Acta Oto-Laryngologica
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Detection of Human Papillomavirus Type 6f Genome in Nasal Papillomatosis H. Ogura, T. Kawakami, T. Fujiwara, A. Sakai, R. Saito, S. Watanabe, Y. Masuda & Y. Yabe To cite this article: H. Ogura, T. Kawakami, T. Fujiwara, A. Sakai, R. Saito, S. Watanabe, Y. Masuda & Y. Yabe (1992) Detection of Human Papillomavirus Type 6f Genome in Nasal Papillomatosis, Acta Oto-Laryngologica, 112:1, 115-119, DOI: 10.3109/00016489209100792 To link to this article: http://dx.doi.org/10.3109/00016489209100792
Published online: 08 Jul 2009.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [Flinders University of South Australia]
Date: 15 March 2016, At: 19:03
Acta Otolaryngol (Stockh) 1992; 112: 115-119
Detection of Human Papillomavirus Type 6f Genome in Nasal Papillomatosis H. OGURA,' T. KAWAKAMI,' T. FUJIWARA,' A. SAKAI,' R. SAIT0,3 S. WATANABE,' Y. MASUDA' and Y. YABE' From the 'Department of Virology, Cancer Institute, and 'Department of Otolaryngology, Okayama University, Medical School, Okayama. and 'Section of Otolaryngology, Saiseikai General Hospilal, Okayama, Japan
Acta Oto-Laryngologica 1992.112:115-119.
Ogura H, Kawakami T, Fujiwara T, Sakai A, Saito R, Watanabe S, Masuda Y, Yabe Y. Detection of human papillomavirus type 6f genome in nasal papillomatosis. Acta Otolaryngol (Stockh) 1992; 112: 115-1 19. Five cases of nasal papillomatosis were studied clinicopathologically and virologically. In a case of recurrent papillomatosis of non-inverted type located on the nasal septum, human papillomavirus (HPV) DNA was detected by dot blot hybridization with an RNA cocktail probe of mucosal HPVs. In Southern blot hybridization, the DNA hybridized with that of HPV types 6 and I 1 but not with those of types 16 and 18. Its restriction endonuclease-cleavage patterns corresponded well to those of HPV type 6f. These results suggested that HPV type 6 would also be associated with nasal non-inverted papillomatosis. Key words: papillomatosis of nasal cavity and paranasal sinus, koilocytosis, dot blot hybridization. Southern blot hybridization, HPV type 6.
INTRODUCTION Nasal inverted papilloma lesion has been shown to contain human papillomavirus (HPV) structural proteins by immunoperoxidase staining ( I ) and has further by DNA analyses been demonstrated to carry HPV genome (2-1 1). So far, HPV types I I (2-7, 9) and 57 (8) in nasal inverted papillomas, and HPV type 16/18 in cancer-associated cases (3, 4, 9, 10) have been Icported. Recently, one group detected HPV type 6 in nasal inverted papillomas by the polymerase chain reaction method (1 1). We examined 5 cases of nasal papillomatosis, and detected an HPV probably of type 6f in a case of non-inverted type. The results are reported in this paper. MATERIAL AND METHODS
Papilloma specimens. These were obtained from 5 patients operatively treated for papillomatosis of the nasal cavities and paranasal sinuses at the Department of Otolaryngology, Okayama University Medical School. As described in Table I, the patients (1 female and 4 males) were 38-60 years of age. The tumors were located in the nasal cavities in 3 cases and both in the nasal cavities and paranasal sinuses in 2 cases. In 3 cases, the tumors recurred after surgical removal. A part of each tumor was fixed and processed for histological examination and the remaining portion was kept frozen at -70°C until DNA extraction. The diagnosis of each tumor was confirmed by histological examination. Dot blot hybridization with tumor homogenates. The dot blot hybridization kit, Vira probe HPV, was obtained from Life Technologies Inc., USA through Toray-Fuji Bionics Inc., Japan. This probe was a "P-labeled RNA cocktail of HPV types 6, 11, 16, 18, 31, 33 and 35. The tumor material was thawed, cut into small pieces with scissors, ground in a precooled mortar with quartz sand, suspended to 3Yo in a lysis solution containing 250 pg/ml proteinase K, 1 O/o sodium dodecyl sulfate and 1 mM ethylenediamine tetraacetate (EDTA) in 10 mM Tris-HCI (pH 8.0),and incubated at 37°C for 6 h. Each homogenate (250 pl) was tested by dot
Acta Oto-Laryngologica 1992.112:115-119.
