L. Gissmann

A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer

DNA of a new papillomavirus type was cloned from a cervical carcinoma biopsy. Two EcoRI clones of 7.8 and 6.9 kb in length were obtained, the latter contained a 900‐bp deletion. The BamHI fragments of both clones were used to characterize the DNA. It represents a distinct type of papillomavirus as determined by its size, its cross‐hybridization with DNA of other papillomavirus types under conditions of low stringency only, the co‐linear alignment of its genome with HPV 6 and HPV 16 prototypes and its occasional occurrence as oligomeric episomes. We tentatively propose to designate it as HPV 18. DNA hybridizing with HPV 18 under stringent conditions was detected in 9/36 cervical carcinomas from Africa and Brazil, in 2/13 cervical tumors from Germany and 1/10 penile carcinomas. Benign tumors (17 cervical dysplasias, 29 genital warts), eight carcinomata in situ and 15 biopsies of normal cervical tissue were devoid of detectable HPV 18 DNA. HPV 18‐related DNA was found, however, in cells of the HeLa, KB and C4‐1 lines all derived from cervical cancer. The state of the viral DNA was investigated in four cervical cancer biopsies. The data reveal that the DNA might be integrated into the host cell genome. One tumor provided evidence for head to tail tandem repeats some of which persisted as circular episomes.

Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridization

Metaphase chromosomes of three cervical cancer cell lines (HeLa, CasKi, SiHa) were subjected to in situ hybridizations with the DNA of human papillomaviruses (HPV) types 16 and 18, respectively. Previous studies have demonstrated multiple copies of HPV 18 DNA in HeLa and of HPV 16 DNA in CasKi cells, but only 1–2 HPV 16 copies in cells of the SiHa line. The viral DNA persists in an integrated state (Schwarz et al 1985). Analysis of the integration sites revealed at least 11 chromosomal sites of HPV 16 integration in CasKi cells. SiHa cells contain integrated HPV 16 DNA in the region q21–q31 of chromosome No. 13. In HeLa cells integration of HPV 18 occurred in chromosome No. 8, band q24. Thus, no evidence was obtained for the existence of preferential chromosomal regions for HPV integration. The data indirectly support a trans-acting function of HPV-mediated cell transformation.

Partial characterization of the proteins of human papilloma viruses (HPV) 1-3.

The protein compositions of full and empty particles of human papilloma viruses 1, 2, and 3 were compared by SDS-gel electrophoresis. The protein patterns revealed a considerable variation in the relative concentrations of three major proteins (VP2, 3, and 4) in individual preparations. In some cases, heavy and light full particles prepared from the same wart showed a similar variability in the concentrations of VP2, 3, and 4. The different protein patterns were interpreted as resulting from conversion of VP2 into VP3 and 4. The molecular relationship of these three proteins was confirmed by BrCN cleavage which led to corresponding oligopeptides. Electron micrographs of empty particles revealed that the only detectable protein components VP3 and 4 are present in typical capsomeres.

Occurrence of HPV genomes in penile smears of healthy men.

SummaryPenile smears of 530 males without any clinically detectable genital papillomavirus infection were subjected to molecular in situ hybridization on filters with different32P-labeled HPV-DNAs. HPV genomes were identified in 31 cases (5.8%). Eight smears reacted with HPV 6/HPV 11 DNA and 5 specimens with HPV 16 and HPV 18 DNA exclusively; in 18 cases HPV 6/HPV 11 and HPV 16/HPV 18 were found in coexistence. With regard to the different age groups, HPV-DNA was found in only 1.7% of smears obtained from persons over 35 years of age, while 7.9% of the samples from men aged 15 to 35 gave positive results. This age distribution indicates that the occurrence of HPV genomes in epithelial cells of the glans may be due to infections by sexual contact rather than to reactivation of persisting viruses.

NACHWEIS UND ORGANISATIONSSTRUKTUR DER DNS MENSCHLICHER PAPILLOMVIREN BEIM KEHLKOPF- UND HYPOPHARYNXKARZINOM

Thirty biopsy specimens from various histological types of human carcinomas of the larynx and hypopharynx were analysed for the presence of human papillomavirus (HPV) DNA: DNA from the individual specimens were tested for the presence of homologous sequences to HPV genotypes 1, 2, 4, 8, 9, 10, 11, 13, 16 and 18. One squamous cell carcinoma of the hypopharynx (postcricoideal area) contained multiple copies of DNA hybridizing under stringent conditions with HPV 16 DNA. The latter DNA has been found to be frequently associated with human genital cancer. HPV 16 DNA was found mostly episomally as oligomeric circles of 7.9 kbp size, and as larger rear-ranged circular molecules. Integration of the viral DNA in the host cell DNA seems quite likely. Integration and rearrangement of viral DNA into cellular DNA may play a role in the induction and maintenance of the transformed state. The presence of sequences reacting under semistringent conditions with HPV DNA was observed in two additional biopsy specimens of this study. This could suggest that additional laryngeal cancers are associated with papilloma virus infections.

