American Joumnal of Pathology, Vol. 141, No. 5, November 1992 Copyright C) American Association of Pathologists
Expression of the L2 and E7 Genes of the Human Papillomavirus Type 16 in Female Genital Dysplasias Eeva Auvinen,* Harry Kujari,t Pertti Arstila,* and Veijo Hukkanen* From the Departments of Virology* and Pathology, t University of Turku, Turku, Finland
The expression of the E7 and L2 genes of HPV 16 was studied in benign and precancerous female genital lesions to evaluate their role in the development of dysplasias. Ninety biopsy specimens from 70 patients, selected on basis of dot blot DNA hybridization, were included in immunohistochemical and in situ hybridization analyses. In the HPV 16 DNA positive cases, L2 mRNA and E7 mRNA were detected in biopsies from 24 and 21 patients, respectively. L2 mRNA was found in eight of 16 cases of condyloma and mild dysplasia and in 13 of 14 cases of moderate to severe dysplasia The figures forE7 mRNA were 6/16 and 13/14, respectively. We found L2 mRNA in four of 12 normal or condylomatous specimens and E7 mRNA in only one of these. The detection rates for L2 and E7 mRNAs increased along with the severity of the lesions (P = 0.0064 and P = 0.0001, respectively). The L2 protein was found in one condyloma and in 12 dysplasias, eight of which were moderate or severe. The L2-antibody-reactive cells were localized in superficial layers of the epithelium. The detection rateforL2 mRNA and especiallyforE7 mRNA increased along with the histopathologic grade of the lesion. (Am JPathol 1992, 141:1217-1224)
To date, almost 70 human papillomavirus (HPV) types have been described, and more than 22 of them have been detected in squamous genital epithelia.1 Only certain HPV types have been shown to be associated with dysplasia and cancer. Particularly HPV type 16 DNA has been identified in cervical cancers and in premalignant lesions,23 whereas HPV types 6 and 1 1 are associated with mere condyloma. Multiple infections are fairly common.4 In approximately 90% of cervical cancers, HPV DNA has been identified,56 and in 50% of those, HPV 16 DNA is present.
Immortalization of keratinocytes with human papillomavirus DNA has been reported in several studies, and the immortalizing ability has been mapped to the E6-E7region.7-11 Furthermore, this immortalizing ability is specific for the HPV types associated with cancer,12"14 and has not been reported for other types of HPV. In most cervical squamous cell cancers and cancer cell lines studied, the HPV 16 genome is integrated into the host cell genome and the E6-E7 region is retained and transcribed, which also suggests that these genes are important in the malignant process.15'16 The relative expression of E6-E7 region of HPV 16 has been found to increase along with the severity of lesions in gynecologic
neoplasias.17 The L2 protein of papillomaviruses is a minor capsid component, which is expressed in highly differentiated cells of benign squamous lesions, where virus production occurs.18 Hence, the expression of the L2 protein is not expected to occur in carcinoma cells.17 L2 mRNA has been identified in higher grade lesions, too, however.19 Homology within the L2 open reading frame (ORF) is considerably lower between various papillomavirus types than that of the Li ORF.20 The anti-L1 -antibodies crossreact between papillomavirus types, whereas the antiL2-antibodies may be considered more type-specific. Human papillomavirus early proteins have been identified in human genital lesions or HPV-containing cancer cell lines by immunoprecipitation or by Western blotting.21 24The role of HPV 16 genes in the development of human genital squamous neoplasias is not thoroughly characterized, however. Despite the intensive research on the molecular mechanisms of human papillomavirus infections, systematic studies on HPV gene expression in clinical material have been few. In the current work, the expression of HPV 16 E7 and L2 ORFs was studied in HPV 16 DNA positive condylomas, dysplasias, and carcinomas to assess their putative role in malignant transformation. Supported by the Cancer Society of Finland and the Turku University Foundation. Accepted for publication May 14, 1992. Address reprint requests to Dr. Eeva Auvinen, Department of Virology, University of Turku, Kiinamyllynkatu 13, SF-20520 Turku, Finland.
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Materials and Methods Biopsy Material Biopsy specimens from cervical, vaginal, and vulvar lesions were fixed with 4% phosphate-buffered formalin, embedded in paraffin, and cut at 4 or 5 Ai on organosiliconized slides25 for in situ hybridization and on poly-Llysine-coated slides for immunohistochemical staining. The specimens were collected from the patient material that was sent for routine HPV dot blot hybridization26'27 to the Department of Virology, University of Turku. All HPV 16-positive patients during 1987 to 1990, from whom adequate biopsy material was available, were included, irrespective of the histopathologic diagnosis. The material consisted of 90 biopsies from 70 patients, selected on the basis of dot blot hybridization results. Included were 50 patients who had been positive for HPV 16 DNA, and, as controls for immunohistochemical analyses, four patients positive for HPV 18 DNA, seven positive for HPV 11 DNA, and nine who had been HPV negative. The tissue specimens were obtained from the Department of Obstetrics and Gynecology, Turku University Hospital, Turku, Finland. The routinely stained sections were reviewed without knowing the primary diagnoses given.