1 16 H. Ogura et al.
Acta Otolaryngol (Stockh) I12
Fig. I. Histopathology of the nasal papillomatosis of case 5 . The epithelium shows squamous metaplasia without inversion into the underlying stroma. Note the koilocytosis as defined by pyknotic nuclei with perinuclear halos in the upper layer.
blot hybridization according to the procedures indicated by the manufacturer of Vira probe HPV. Extraction and digestion ofDNA. DNA was extracted from tumor homogenates and purified by the method described by zur Hausen et al. (12). Restriction endonucleases were purchased from Boehringer Mannheim, Germany, o r Takara-Shuzo Co., Kyoto, Japan. Digestion with these enzymes was performed according to the methods indicated by the manufacturers. DNA labeling and Southern blot hybridization. HPV type 1 I DNA was cloned from condyloma acuminatum in this laboratory. The DNAs of HPV types 6b, 16 and 18 were generously given by Drs zur Hausen and de Villiers, Heidelberg, Germany. These HPV DNAs were separated from vector pBR322 and labeled with digoxigenin-deoxyuridinetriphosphate using the kit prepared by Boehringer Mannheim, Germany. Electrophoresis of DNA (3 kg) was done
Table 1. Detection of HPV genome in nasal papillomatosis F,female; M. male; nc, nasal cavity; ps. paranasal sinus; DB, dot blot; SB, Southern blot Histology
Case
Age
Sex
Frequency of operation
Location of lesion
I
38
F
3"
nc PS nc PS nc nc nc
2
3 4
5
40 49 53 60
M
M M M
1
3" 1 2"
" Operated repeatedly because of recurrences.
Inverted growth
Koilocytosis
+ + + + +
+ +
-
I
Hybridization
DB
SB
-
-
-
-
-
-
+
+
-
-
-
+
Acta Otolaryngol (Stockh) I 12
HPV-6 in nasal papillomatosis I 1 7
Fig. 2. A sample of autoradiogram after dot blot hybridization with Vira probe HPV: Negative hybridization with DNAs of pBR322 (A) and case 4 (B). and positive with DNA of case 5 ( C ) .
Acta Oto-Laryngologica 1992.112:115-119.
in 0.8 Oo/ agarose gel in TEA-NaCI buffer (50 mM Tris-HCI, pH 8.0, containing 20 mM sodium acetate, 2 mM NazEDTA and 18 mM NaCI). After gel electrophoresis, DNA was transferred to a Nytran membrane (Schleicher & Schuell, Dassel. Germany) by Southern blotting (13). After hybridization under stringent conditions in 50% formamide, at 42°C overnight, hybrids were detected by enzyme-linked immunoassay according to the manual prepared by the manufacturer. RESULTS
Histopatho1og.v of papillomas. Histological examinations of papillomas showed inverted growth in 3 cases and non-inverted growth in 2 cases (Table I). Inverted papillomas were characterized by squamous metaplasia, epithelial hyperplasia and inverting fingers of growth into underlying stroma. Non-inverted papillomas showed exophytic growth with squamous metaplasia and epithelial hyperplasia. Koilocytosis as defined by pyknotic nuclei with perinuclear halos was observed in tumors of cases 1 and 5 (Fig. I ) . Detection of HPV genome. Examination of tumor homogenates by dot blot hybridization with Vira probe HPV was positive in one (case 5) of 5 cases (Table I and Fig. 2). DNAs extracted and purified from tumor homogenates were examined by Southern blot hybridization with digoxigenin-labeled mixed DNA probe of HPV types 6b. 1 I , 16 and 18, after Bum HI digestion. Only that of case 5 was positive and showed a single band at the site of approximately 7.9 kilobases (Kb) (Fig. 3). Those of the remaining 6 samples of 4 cases showed no
Fig. 3. Detection o f HPV genome in Barn HI-digested DNAs by Southern blot hybridization with the mixture of digoxigeninlabeled DNAs of HPV types 6. 1 I , 16 and 18. A band of approximately 7.9 Kb was detected in the DNA of case 5 (lane 7). No band was detected in the DNAs of other nasal papillomatosis (6 samples from 4 cases, lanes 1-6). The position of the molecular size marker (in Kb) is on the right.