Association of HPV with human genital tumors

Human papillomaviruses (HPV) are clearly responsible for the induction of genital lesions like condylomata acuminata, bowenoid papules, and flat condylomas. Moreover, the DNA of particular virus types (HPV 16 and 18) is found in a substantial number of invasively growing squamous cell carcinomas of the genital tract, suggesting an etiologic involvement of these viruses in tumor development. Since HPV 16 and 18 as well as other papillomaviruses (HPV 6 or 11) usually present within the benign genital warts can be found in dysplastic lesions of the uterine cervix known as putative precancerous lesions, determination of the virus type might be of diagnostic relevance. Since no type-specific serologic reagents are available, viruses can be identified by nucleic acid hybridization using radioactively labeled HPV DNAs that have been molecularly cloned as probes.

Analysis of the biological role of human papilloma virus (HPV)-encoded transcripts in cervical carcinoma cells by antisense RNA.

In cervical carcinomas and cell lines derived from them, human papilloma virus type 16 or 18 DNA has been found integrated into the host cell genome [6]. The chromosomal localization differs in different cell lines, as has been shown by in situ hybridization techniques (Mincheva et al., submitted for publication). The circular viral DNA is always disrupted in the E1/E2 open reading frame (ORF) by the integration event, and parts of the late viral genes can be deleted [6]. In contrast, the noncoding region containing promoter and enhancer sequences and the ORFs E6 and E7 are preserved. Northern blot analysis revealed that in all cell lines tested so far these ORF E6 and E7 are consistently transcribed into mRNA. Sequence analysis of cDNA clones derived from the three HPV 18-positive cell lines HeLa, SW 756, and C4-I revealed that the mRNAs consist of a 5′ viral sequence and a 3′ cellular sequence encoded by the flanking host cell DNA. Only the viral sequences give rise to major ORFs which may code for three putative proteins: E6, E7, and a spliced form of E6 (E6*) [5]. Therefore, it was speculated that these proteins are required for the characteristic growth pattern of these cervical carcinoma cells.

Human Papillomavirus Early Gene Products and Maintenance of the Transformed State of Cervical Cancer Cells in Vitro

There are several lines of evidence from epidemiological as well as from laboratory experimental work linking human papillomaviruses (HPV) to the development of squamous cell cancer of the uterine cervix: n n1. n nThe association of a sexually transmissible infectious agent is strongly suggested by epidemiological observations (for review, see Doll 1986) n n n n n2. n nCervical intraepithelial neoplasias (CIN), considered as putative precursor lesions of cervical cancer, are regularly associated with HPVs. Recently, the development of this kind of lesion was induced by infection of normal human epithelium in vitro and subsequent grafting into nude mice (Kreider et al. 1985). From such lesions papillomavirus particles can be purified which are efficient in a second round of infection. Thus the Koch’s postulate for a causal relationship of the infectious agent and the disease have been fulfilled. n n n n n3. n nHPVs exhibit a transforming potential in vitro. Primary or established rodent cells can be transformed by transfection with the HPV early gene E7 (Bedell et al. 1987; Kanda et al. 1988; L. Laimins, personal communication), and a synergistic effect was observed with the activated human ras oncogene (Matlashewski et al. 1987). In addition, human keratinocytes, which represent the natural target cells for papillomavirus infection, are immortalized by HPV-16 or HPV-18 DNA, respectively (Drst et al. 1987; Pirisi et al. 1987; P. Kaur and J. McDougall, personal communication).

Analysis of Human Genital Warts (Condylomata Acuminata) and Other Genital Tumors for Human Papillomavirus Type 6 DNA

32P-labelled cloned HPV 6 DNA was used as probe to analyze human genital tumors for DNA sequences homologous to HPV 6 DNA. Ninety three percent of all condylomata acuminata (41 out of 44) were found to harbor HPV 6 DNA. Of the remaining three, one contained HPV 1 DNA. No papillomavirus DNA was identified in the two other tumors. All three invasively growing giant condylomata acuminata (Buschke-Löwenstein tumors) investigated also contained HPV 6 DNA. Two out of six atypical condylomata of the cervix hybridized with HPV 6 DNA under stringent conditions, one only under conditions of low stringency. All DNA preparations from malignant tumors studies (54 cervical carcinomas, 10 penile carcinomas, two vulvar carcinomas) failed to anneal with HPV 6 DNA, even under conditions of low stringency. Although all HPV 6-positive condylomata acuminata analyzed in this study revealed HPV 6 DNA of regular molecular weight (5.1 x 10(6)), two of the Buschke-Löwenstein tumors, as well as one of the two positive atypical condylomata of the cervix, contained HPV 6 DNA with a remarkable size classes occurred in a supercoiled form without evidence for integration into host cell DNA.

B-Lymphotropic Papovavirus (LPV)—Infections of Man?

A possibly new subgroup of papovaviruses has been isolated which appears to be characterized by its highly restricted host range which does not seem to extend beyond proliferating lymphoblasts. A virus revealing this host range derived from transformed African green monkey (AGM) lymphoblasts in described.