Cloning Procedures Figure 1 illustrates the genomic localization of the hybridization probes and of the HPV sequences used for expression of fusion proteins. The 1776-base pair (bp) Pstl fragment of the HPV 16 genome, comprising the 3' terminal part of the Li ORF, the noncoding region, and the E6 and E7 ORFs, was subcloned into pGEM5Zf( +) vector (Promega, Biotec, Madison, WI) to prepare singlestranded RNA probes for detection of HPV 16 E7 mRNA using in situ hybridization. Similarly, the 1063-bp Pstl fragment, comprising the 3' terminal part of the E2 ORF and the 5' terminal part of the L2 ORF, was cloned into
E6
E1
LI
L2 clone
E7 cbn
PSII
Pstl
7007
879
S7p2
Pstl 3696
e
Pstl
St,4759 4468 L2 nt09gn Alul Pstl 447a 4759
Figure 1. Genomic localization of thefragments of HPV 16 DNA used for synthesis of in situ hybridization R.NA probes and for production offusion protein for use as immunization antigen. The horizontal line on the top ofthefigure represents the genomic DNA ofHPV 16 The open boxes represent the open readingframes The arrows at the E7 and L2 clones indicate the restriction enzyme cleavage sites used for linearization of the templates for RNA probes.
pGEM1 (Promega Biotec). The 281 bp AIul-Pstl fragment from the L2 ORF was cloned into pEX128 using Smal-Pstl sites. In-frame ligation and the correct orientation of the subclones was verified by restriction mapping and sequencing by the Sanger dideoxy method"9 using Sequenase(!3' Version 2.0 kit (US Biochemicals, Cleveland, OH).
In Situ Hybridization The probes used for in situ DNA hybridization were HPV 1 1, HPV 16, and HPV 18 DNA purified from the vector. The DNA was labeled in a random priming reaction using 35S-dATPaxS (1000 Ci/mmol, Amersham, Buckinghamshire, UK) to specific activities of 0.8 to 2.0 x 109 dpm/,ug template. For preparation of single-stranded RNAprobes, the E7 pGEM clone was linearized at the Sspl site at nucleotide (nt) 720 and the transcription was performed from the SP6 promoter for production of a 159 nt probe. The control probe was prepared from the complementary strand of the same clone using the T7 promoter. The L2 pGEM clone was linearized with Stul at nt 4468 and transcription was done from the SP6 promoter to produce a 291 nt long probe for detection of L2 mRNA, and the control probe was transcribed from the T7 promoter. The probes for detection of mRNA were labeled with 35S-UTPaS (1000 Ci/mmol, Amersham) to specific activities of 3.0-4.2 x 109 dpm/,g template. Unlabeled UTP was added in the transcription mixture to a final concentration of 10 ,umolA. In situ hybridization for detection of DNA and mRNA in tissue sections was performed as described by Hukkanen et al,30 with slight modifications. The concentration of proteinase K was 40 p6g/ml. The nonspecific blocking nucleic acid in the hybridization buffer was singlestranded herring sperm DNA (Sigma, St. Louis, MO, 0.5 mg/ml), and transfer RNA (Sigma, 0.5 mg/ml, for RNA probes). One control slide for each biopsy specimen was pretreated before acetylation with 100 ,ug/ml RNase A for 2 hours at 37°C to control the RNA specificity of the reaction. The slides were washed according to previously described protocols,31 with some modifications. For detection of mRNA, an additional washing in 60 ,ug/ml RNase A and 10 U/mI RNase Ti in 10 mmolA TRIS pH 7.5, 300 mmolA NaCI was performed for 40 minutes at 370C. The dehydrated slides were dipped in Kodak NTB-2 emulsion, exposed for 7 to 14 days, developed, and counterstained using hematoxylin and eosin.
Expression of Fusion Proteins and Production of Antisera Bacteria from the Escherichia coli strain pop2136 were transformed with the recombinant plasmid described in
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"Cloning procedures," encoding the 281 bp fragment from the L2 ORF, and grown up at 300C to the optical density of 0.6 at 550 nm. Production of the fusion protein was induced by increasing the temperature to 420C, and the bacteria were grown for an additional 2 hours. The bacteria were lysed using 2 mg/ml lysozyme and 0.7% Triton X-100. DNA was sheared by passing it several times through an 18-gauge needle. The bacterial lysate was run in a 7.5% sodium dodecyl sulfate (SDS)polyacrylamide gel, the fusion protein band recovered, the protein electroeluted from the gel with NaHCO3 or NH4HCO3 buffer using ISCO electrophoretic concentrator (Isco Inc., Lincoln, NE), and used for immunization of rabbits. The rabbits were immunized with approximately 100 ,ug fusion protein in Freund's complete adjuvant and boosted six to seven times with 100 ,ug protein in Freund's incomplete adjuvant at 1-month intervals.
Immunohistochemical Detection of the L2 Antigen The detection of HPV 16 L2 antigen in tissue sections was performed using Vectastain ABC kit PK-4001 (Vector Laboratories, Burlingame, CA). Rabbit anti-bovine papillomavirus (BPV) antibody, raised against disrupted BPV 1 particles (Dako, Glostrup, Denmark), was used as a cross-reactive positive control and rabbit preimmune serum served as a negative control for each specimen. The serum dilutions were from 1/100 to 1/2500. The sections were stained with hematoxylin. To evaluate the presence of cross-reactive antibodies in the antisera, the antisera were in some experiments absorbed with a bacterial lysate from induced E. cofi bacteria that produce the P-galactosidase domain of the fusion protein but not the HPV 16 domain. The sera were diluted, incubated with the bacterial lysate at 37°C for 1 hour, and centrifuged. The supernatant was used for immunohistochemistry.
Statistical Methods Statistical computations were performed with BMDP statistical software package run on a VAX/VMS mainframe computer. Test for linear trend was used to assess the distribution of the mRNA positive hybridization results along the axis from normal through dysplasias of different degrees, and test for homogeneity was used to evaluate the possible differences in the occurrence in the L2 mRNA positivity as compared with that of E7. The histologic variable was first dichotomized at the level of mild
dysplasia in the axis from normal to severe dysplasia, ignoring the few lesions more severe than grade Ill dysplasias. All P-values given are two tailed.