1 18 H. Ogura et al.
Acla Otolaryngol (Stockh) I12
Acta Oto-Laryngologica 1992.112:115-119.
Fig. 4. Endonuclease-cleavage patterns of HPV DNA of case 5 detected by Southern blot hybridization with digoxigenin-labeled HPV 6b DNA. Lanes: 1, digested with Bum HI; 2, digested with Hind 11; 3, digested with Ps[I; 4, non-digested; 5, digested with Eco RI; 6, digested with Hpa 1.
detectable bands. When examined with the probe DNA of each of these HPV types, the DNA from case 5 hybridized with the DNA of HPV types 6b and 11 but not with types 16 and 18 (data not shown). The DNA of case 5 was further digested with other restriction endonucleases and examined by Southern blot hybridization with the DNA of HPV type 6b (Fig. 4). A band of approximately 7.6 Kb was detected after Hind I1 digestion (Fig. 4, lane 2). Four bands of approximately 3.5, 1.6, 1.4 and 1.0 Kb were detected after Pst I digestion (Fig. 4, lane 3). Two bands (forms I and 11) were detected in the non-digested DNA of case 5 (Fig. 4, lane 4), and a band of approximately 7.9 Kb corresponding to form 111 was detected after Eco RI and Hpa I digestion (Fig. 4, lanes 5 and 6).
DISCUSSION DNA detectable by dot blot hybridization with Vira probe HPV was present in one (case 5 ) of 5 cases of nasal papillomatosis. This DNA from case 5 hybridized with those of HPV types 6b and 1 1, but not with those of types 16 and 18. When digested with Barn HI, Hind I1 and Pst I, the DNA of case 5 yielded fragments characteristic of HPV type 6 (14-16). Further, the cleavage patterns with Eco RI, Hpa I and Pst I corresponded to those of HPV type 6f ( I 7, 18). These findings suggested that the HPV detected in case 5 would be type 6f. Case 5 of the present study was a nasal non-inverted papillomatosis located on the nasal septum. Accordingly we have demonstrated that HPV is associated with non-inverted nasal papillomatosis as well. In our study, 4 out of 5 cases were HPV negative, although one (case 1) of them showed koilocytosis, two (case 1 and 3) were recurrent and two (case 1 and 2) multicentric, suggesting the infection with HPVs. The negative results in these cases might be due to the presence of HPVs unrelated to those used as probes in the present study.
ACKNOWLEDGEMENTS We are grateful to Drs H. zur Hausen and E. M. de Villiers for kindly providing us with DNAs of HPV types 6b, 16 and 18.
Acta Otolaryngol (Stockh) I12
HPV-6 in nasal papillomatosis
REFERENCES
Acta Oto-Laryngologica 1992.112:115-119.
