gynecol. Oncol. 65:121-129.SCHEFFNER M., WERNESS B. A., HUIBREGTSE J. M., LEVINEA. J., HOWLEY P. M. 1990. The E6 oncoprotein encoded byhuman papillomavirus type 16 and 18 promotes the degrada-tion of p53. Cell 63: 1129-1136.WOODWORTH C. D., DONIGER J., DIPAOLA J. A. 1989. Im-mortalization of human keratinocytes by various human pa-pillomavirus DNAs corresponds to their association withcervical carcinoma. J. Virol. 63: 159-164.YOSHINOUCHI M., HONGO A., NAKAMURA K., KODAMA J.,ITOH S., SAKAI H., KUDO T. 1999. Analysis by multiplexPCR of the physical status of human papillomavirus type 16DNA in cervical cancer. J. Clin. Microbiol. 37: 3514-3517.ZUR HAUSEN H. 2000. Papillomaviruses causing cancer: eva-sion from host-cell control in early events in carcinogenesis.J. Natl. Cancer Inst. 92: 690-698.ZURHAUSEN H. 2002. Papillomaviruses and cancer: from ba-sic studies to clinical application. Nat. Rev. Cancer 2:342-350.Physical State of HPV 16 271

Physical state of human papillomavirus type 16 in cervical cell DNA

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Physical state of human papillomavirus type 16 in cervical cell DNA

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