Results In total, 90 biopsy specimens from 70 patients were included in this study. Based on the dot hybridization results,26 50 patients were included, who had been positive for HPV 16 DNA, and as controls for immunohistochemical analyses, four patients positive for HPV 18 DNA, seven for HPV 11 DNA, and nine who had been HPV-dot hybridization negative. We used formalin-fixed paraffinembedded specimens, which had been taken essentially at the same time as the specimens for dot blot hybridization. In further verification by in situ DNA hybridization, the paraffin-embedded biopsies from 38/50 patients were found positive for HPV 16 DNA (Table 1). One HPV 16 positive specimen was also positive for HPV 11 DNA. Four of the patients had normal histologic findings, nine had condyloma, 22 had dysplasia, and three had carcinoma. The expression of the E7 and L2 genes was studied in 36 of the 38 patients, who were positive for HPV 16 DNA in both dot blot and in situ hybridization tests (Table 1). The presence of L2 mRNA was observed in 24 (Table 1; Figure 2B), and the presence of E7 mRNA in 21 specimens (Table 1; Figure 2D). HPV 16 DNA and HPV 16 L2 and E7 mRNA were present only in the middle and superficial layers of the epithelium (Figure 2B, D), and not in basal layer or stroma. No specific reaction was seen with the complementary strand probes (Figure 2A, C). Expression of L2 mRNA was found in histologically normal tissue, in condylomas, in all grades of dysplasia, and in in situ carcinomas (Table 1, #36 and #37). In the only invasive carcinoma included (Table 1, #38) expression of L2 mRNA was not detected. The expression of E7 mRNA was seen in one condyloma, dysplasias, and in situ carcinomas. In invasive carcinoma E7 mRNA was not detected. Two of the three specimens, where the L2 ORF but not the E7 was expressed, were classified as condyloma, and one had mild inflammation only, regarded as histologically normal. The detection rate for the L2 mRNA increased along with the severity of the lesions (P = 0.0064; test for linear trend). For E7 mRNA, the increase in detection rate was even more evident (P = 0.0001). Using the test of homogeneity, no significant differences were found in the occurrence in the L2 mRNA positivity as compared with that of E7 mRNA. However, L2 mRNA was detected in four of 12 tissue specimens without dysplastic changes, but E7 mRNA in only one of these.
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Table 1. E7 and L2 Gene Expression in the Lesions Positive for HPV 16 DNA by In Situ Hybridization E7 mRNA L2 mRNA Histology Case #
Normal Normal Normal Normal Condyloma planum Condyloma planum Condyloma planum Condyloma planum
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
NT*
L2 antigen
NT
+
Condyloma planum Condyloma planum Condyloma planum Condyloma planum Condyloma planumt Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Severe dysplasia Bowenoid papulosis Carcinoma in situ Carcinoma
+ + +
+ +
+ + + NT
+ + + NT
+ +
+ +
+ + + +
+ + + + + + +
+ + + + + + + + + + +
+ + + + + +
+
+ + + + + + +
+
+
+ + +
+
NT, not tested.
t This patient was also positive for HPV 11 DNA.
All the 90 biopsy specimens from 70 patients were analyzed by immunohistochemistry for L2 antigen. For control purposes, antiserum to BPV 1 was included. Specimens from 13 patients, positive also for HPV 16 DNA, reacted with anti-HPV 16 L2-antiserum (Table 1). Ten of those were positive for L2 and E7 mRNA by in situ hybridization. Two specimens that were negative for L2 and E7 mRNA and one specimen in which no tissue was available for mRNA analysis were also positive for the L2 antigen. The L2-antigen was localized exclusively in the nuclei of individual cells in the middle and superficial layepithelium (Figure 2F). No reactivity was seen with rabbit preimmune serum. Four of the L2 antigen-positive specimens reacted with anti-BPV-antibodies. This staining was nuclear as well (Figure 2E). Two in situ HPV 11, 16, and 18 DNAnegative specimens and one HPV 11-positive specimen reacted also with the anti-BPV antiserum. The anti-BPV antibodies are mainly directed against the major capsid ers of the
protein Li, which cross-reacts between papillomavirus types and even between papillomaviruses from different animal species.20 One of the 13 patients whose specimens reacted with the anti-L2 antiserum had only condyloma, four had mild dysplasia, seven had moderate dysplasia, and one had severe dysplasia. Presence of the L2 antigen could not be shown in histologically normal or in carcinoma specimens. The anti-L2 antiserum reacted specifically only with the L2 protein of HPV 16. No reaction was seen in the lesions harboring the HPV 1 1 or HPV 18 genome or in the HPV-negative lesions included in this study. The absorption of the antiserum with the bacterial lysate did not change its reactivity; it merely decreased the background to some extent. The specificity of the anti-L2 antiserum as well as the reactivity of the rabbit preimmune serum were further tested by incubating them with heterologous tissue sections, where no reaction with anti-papillomavirus sera
t.-
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Figure 2. Expression of L2 and E7 genes in a HPV 16 DNA-positive lesion classified as moderate dysplasia. In situ hybridization with HPV 16 L2 control RNIA probe (A), RNA probefor 12 mRNVA (B), E7 control R~NA probe (C), RNA probefor E7 mRNA (D). Immunohistocbemical reaction with anti-BP V-antibodies, serum dilution 12500 (E) and anti-HPV 16 L2-antibodies, serum dilution 1:500 (F). The scale bar in (A) represents 1 00 pLm. The arrow in (B) indicates L2 mRNA positive cells. The arrou' in (F) indicates a cell positive for the L2 protein.
should be expected. Faint diffuse unspecific staining was seen in cells of squamous epithelium, in sweat glands, in mammary gland lobulus cells, and in the endothelia of arterioles. Identical reactions in the control tissues were seen with the preimmune serum. The unspecific staining in control tissues was more diffuse and not as intensive as the specific staining with anti-L2-antiserum in the individual cells of positive lesions. The specific L2 staining
pattern was easily distinguished from the unspecific staining of the remaining squamous epithelium.