1. Syrjanen KJ, Pyrhonen S, Syrjanen SM. Evidence suggesting human papillomavirus (HPV) etiology for
the squamous cell papilloma of the paranasal sinus. Arch Geschwulstforsch 1983; 53: 77-82. 2. de Villiers EM, Weidauer H, Le JY, Neumann C, zur Hausen H. Papillomviren in benignen und malignen Tumoren des Mundes und des oberen Respirationstraktes. Laryng Rhinol Otol 1986; 65: 177-9. 3. Brandsma JL, Steinberg BM, Abramson AL, Winkler B. Presence of human papillomavirus type 16 related sequences in verrucous carcinoma of the larynx. Cancer Res 1986; 46: 2185-8. 4. Syrjanen S, Happonen R-P. Virolainen E, Siivonen L. Syrjanen K. Detection of human papillomavirus (HPV) structural antigens and DNA types in inverted papillomas and squamous cell carcinomas of the nasal cavities and paranasal sinuses. Acta Otolaryngol (Stockh) 1987; 104: 334-41. 5. Respler DS, Jahn A, Pater A, Pater MM. Isolation and characterization of papillomavirus DNA from nasal inverting (Schneiderian) papillomas. Ann Otol Rhinol Laryngol 1987; 86: 170-3. 6. Weber RS, Shillitoe EJ, Robbins KT et al. Prevalence of human papillomavirus in inverted nasal papillomas. Arch Otolaryngol Head Neck Surg 1988; 114: 23-6. 7. Pater A, Gardner H, Respler DS, Jahn A, Pater M M . Isolation and characterization of a variant of human papillomavirus type I I from a nasal inverting (Schneiderian) papilloma. J Med Virol 1988; 25: 149-56. 8. de Villiers EM, Hirsch-Behnam A, von Knebel-Doeberitz C, Neumann C, zur Hausen H. Two newly identified human papillomavirus types (HPV 40 and 57) isolated from mucosal lesions. Virology 1989; 171: 248-53. 9. Furuta Y, Inuyama Y, Nagashima K. Detection of human papillomavirus genome in nasolaryngeal papillomas using digoxigenin labeled DNA probes. J Otolaryngol Jpn 1989; 92: 2055-63. 10. Brandwein M, Steinberg B, Thung S, Biller H, Dilorenzo T, Galli R. Human papillomavirus 6/11 and 16/18 in Schneiderian inverted papillomas. Cancer 1989; 63: 1708-13. 1 1 . Bryan RL, Bevan IS. Crocker J, Young LS. Detection of HPV 6 and 1 1 in tumours of the upper respiratory tract using the polymerase chain reaction. Clin Otolaryngol 1990; 15: 177-80. 12. zur Hausen H, Schulte-Holthausen H, Klein G et al. EBV DNA in biopsies of Burkitt tumors and anaplastic carcinomas of the nasopharynx. Nature 1970; 228: 1056-8. 13. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: 503-17. 14. Gissmann L, Wolnik L, lkenberg H, Koldovsky U, Schnurch HG, zur Hausen H. Human papillomavirus type 6 and I I DNA sequences in genital and laryngeal papillomas and in some cervical cancers. Proc Natl Acad Sci USA 1983: 80: 560-3. 15. de Villiers EM, Gissmann L, zur Hausen H. Molecular cloning of viral DNA from human genital warts. J Virol 1981; 40: 932-5. 16. Gissmann L, Diehl V, Schultz-Coulon HJ, zur Hausen H. Molecular cloning and characterization of human papillomavirus DNA derived from a laryngeal papilloma. J Virol 1982; 44: 393-400. 17. Mounts P, Shah K, Kashima H. Viral etiology ofjuvenile- and adult-onset squamous papilloma of the larynx. Proc Natl Acad Sci USA 1982; 79: 5425-9. 18. Mounts P, Kashima H. Association of human papillomavirus subtype and clinical course in respiratory papillomatosis. Laryngoscope 1984; 94: 28-33. Manuscript received April 2. 1991: accepted June 13, 1991 Address for correspondence: H. Ogura, Department of Virology Cancer Institute, Okayama University Medical School, 2-5-1 Shikatacho, 700 Okayama, Japan
1 19
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Detection of Human Papillomavirus Type 6f Genome in Nasal Papillomatosis H. Ogura, T. Kawakami, T. Fujiwara, A. Sakai, R. Saito, S. Watanabe, Y. Masuda & Y. Yabe To cite this article: H. Ogura, T. Kawakami, T. Fujiwara, A. Sakai, R. Saito, S. Watanabe, Y. Masuda & Y. Yabe (1992) Detection of Human Papillomavirus Type 6f Genome in Nasal Papillomatosis, Acta Oto-Laryngologica, 112:1, 115-119, DOI: 10.3109/00016489209100792 To link to this article: http://dx.doi.org/10.3109/00016489209100792