Discussion Human papillomaviruses are associated with cervical cancer, and of the different HPV types, the association of HPV 16 with cancer is most evident.32 In the current work,
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we were interested to evaluate the role of HPV 16 gene expression in progression of HPV 16-associated neoplasias by studying the correlation between HPV gene expression and the histologic grade of the lesion. The E6 and E7 genes of HPV encode transforming proteins, which have the capability to bind cellular antioncogenes p53 and pRB, respectively.33'34 Transcription of the E7 gene is characteristic for cervical cancer cell lines and tumors, where the viral DNA is integrated into the host cell genome.15'16 E7 mRNA expression of HPV 16 has been observed in premalignant lesions irrespective of the histopathologic grade of dysplasia,19 although some investigators have reported an increase in expression of the E6-E7 region and a decrease in E4, E5, and Li gene expression in higher-grade lesions.17 In a study of HPV-16-positive cervical and vulvar precancers, the most abundant signal from the early region was from the E4 and E5 ORFs, whereas the major late signal originated from the Li ORF.35 The expression of the capsid proteins Li and L2 is confined to highly differentiated superficial cells of the squamous epithelium, where virus production occurs.18 In the current work, we studied the expression of the HPV 16 E7 and L2 genes in a material consisting mainly of condylomas and dysplasias, and one case of invasive cancer, representing the HPV 16 positive cases diagnosed at our department in 1987 to 1990, from whom adequate biopsy specimen was available. The presence of HPV 16 DNA was verified by in situ hybridization in 38 of 50 dot blot positive patients, which may be explained by the higher sensitivity of the dot hybridization test. The hybridization tests were done using adjacent tissue samples from the same lesion, and the verified HPV 16 positive specimens were subjected to mRNA analysis. The expression of the L2 gene was detected more frequently than the expression of the E7 gene. One of the three patients in which the L2 but not E7 expression was detected had only nonspecific inflammation, and two had condyloma planum. In the only invasive carcinoma, the expression of L2 or E7 mRNA was not detected. Crum et aI19 have described that the E6-E7 region and the E2E5-L2 region of HPV 16 are expressed in similar proportions in genital precancers. In their work, the expression pattern did not have direct correlation to histopathologic grade of the lesions. These probes were different from ours, however, which detected the E7 and L2 transcripts. In our material, the E7 and L2 mRNAs were as well found in all grades of dysplasia, with emphasis on the moderate/severe dysplasia group. A considerable portion of the RNA that we detected using the L2 probe was mRNA for the L2 protein, because we found also L2 antigen in these tissue specimens. Some of the L2 RNA signal may have been originated from readthrough transcripts from
early promoters of HPV 16, however. The L2 antigen was detected in a large proportion of the mild and moderate dysplasias, whereas, surprisingly, its expression in condylomas was rare. In dysplastic cells, the copy number of the viral genome is high enough to enable expression of the HPV proteins at detectable levels. In cancer-derived CaSki cells, where several hundred copies of integrated HPV 16 DNA per cell are present, and in a cervical carcinoma containing several copies of mainly episomal HPV 16 DNA per cell, viral RNA has predominantly been mapped to the early region of the genome.16 The major viral transcript was from the E7 region in CaSki cells, and the corresponding protein was detected in the cells by immunoprecipitation.16 Interestingly, both in the CaSki cells and in the cervical carcinoma, transcripts were found that were spliced from the E6-E7 region to the E2-E4 region. In the cervical carcinoma that contained mainly episomal HPV DNA, a transcript capable of encoding the intact E2 protein was identified. In cancers and in cancer-derived cell lines, integration of the HPV genome has usually occurred within the E1-E2 region, which disrupts the regulating function of the E2 protein.36 The 3' terminal domain of the E2 ORF encodes a transcriptional repressor, and if expression of such a protein is inhibited by e.g. disruption of the EZ ORF by integration, it is possible that transcription of the E6 and E7 genes from the early promoter is enhanced, and this results in a transformed phenotype of the cells.37 No correlation between the expression of either early or late ORFs and the histopathologic grade of the lesions was found in a work where the expression of several HPV 16 ORFs was studied by PCR.38 Moreover, no association between the transcription pattern and the physical state of the viral DNA was found. RNA splicing pattern was not shown to correlate with tumor progression. These results suggest that either quantitative differences in the expression of different viral genes or differences in the regulation of initiation or termination of transcription may play a role in the carcinogenic process.i8 In the current work, L2 and, particularly, E7 gene expression correlated with increasing grade of dysplasia. This is in accordance with the putative role of the E7 gene of HPV 16 in malignant transformation. The L2 mRNA and protein were expressed mostly in the superficial cell layers of dysplastic epithelia, which is the probable location for virus maturation. These results, together with those of other authors, support the hypothesis that viral proteins have a role in malignant transformation of cells. Additionally, cellular functions, such as the expression of p53 and pRB tumor suppressors, or the expression of oncogenes, are involved and they further complement the function of viral proteins in the malignant process.
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Acknowledgments The authors thank Mrs. Merja Virtanen for technical assistance, Dr. Markku Kallajoki for helpful discussions, and Professor Lutz Gissmann (Deutsches Krebsforschungszentrum, Heidelberg, Germany) for the HPV 1 1, HPV 16, and HPV 18 clones used in this study:'
14.
15.