Published online: 08 Jul 2009.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [Flinders University of South Australia]
Date: 15 March 2016, At: 19:03
Acta Otolaryngol (Stockh) 1992; 112: 115-119
Detection of Human Papillomavirus Type 6f Genome in Nasal Papillomatosis H. OGURA,' T. KAWAKAMI,' T. FUJIWARA,' A. SAKAI,' R. SAIT0,3 S. WATANABE,' Y. MASUDA' and Y. YABE' From the 'Department of Virology, Cancer Institute, and 'Department of Otolaryngology, Okayama University, Medical School, Okayama. and 'Section of Otolaryngology, Saiseikai General Hospilal, Okayama, Japan
Acta Oto-Laryngologica 1992.112:115-119.
Ogura H, Kawakami T, Fujiwara T, Sakai A, Saito R, Watanabe S, Masuda Y, Yabe Y. Detection of human papillomavirus type 6f genome in nasal papillomatosis. Acta Otolaryngol (Stockh) 1992; 112: 115-1 19. Five cases of nasal papillomatosis were studied clinicopathologically and virologically. In a case of recurrent papillomatosis of non-inverted type located on the nasal septum, human papillomavirus (HPV) DNA was detected by dot blot hybridization with an RNA cocktail probe of mucosal HPVs. In Southern blot hybridization, the DNA hybridized with that of HPV types 6 and I 1 but not with those of types 16 and 18. Its restriction endonuclease-cleavage patterns corresponded well to those of HPV type 6f. These results suggested that HPV type 6 would also be associated with nasal non-inverted papillomatosis. Key words: papillomatosis of nasal cavity and paranasal sinus, koilocytosis, dot blot hybridization. Southern blot hybridization, HPV type 6.
INTRODUCTION Nasal inverted papilloma lesion has been shown to contain human papillomavirus (HPV) structural proteins by immunoperoxidase staining ( I ) and has further by DNA analyses been demonstrated to carry HPV genome (2-1 1). So far, HPV types I I (2-7, 9) and 57 (8) in nasal inverted papillomas, and HPV type 16/18 in cancer-associated cases (3, 4, 9, 10) have been Icported. Recently, one group detected HPV type 6 in nasal inverted papillomas by the polymerase chain reaction method (1 1). We examined 5 cases of nasal papillomatosis, and detected an HPV probably of type 6f in a case of non-inverted type. The results are reported in this paper. MATERIAL AND METHODS
Papilloma specimens. These were obtained from 5 patients operatively treated for papillomatosis of the nasal cavities and paranasal sinuses at the Department of Otolaryngology, Okayama University Medical School. As described in Table I, the patients (1 female and 4 males) were 38-60 years of age. The tumors were located in the nasal cavities in 3 cases and both in the nasal cavities and paranasal sinuses in 2 cases. In 3 cases, the tumors recurred after surgical removal. A part of each tumor was fixed and processed for histological examination and the remaining portion was kept frozen at -70°C until DNA extraction. The diagnosis of each tumor was confirmed by histological examination. Dot blot hybridization with tumor homogenates. The dot blot hybridization kit, Vira probe HPV, was obtained from Life Technologies Inc., USA through Toray-Fuji Bionics Inc., Japan. This probe was a "P-labeled RNA cocktail of HPV types 6, 11, 16, 18, 31, 33 and 35. The tumor material was thawed, cut into small pieces with scissors, ground in a precooled mortar with quartz sand, suspended to 3Yo in a lysis solution containing 250 pg/ml proteinase K, 1 O/o sodium dodecyl sulfate and 1 mM ethylenediamine tetraacetate (EDTA) in 10 mM Tris-HCI (pH 8.0),and incubated at 37°C for 6 h. Each homogenate (250 pl) was tested by dot
Acta Oto-Laryngologica 1992.112:115-119.
1 16 H. Ogura et al.
Acta Otolaryngol (Stockh) I12
Fig. I. Histopathology of the nasal papillomatosis of case 5 . The epithelium shows squamous metaplasia without inversion into the underlying stroma. Note the koilocytosis as defined by pyknotic nuclei with perinuclear halos in the upper layer.
blot hybridization according to the procedures indicated by the manufacturer of Vira probe HPV. Extraction and digestion ofDNA. DNA was extracted from tumor homogenates and purified by the method described by zur Hausen et al. (12). Restriction endonucleases were purchased from Boehringer Mannheim, Germany, o r Takara-Shuzo Co., Kyoto, Japan. Digestion with these enzymes was performed according to the methods indicated by the manufacturers. DNA labeling and Southern blot hybridization. HPV type 1 I DNA was cloned from condyloma acuminatum in this laboratory. The DNAs of HPV types 6b, 16 and 18 were generously given by Drs zur Hausen and de Villiers, Heidelberg, Germany. These HPV DNAs were separated from vector pBR322 and labeled with digoxigenin-deoxyuridinetriphosphate using the kit prepared by Boehringer Mannheim, Germany. Electrophoresis of DNA (3 kg) was done
Table 1. Detection of HPV genome in nasal papillomatosis F,female; M. male; nc, nasal cavity; ps. paranasal sinus; DB, dot blot; SB, Southern blot Histology
Case
Age
Sex
Frequency of operation
Location of lesion
I
38
F
3"
nc PS nc PS nc nc nc
2
3 4
5
40 49 53 60
M
M M M
1
3" 1 2"
" Operated repeatedly because of recurrences.
Inverted growth
Koilocytosis
+ + + + +
+ +
-
I
Hybridization
DB
SB
-
-
-
-
-
-
+
+
-
-
-
+
Acta Otolaryngol (Stockh) I 12
HPV-6 in nasal papillomatosis I 1 7
Fig. 2. A sample of autoradiogram after dot blot hybridization with Vira probe HPV: Negative hybridization with DNAs of pBR322 (A) and case 4 (B). and positive with DNA of case 5 ( C ) .
Acta Oto-Laryngologica 1992.112:115-119.
in 0.8 Oo/ agarose gel in TEA-NaCI buffer (50 mM Tris-HCI, pH 8.0, containing 20 mM sodium acetate, 2 mM NazEDTA and 18 mM NaCI). After gel electrophoresis, DNA was transferred to a Nytran membrane (Schleicher & Schuell, Dassel. Germany) by Southern blotting (13). After hybridization under stringent conditions in 50% formamide, at 42°C overnight, hybrids were detected by enzyme-linked immunoassay according to the manual prepared by the manufacturer. RESULTS
Histopatho1og.v of papillomas. Histological examinations of papillomas showed inverted growth in 3 cases and non-inverted growth in 2 cases (Table I). Inverted papillomas were characterized by squamous metaplasia, epithelial hyperplasia and inverting fingers of growth into underlying stroma. Non-inverted papillomas showed exophytic growth with squamous metaplasia and epithelial hyperplasia. Koilocytosis as defined by pyknotic nuclei with perinuclear halos was observed in tumors of cases 1 and 5 (Fig. I ) . Detection of HPV genome. Examination of tumor homogenates by dot blot hybridization with Vira probe HPV was positive in one (case 5) of 5 cases (Table I and Fig. 2). DNAs extracted and purified from tumor homogenates were examined by Southern blot hybridization with digoxigenin-labeled mixed DNA probe of HPV types 6b. 1 I , 16 and 18, after Bum HI digestion. Only that of case 5 was positive and showed a single band at the site of approximately 7.9 kilobases (Kb) (Fig. 3). Those of the remaining 6 samples of 4 cases showed no
Fig. 3. Detection o f HPV genome in Barn HI-digested DNAs by Southern blot hybridization with the mixture of digoxigeninlabeled DNAs of HPV types 6. 1 I , 16 and 18. A band of approximately 7.9 Kb was detected in the DNA of case 5 (lane 7). No band was detected in the DNAs of other nasal papillomatosis (6 samples from 4 cases, lanes 1-6). The position of the molecular size marker (in Kb) is on the right.