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34. Werness BA, Levine AJ, Howley PM: Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990, 248:76-79 35. Crum CP, Nuovo G, Friedman D, Silverstein SJ: Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers. J Virol 1988, 62:84-90 36. Phelps WC, Howley PM: Transcriptional trans-activation by the human papillomavirus type 16 E2 gene product. J Virol 1987, 61:1630-1638 37. Giri I, Yaniv M: Structural and mutational analysis of E2 transactivating proteins of papillomaviruses reveals three distinct functional domains. EMBO J 1988, 7:2823-2829 38. Sherman L, Alloul N, Golan I, Durst M, Baram A: Expression and splicing patterns of human papillomavirus type-16 mRNAs in pre-cancerous lesions and carcinomas of the cervix, in human keratinocytes immortalized by HPV 16, and in cell lines established from cervical cancers. Int J Cancer 1992, 50:356-364
Expression of the L2 and E7 Genes of the Human Papillomavirus Type 16 in Female Genital Dysplasias Eeva Auvinen,* Harry Kujari,t Pertti Arstila,* and Veijo Hukkanen* From the Departments of Virology* and Pathology, t University of Turku, Turku, Finland
The expression of the E7 and L2 genes of HPV 16 was studied in benign and precancerous female genital lesions to evaluate their role in the development of dysplasias. Ninety biopsy specimens from 70 patients, selected on basis of dot blot DNA hybridization, were included in immunohistochemical and in situ hybridization analyses. In the HPV 16 DNA positive cases, L2 mRNA and E7 mRNA were detected in biopsies from 24 and 21 patients, respectively. L2 mRNA was found in eight of 16 cases of condyloma and mild dysplasia and in 13 of 14 cases of moderate to severe dysplasia The figures forE7 mRNA were 6/16 and 13/14, respectively. We found L2 mRNA in four of 12 normal or condylomatous specimens and E7 mRNA in only one of these. The detection rates for L2 and E7 mRNAs increased along with the severity of the lesions (P = 0.0064 and P = 0.0001, respectively). The L2 protein was found in one condyloma and in 12 dysplasias, eight of which were moderate or severe. The L2-antibody-reactive cells were localized in superficial layers of the epithelium. The detection rateforL2 mRNA and especiallyforE7 mRNA increased along with the histopathologic grade of the lesion. (Am JPathol 1992, 141:1217-1224)
To date, almost 70 human papillomavirus (HPV) types have been described, and more than 22 of them have been detected in squamous genital epithelia.1 Only certain HPV types have been shown to be associated with dysplasia and cancer. Particularly HPV type 16 DNA has been identified in cervical cancers and in premalignant lesions,23 whereas HPV types 6 and 1 1 are associated with mere condyloma. Multiple infections are fairly common.4 In approximately 90% of cervical cancers, HPV DNA has been identified,56 and in 50% of those, HPV 16 DNA is present.
Immortalization of keratinocytes with human papillomavirus DNA has been reported in several studies, and the immortalizing ability has been mapped to the E6-E7region.7-11 Furthermore, this immortalizing ability is specific for the HPV types associated with cancer,12"14 and has not been reported for other types of HPV. In most cervical squamous cell cancers and cancer cell lines studied, the HPV 16 genome is integrated into the host cell genome and the E6-E7 region is retained and transcribed, which also suggests that these genes are important in the malignant process.15'16 The relative expression of E6-E7 region of HPV 16 has been found to increase along with the severity of lesions in gynecologic
neoplasias.17 The L2 protein of papillomaviruses is a minor capsid component, which is expressed in highly differentiated cells of benign squamous lesions, where virus production occurs.18 Hence, the expression of the L2 protein is not expected to occur in carcinoma cells.17 L2 mRNA has been identified in higher grade lesions, too, however.19 Homology within the L2 open reading frame (ORF) is considerably lower between various papillomavirus types than that of the Li ORF.20 The anti-L1 -antibodies crossreact between papillomavirus types, whereas the antiL2-antibodies may be considered more type-specific. Human papillomavirus early proteins have been identified in human genital lesions or HPV-containing cancer cell lines by immunoprecipitation or by Western blotting.21 24The role of HPV 16 genes in the development of human genital squamous neoplasias is not thoroughly characterized, however. Despite the intensive research on the molecular mechanisms of human papillomavirus infections, systematic studies on HPV gene expression in clinical material have been few. In the current work, the expression of HPV 16 E7 and L2 ORFs was studied in HPV 16 DNA positive condylomas, dysplasias, and carcinomas to assess their putative role in malignant transformation. Supported by the Cancer Society of Finland and the Turku University Foundation. Accepted for publication May 14, 1992. Address reprint requests to Dr. Eeva Auvinen, Department of Virology, University of Turku, Kiinamyllynkatu 13, SF-20520 Turku, Finland.
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Materials and Methods Biopsy Material Biopsy specimens from cervical, vaginal, and vulvar lesions were fixed with 4% phosphate-buffered formalin, embedded in paraffin, and cut at 4 or 5 Ai on organosiliconized slides25 for in situ hybridization and on poly-Llysine-coated slides for immunohistochemical staining. The specimens were collected from the patient material that was sent for routine HPV dot blot hybridization26'27 to the Department of Virology, University of Turku. All HPV 16-positive patients during 1987 to 1990, from whom adequate biopsy material was available, were included, irrespective of the histopathologic diagnosis. The material consisted of 90 biopsies from 70 patients, selected on the basis of dot blot hybridization results. Included were 50 patients who had been positive for HPV 16 DNA, and, as controls for immunohistochemical analyses, four patients positive for HPV 18 DNA, seven positive for HPV 11 DNA, and nine who had been HPV negative. The tissue specimens were obtained from the Department of Obstetrics and Gynecology, Turku University Hospital, Turku, Finland. The routinely stained sections were reviewed without knowing the primary diagnoses given.
Cloning Procedures Figure 1 illustrates the genomic localization of the hybridization probes and of the HPV sequences used for expression of fusion proteins. The 1776-base pair (bp) Pstl fragment of the HPV 16 genome, comprising the 3' terminal part of the Li ORF, the noncoding region, and the E6 and E7 ORFs, was subcloned into pGEM5Zf( +) vector (Promega, Biotec, Madison, WI) to prepare singlestranded RNA probes for detection of HPV 16 E7 mRNA using in situ hybridization. Similarly, the 1063-bp Pstl fragment, comprising the 3' terminal part of the E2 ORF and the 5' terminal part of the L2 ORF, was cloned into
E6
E1
LI
L2 clone
E7 cbn
PSII
Pstl
7007
879
S7p2
Pstl 3696
e
Pstl
St,4759 4468 L2 nt09gn Alul Pstl 447a 4759
Figure 1. Genomic localization of thefragments of HPV 16 DNA used for synthesis of in situ hybridization R.NA probes and for production offusion protein for use as immunization antigen. The horizontal line on the top ofthefigure represents the genomic DNA ofHPV 16 The open boxes represent the open readingframes The arrows at the E7 and L2 clones indicate the restriction enzyme cleavage sites used for linearization of the templates for RNA probes.
pGEM1 (Promega Biotec). The 281 bp AIul-Pstl fragment from the L2 ORF was cloned into pEX128 using Smal-Pstl sites. In-frame ligation and the correct orientation of the subclones was verified by restriction mapping and sequencing by the Sanger dideoxy method"9 using Sequenase(!3' Version 2.0 kit (US Biochemicals, Cleveland, OH).
In Situ Hybridization The probes used for in situ DNA hybridization were HPV 1 1, HPV 16, and HPV 18 DNA purified from the vector. The DNA was labeled in a random priming reaction using 35S-dATPaxS (1000 Ci/mmol, Amersham, Buckinghamshire, UK) to specific activities of 0.8 to 2.0 x 109 dpm/,ug template. For preparation of single-stranded RNAprobes, the E7 pGEM clone was linearized at the Sspl site at nucleotide (nt) 720 and the transcription was performed from the SP6 promoter for production of a 159 nt probe. The control probe was prepared from the complementary strand of the same clone using the T7 promoter. The L2 pGEM clone was linearized with Stul at nt 4468 and transcription was done from the SP6 promoter to produce a 291 nt long probe for detection of L2 mRNA, and the control probe was transcribed from the T7 promoter. The probes for detection of mRNA were labeled with 35S-UTPaS (1000 Ci/mmol, Amersham) to specific activities of 3.0-4.2 x 109 dpm/,g template. Unlabeled UTP was added in the transcription mixture to a final concentration of 10 ,umolA. In situ hybridization for detection of DNA and mRNA in tissue sections was performed as described by Hukkanen et al,30 with slight modifications. The concentration of proteinase K was 40 p6g/ml. The nonspecific blocking nucleic acid in the hybridization buffer was singlestranded herring sperm DNA (Sigma, St. Louis, MO, 0.5 mg/ml), and transfer RNA (Sigma, 0.5 mg/ml, for RNA probes). One control slide for each biopsy specimen was pretreated before acetylation with 100 ,ug/ml RNase A for 2 hours at 37°C to control the RNA specificity of the reaction. The slides were washed according to previously described protocols,31 with some modifications. For detection of mRNA, an additional washing in 60 ,ug/ml RNase A and 10 U/mI RNase Ti in 10 mmolA TRIS pH 7.5, 300 mmolA NaCI was performed for 40 minutes at 370C. The dehydrated slides were dipped in Kodak NTB-2 emulsion, exposed for 7 to 14 days, developed, and counterstained using hematoxylin and eosin.
Expression of Fusion Proteins and Production of Antisera Bacteria from the Escherichia coli strain pop2136 were transformed with the recombinant plasmid described in
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"Cloning procedures," encoding the 281 bp fragment from the L2 ORF, and grown up at 300C to the optical density of 0.6 at 550 nm. Production of the fusion protein was induced by increasing the temperature to 420C, and the bacteria were grown for an additional 2 hours. The bacteria were lysed using 2 mg/ml lysozyme and 0.7% Triton X-100. DNA was sheared by passing it several times through an 18-gauge needle. The bacterial lysate was run in a 7.5% sodium dodecyl sulfate (SDS)polyacrylamide gel, the fusion protein band recovered, the protein electroeluted from the gel with NaHCO3 or NH4HCO3 buffer using ISCO electrophoretic concentrator (Isco Inc., Lincoln, NE), and used for immunization of rabbits. The rabbits were immunized with approximately 100 ,ug fusion protein in Freund's complete adjuvant and boosted six to seven times with 100 ,ug protein in Freund's incomplete adjuvant at 1-month intervals.
Immunohistochemical Detection of the L2 Antigen The detection of HPV 16 L2 antigen in tissue sections was performed using Vectastain ABC kit PK-4001 (Vector Laboratories, Burlingame, CA). Rabbit anti-bovine papillomavirus (BPV) antibody, raised against disrupted BPV 1 particles (Dako, Glostrup, Denmark), was used as a cross-reactive positive control and rabbit preimmune serum served as a negative control for each specimen. The serum dilutions were from 1/100 to 1/2500. The sections were stained with hematoxylin. To evaluate the presence of cross-reactive antibodies in the antisera, the antisera were in some experiments absorbed with a bacterial lysate from induced E. cofi bacteria that produce the P-galactosidase domain of the fusion protein but not the HPV 16 domain. The sera were diluted, incubated with the bacterial lysate at 37°C for 1 hour, and centrifuged. The supernatant was used for immunohistochemistry.
Statistical Methods Statistical computations were performed with BMDP statistical software package run on a VAX/VMS mainframe computer. Test for linear trend was used to assess the distribution of the mRNA positive hybridization results along the axis from normal through dysplasias of different degrees, and test for homogeneity was used to evaluate the possible differences in the occurrence in the L2 mRNA positivity as compared with that of E7. The histologic variable was first dichotomized at the level of mild
dysplasia in the axis from normal to severe dysplasia, ignoring the few lesions more severe than grade Ill dysplasias. All P-values given are two tailed.