1 18 H. Ogura et al.
Acla Otolaryngol (Stockh) I12
Acta Oto-Laryngologica 1992.112:115-119.
Fig. 4. Endonuclease-cleavage patterns of HPV DNA of case 5 detected by Southern blot hybridization with digoxigenin-labeled HPV 6b DNA. Lanes: 1, digested with Bum HI; 2, digested with Hind 11; 3, digested with Ps[I; 4, non-digested; 5, digested with Eco RI; 6, digested with Hpa 1.
detectable bands. When examined with the probe DNA of each of these HPV types, the DNA from case 5 hybridized with the DNA of HPV types 6b and 11 but not with types 16 and 18 (data not shown). The DNA of case 5 was further digested with other restriction endonucleases and examined by Southern blot hybridization with the DNA of HPV type 6b (Fig. 4). A band of approximately 7.6 Kb was detected after Hind I1 digestion (Fig. 4, lane 2). Four bands of approximately 3.5, 1.6, 1.4 and 1.0 Kb were detected after Pst I digestion (Fig. 4, lane 3). Two bands (forms I and 11) were detected in the non-digested DNA of case 5 (Fig. 4, lane 4), and a band of approximately 7.9 Kb corresponding to form 111 was detected after Eco RI and Hpa I digestion (Fig. 4, lanes 5 and 6).
DISCUSSION DNA detectable by dot blot hybridization with Vira probe HPV was present in one (case 5 ) of 5 cases of nasal papillomatosis. This DNA from case 5 hybridized with those of HPV types 6b and 1 1, but not with those of types 16 and 18. When digested with Barn HI, Hind I1 and Pst I, the DNA of case 5 yielded fragments characteristic of HPV type 6 (14-16). Further, the cleavage patterns with Eco RI, Hpa I and Pst I corresponded to those of HPV type 6f ( I 7, 18). These findings suggested that the HPV detected in case 5 would be type 6f. Case 5 of the present study was a nasal non-inverted papillomatosis located on the nasal septum. Accordingly we have demonstrated that HPV is associated with non-inverted nasal papillomatosis as well. In our study, 4 out of 5 cases were HPV negative, although one (case 1) of them showed koilocytosis, two (case 1 and 3) were recurrent and two (case 1 and 2) multicentric, suggesting the infection with HPVs. The negative results in these cases might be due to the presence of HPVs unrelated to those used as probes in the present study.
ACKNOWLEDGEMENTS We are grateful to Drs H. zur Hausen and E. M. de Villiers for kindly providing us with DNAs of HPV types 6b, 16 and 18.
Acta Otolaryngol (Stockh) I12
HPV-6 in nasal papillomatosis
REFERENCES
Acta Oto-Laryngologica 1992.112:115-119.