Results In total, 90 biopsy specimens from 70 patients were included in this study. Based on the dot hybridization results,26 50 patients were included, who had been positive for HPV 16 DNA, and as controls for immunohistochemical analyses, four patients positive for HPV 18 DNA, seven for HPV 11 DNA, and nine who had been HPV-dot hybridization negative. We used formalin-fixed paraffinembedded specimens, which had been taken essentially at the same time as the specimens for dot blot hybridization. In further verification by in situ DNA hybridization, the paraffin-embedded biopsies from 38/50 patients were found positive for HPV 16 DNA (Table 1). One HPV 16 positive specimen was also positive for HPV 11 DNA. Four of the patients had normal histologic findings, nine had condyloma, 22 had dysplasia, and three had carcinoma. The expression of the E7 and L2 genes was studied in 36 of the 38 patients, who were positive for HPV 16 DNA in both dot blot and in situ hybridization tests (Table 1). The presence of L2 mRNA was observed in 24 (Table 1; Figure 2B), and the presence of E7 mRNA in 21 specimens (Table 1; Figure 2D). HPV 16 DNA and HPV 16 L2 and E7 mRNA were present only in the middle and superficial layers of the epithelium (Figure 2B, D), and not in basal layer or stroma. No specific reaction was seen with the complementary strand probes (Figure 2A, C). Expression of L2 mRNA was found in histologically normal tissue, in condylomas, in all grades of dysplasia, and in in situ carcinomas (Table 1, #36 and #37). In the only invasive carcinoma included (Table 1, #38) expression of L2 mRNA was not detected. The expression of E7 mRNA was seen in one condyloma, dysplasias, and in situ carcinomas. In invasive carcinoma E7 mRNA was not detected. Two of the three specimens, where the L2 ORF but not the E7 was expressed, were classified as condyloma, and one had mild inflammation only, regarded as histologically normal. The detection rate for the L2 mRNA increased along with the severity of the lesions (P = 0.0064; test for linear trend). For E7 mRNA, the increase in detection rate was even more evident (P = 0.0001). Using the test of homogeneity, no significant differences were found in the occurrence in the L2 mRNA positivity as compared with that of E7 mRNA. However, L2 mRNA was detected in four of 12 tissue specimens without dysplastic changes, but E7 mRNA in only one of these.
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Table 1. E7 and L2 Gene Expression in the Lesions Positive for HPV 16 DNA by In Situ Hybridization E7 mRNA L2 mRNA Histology Case #
Normal Normal Normal Normal Condyloma planum Condyloma planum Condyloma planum Condyloma planum
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
NT*
L2 antigen
NT
+
Condyloma planum Condyloma planum Condyloma planum Condyloma planum Condyloma planumt Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Mild dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Moderate dysplasia Severe dysplasia Bowenoid papulosis Carcinoma in situ Carcinoma
+ + +
+ +
+ + + NT
+ + + NT
+ +
+ +
+ + + +
+ + + + + + +
+ + + + + + + + + + +
+ + + + + +
+
+ + + + + + +
+
+
+ + +
+
NT, not tested.
t This patient was also positive for HPV 11 DNA.
All the 90 biopsy specimens from 70 patients were analyzed by immunohistochemistry for L2 antigen. For control purposes, antiserum to BPV 1 was included. Specimens from 13 patients, positive also for HPV 16 DNA, reacted with anti-HPV 16 L2-antiserum (Table 1). Ten of those were positive for L2 and E7 mRNA by in situ hybridization. Two specimens that were negative for L2 and E7 mRNA and one specimen in which no tissue was available for mRNA analysis were also positive for the L2 antigen. The L2-antigen was localized exclusively in the nuclei of individual cells in the middle and superficial layepithelium (Figure 2F). No reactivity was seen with rabbit preimmune serum. Four of the L2 antigen-positive specimens reacted with anti-BPV-antibodies. This staining was nuclear as well (Figure 2E). Two in situ HPV 11, 16, and 18 DNAnegative specimens and one HPV 11-positive specimen reacted also with the anti-BPV antiserum. The anti-BPV antibodies are mainly directed against the major capsid ers of the
protein Li, which cross-reacts between papillomavirus types and even between papillomaviruses from different animal species.20 One of the 13 patients whose specimens reacted with the anti-L2 antiserum had only condyloma, four had mild dysplasia, seven had moderate dysplasia, and one had severe dysplasia. Presence of the L2 antigen could not be shown in histologically normal or in carcinoma specimens. The anti-L2 antiserum reacted specifically only with the L2 protein of HPV 16. No reaction was seen in the lesions harboring the HPV 1 1 or HPV 18 genome or in the HPV-negative lesions included in this study. The absorption of the antiserum with the bacterial lysate did not change its reactivity; it merely decreased the background to some extent. The specificity of the anti-L2 antiserum as well as the reactivity of the rabbit preimmune serum were further tested by incubating them with heterologous tissue sections, where no reaction with anti-papillomavirus sera
t.-
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.. . .
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1
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Figure 2. Expression of L2 and E7 genes in a HPV 16 DNA-positive lesion classified as moderate dysplasia. In situ hybridization with HPV 16 L2 control RNIA probe (A), RNA probefor 12 mRNVA (B), E7 control R~NA probe (C), RNA probefor E7 mRNA (D). Immunohistocbemical reaction with anti-BP V-antibodies, serum dilution 12500 (E) and anti-HPV 16 L2-antibodies, serum dilution 1:500 (F). The scale bar in (A) represents 1 00 pLm. The arrow in (B) indicates L2 mRNA positive cells. The arrou' in (F) indicates a cell positive for the L2 protein.
should be expected. Faint diffuse unspecific staining was seen in cells of squamous epithelium, in sweat glands, in mammary gland lobulus cells, and in the endothelia of arterioles. Identical reactions in the control tissues were seen with the preimmune serum. The unspecific staining in control tissues was more diffuse and not as intensive as the specific staining with anti-L2-antiserum in the individual cells of positive lesions. The specific L2 staining
pattern was easily distinguished from the unspecific staining of the remaining squamous epithelium.