1. Syrjanen KJ, Pyrhonen S, Syrjanen SM. Evidence suggesting human papillomavirus (HPV) etiology for
the squamous cell papilloma of the paranasal sinus. Arch Geschwulstforsch 1983; 53: 77-82. 2. de Villiers EM, Weidauer H, Le JY, Neumann C, zur Hausen H. Papillomviren in benignen und malignen Tumoren des Mundes und des oberen Respirationstraktes. Laryng Rhinol Otol 1986; 65: 177-9. 3. Brandsma JL, Steinberg BM, Abramson AL, Winkler B. Presence of human papillomavirus type 16 related sequences in verrucous carcinoma of the larynx. Cancer Res 1986; 46: 2185-8. 4. Syrjanen S, Happonen R-P. Virolainen E, Siivonen L. Syrjanen K. Detection of human papillomavirus (HPV) structural antigens and DNA types in inverted papillomas and squamous cell carcinomas of the nasal cavities and paranasal sinuses. Acta Otolaryngol (Stockh) 1987; 104: 334-41. 5. Respler DS, Jahn A, Pater A, Pater MM. Isolation and characterization of papillomavirus DNA from nasal inverting (Schneiderian) papillomas. Ann Otol Rhinol Laryngol 1987; 86: 170-3. 6. Weber RS, Shillitoe EJ, Robbins KT et al. Prevalence of human papillomavirus in inverted nasal papillomas. Arch Otolaryngol Head Neck Surg 1988; 114: 23-6. 7. Pater A, Gardner H, Respler DS, Jahn A, Pater M M . Isolation and characterization of a variant of human papillomavirus type I I from a nasal inverting (Schneiderian) papilloma. J Med Virol 1988; 25: 149-56. 8. de Villiers EM, Hirsch-Behnam A, von Knebel-Doeberitz C, Neumann C, zur Hausen H. Two newly identified human papillomavirus types (HPV 40 and 57) isolated from mucosal lesions. Virology 1989; 171: 248-53. 9. Furuta Y, Inuyama Y, Nagashima K. Detection of human papillomavirus genome in nasolaryngeal papillomas using digoxigenin labeled DNA probes. J Otolaryngol Jpn 1989; 92: 2055-63. 10. Brandwein M, Steinberg B, Thung S, Biller H, Dilorenzo T, Galli R. Human papillomavirus 6/11 and 16/18 in Schneiderian inverted papillomas. Cancer 1989; 63: 1708-13. 1 1 . Bryan RL, Bevan IS. Crocker J, Young LS. Detection of HPV 6 and 1 1 in tumours of the upper respiratory tract using the polymerase chain reaction. Clin Otolaryngol 1990; 15: 177-80. 12. zur Hausen H, Schulte-Holthausen H, Klein G et al. EBV DNA in biopsies of Burkitt tumors and anaplastic carcinomas of the nasopharynx. Nature 1970; 228: 1056-8. 13. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: 503-17. 14. Gissmann L, Wolnik L, lkenberg H, Koldovsky U, Schnurch HG, zur Hausen H. Human papillomavirus type 6 and I I DNA sequences in genital and laryngeal papillomas and in some cervical cancers. Proc Natl Acad Sci USA 1983: 80: 560-3. 15. de Villiers EM, Gissmann L, zur Hausen H. Molecular cloning of viral DNA from human genital warts. J Virol 1981; 40: 932-5. 16. Gissmann L, Diehl V, Schultz-Coulon HJ, zur Hausen H. Molecular cloning and characterization of human papillomavirus DNA derived from a laryngeal papilloma. J Virol 1982; 44: 393-400. 17. Mounts P, Shah K, Kashima H. Viral etiology ofjuvenile- and adult-onset squamous papilloma of the larynx. Proc Natl Acad Sci USA 1982; 79: 5425-9. 18. Mounts P, Kashima H. Association of human papillomavirus subtype and clinical course in respiratory papillomatosis. Laryngoscope 1984; 94: 28-33. Manuscript received April 2. 1991: accepted June 13, 1991 Address for correspondence: H. Ogura, Department of Virology Cancer Institute, Okayama University Medical School, 2-5-1 Shikatacho, 700 Okayama, Japan
1 19