Discussion Human papillomaviruses are associated with cervical cancer, and of the different HPV types, the association of HPV 16 with cancer is most evident.32 In the current work,
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we were interested to evaluate the role of HPV 16 gene expression in progression of HPV 16-associated neoplasias by studying the correlation between HPV gene expression and the histologic grade of the lesion. The E6 and E7 genes of HPV encode transforming proteins, which have the capability to bind cellular antioncogenes p53 and pRB, respectively.33'34 Transcription of the E7 gene is characteristic for cervical cancer cell lines and tumors, where the viral DNA is integrated into the host cell genome.15'16 E7 mRNA expression of HPV 16 has been observed in premalignant lesions irrespective of the histopathologic grade of dysplasia,19 although some investigators have reported an increase in expression of the E6-E7 region and a decrease in E4, E5, and Li gene expression in higher-grade lesions.17 In a study of HPV-16-positive cervical and vulvar precancers, the most abundant signal from the early region was from the E4 and E5 ORFs, whereas the major late signal originated from the Li ORF.35 The expression of the capsid proteins Li and L2 is confined to highly differentiated superficial cells of the squamous epithelium, where virus production occurs.18 In the current work, we studied the expression of the HPV 16 E7 and L2 genes in a material consisting mainly of condylomas and dysplasias, and one case of invasive cancer, representing the HPV 16 positive cases diagnosed at our department in 1987 to 1990, from whom adequate biopsy specimen was available. The presence of HPV 16 DNA was verified by in situ hybridization in 38 of 50 dot blot positive patients, which may be explained by the higher sensitivity of the dot hybridization test. The hybridization tests were done using adjacent tissue samples from the same lesion, and the verified HPV 16 positive specimens were subjected to mRNA analysis. The expression of the L2 gene was detected more frequently than the expression of the E7 gene. One of the three patients in which the L2 but not E7 expression was detected had only nonspecific inflammation, and two had condyloma planum. In the only invasive carcinoma, the expression of L2 or E7 mRNA was not detected. Crum et aI19 have described that the E6-E7 region and the E2E5-L2 region of HPV 16 are expressed in similar proportions in genital precancers. In their work, the expression pattern did not have direct correlation to histopathologic grade of the lesions. These probes were different from ours, however, which detected the E7 and L2 transcripts. In our material, the E7 and L2 mRNAs were as well found in all grades of dysplasia, with emphasis on the moderate/severe dysplasia group. A considerable portion of the RNA that we detected using the L2 probe was mRNA for the L2 protein, because we found also L2 antigen in these tissue specimens. Some of the L2 RNA signal may have been originated from readthrough transcripts from
early promoters of HPV 16, however. The L2 antigen was detected in a large proportion of the mild and moderate dysplasias, whereas, surprisingly, its expression in condylomas was rare. In dysplastic cells, the copy number of the viral genome is high enough to enable expression of the HPV proteins at detectable levels. In cancer-derived CaSki cells, where several hundred copies of integrated HPV 16 DNA per cell are present, and in a cervical carcinoma containing several copies of mainly episomal HPV 16 DNA per cell, viral RNA has predominantly been mapped to the early region of the genome.16 The major viral transcript was from the E7 region in CaSki cells, and the corresponding protein was detected in the cells by immunoprecipitation.16 Interestingly, both in the CaSki cells and in the cervical carcinoma, transcripts were found that were spliced from the E6-E7 region to the E2-E4 region. In the cervical carcinoma that contained mainly episomal HPV DNA, a transcript capable of encoding the intact E2 protein was identified. In cancers and in cancer-derived cell lines, integration of the HPV genome has usually occurred within the E1-E2 region, which disrupts the regulating function of the E2 protein.36 The 3' terminal domain of the E2 ORF encodes a transcriptional repressor, and if expression of such a protein is inhibited by e.g. disruption of the EZ ORF by integration, it is possible that transcription of the E6 and E7 genes from the early promoter is enhanced, and this results in a transformed phenotype of the cells.37 No correlation between the expression of either early or late ORFs and the histopathologic grade of the lesions was found in a work where the expression of several HPV 16 ORFs was studied by PCR.38 Moreover, no association between the transcription pattern and the physical state of the viral DNA was found. RNA splicing pattern was not shown to correlate with tumor progression. These results suggest that either quantitative differences in the expression of different viral genes or differences in the regulation of initiation or termination of transcription may play a role in the carcinogenic process.i8 In the current work, L2 and, particularly, E7 gene expression correlated with increasing grade of dysplasia. This is in accordance with the putative role of the E7 gene of HPV 16 in malignant transformation. The L2 mRNA and protein were expressed mostly in the superficial cell layers of dysplastic epithelia, which is the probable location for virus maturation. These results, together with those of other authors, support the hypothesis that viral proteins have a role in malignant transformation of cells. Additionally, cellular functions, such as the expression of p53 and pRB tumor suppressors, or the expression of oncogenes, are involved and they further complement the function of viral proteins in the malignant process.
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Acknowledgments The authors thank Mrs. Merja Virtanen for technical assistance, Dr. Markku Kallajoki for helpful discussions, and Professor Lutz Gissmann (Deutsches Krebsforschungszentrum, Heidelberg, Germany) for the HPV 1 1, HPV 16, and HPV 18 clones used in this study:'
14.
15.
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34. Werness BA, Levine AJ, Howley PM: Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990, 248:76-79 35. Crum CP, Nuovo G, Friedman D, Silverstein SJ: Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers. J Virol 1988, 62:84-90 36. Phelps WC, Howley PM: Transcriptional trans-activation by the human papillomavirus type 16 E2 gene product. J Virol 1987, 61:1630-1638 37. Giri I, Yaniv M: Structural and mutational analysis of E2 transactivating proteins of papillomaviruses reveals three distinct functional domains. EMBO J 1988, 7:2823-2829 38. Sherman L, Alloul N, Golan I, Durst M, Baram A: Expression and splicing patterns of human papillomavirus type-16 mRNAs in pre-cancerous lesions and carcinomas of the cervix, in human keratinocytes immortalized by HPV 16, and in cell lines established from cervical cancers. Int J Cancer 1992, 50:356-364
