Yoshimi Tomita

Integration and transcription of human papillomavirus type 16 and 18 sequences in cell lines derived from cervical carcinomas.

Five cell lines, SKG-I, SKG-II, SKG-IIIb, QG-U and QG-H derived from cervical carcinomas of Japanese patients, were examined for the presence of human papillomavirus (HPV) DNA and the expression of viral mRNA. The DNA of HPV type 16 was shown to be linked covalently with SKG-IIb, QG-U and QG-H cell DNA, and HPV 18 DNA with SKG-I and SKG-II cell DNA. Although different regions of the HPV genome were integrated in these cell lines, the non-coding region and an early region including the E6 and E7 open reading frames (ORFs) were conserved in all cell lines. The complete genome of HPV 16 was found in QG-H cells by digestion of the DNA with a single-cut restriction enzyme. The other early region ORFs E1, E2, E4 and E5 were interrupted by flanking host cell DNA, suggesting that the integration into host cell DNA occurs preferentially in this region. HPV-specific mRNA species were detected in all five cell lines. In the three cell lines containing the HPV 16 genome, mRNAs hybridized with the early region of the genome, covering the entire E6 and E7 ORFs and a minor part of the E1 ORF, although the amount and size of the major mRNAs varied in these cell lines. These mRNAs did not hybridize with the late region of the HPV genome containing the L1 and L2 ORFs. In SKG-II, SKG-IIIb and QG-H cells we also detected c-myc and c-Ha-ras mRNA expression at about nine times the level of that in normal cells.

Detection of human papillomavirus type 16 DNA and evidence for integration into the cell DNA in cervical dysplasia

The presence of human papillomavirus (HPV) type 16 DNA in biopsies from precancerous lesions and from early lesions of human cervical cancer, and the integration of virus DNA into host cell DNA were analysed by dot blot and Southern blot hybridizations. HPV 16 DNA was detected in 23% of mild dysplasias, 32% of moderate dysplasias, 55% of severe dysplasias and 62% of carcinomas in situ by dot blot hybridization. Digestion of the DNA with restriction enzymes PstI and BamHI followed by Southern blot analysis revealed the presence of some typical restriction fragments of HPV 16 DNA in most virus-positive samples. In addition, we detected submolar fragments which might represent virus-cell junction sequences in 86% of dysplasias, suggesting that the integration of HPV 16 DNA could occur in the precancerous stage.

Detection of human papillomavirus in head and neck tumors with DNA hybridization and immunohistochemical analysis

The presence of human papillomavirus (HPV) DNA in oral, sinus, pharynx, and larynx lesions of Japanese patients was studied by Southern blot hybridization under less stringent (25% formamide, 42 degrees C) and stringent (50% formamide, 42 degrees C) conditions. Three samples from 10 benign tumors, and 3 of 30 malignant tumors, contained HPV DNA or HPV-related sequences. The HPV DNAs harbored in three laryngeal papillomas were HPV-11, -6, and -6 or -11, respectively. The HPV DNA and viral capsid antigens were easily detected by in situ hybridization, Western blotting, and peroxidase-antiperoxidase staining. However, neither the typical restriction pattern of HPV DNA nor viral antigen was identified in the malignant tumors, suggesting that subgenomic fragments remained integrated in the host cell DNA.

Structure and Expression of an Integrated Human Papillomavirus Type 16 Genome Amplified in a Cervical Carcinoma Cell Line

A cellular sequence containing the integrated human papillomavirus type 16 genome in a cervical carcinoma cell line QG-U was cloned and analysed. The transcriptionally active viral genome disrupted at the E2 and L2 open reading frames was amplified with its flanking sequences.

Detection of human papillomavirus DNA in genital warts, cervical dysplasias and neoplasias

Biopsies from human genital lesions in Japan (108 samples), including condyloma acuminata, squamous metaplasia, dysplasia, and cervical cancer, were screened for the presence of human papillomavirus (HPV) 6-, 11-, 16-, and 18-related DNAs by spot hybridization and Southern blot hybridization under stringent conditions. By spot hybridization, HPV 6/11-related DNA was found in 92.9% (13/14) of condyloma acuminata and in 6.7% (1/15) of cervical cancer biopsies. HPV 16/18-related DNA was found in 37.7% (5/14) of cervical cancer biopsies and was exclusively associated with invasive squamous cell cancer, 66.7% (8/12), and with cervical carcinoma in situ, 50% (9/18). Some sample DNAs were further characterized by restriction enzyme digestion followed by Southern blot analysis, and we confirmed the presence of HPV-specific DNA fragments and mixed infection with both HPV 6/11- and HPV 16/18-related HPVs in one lesion.

Expression of the human papillomavirus type 6b L2 open reading frame in Escherichia coli: L2-β-galactosidase fusion proteins and their antigenic properties

Human papillomavirus (HPV) type 6b genome contains two large open reading frames (ORFs), designated L1 and L2, in a putative late region. These ORFs are expected to code for viral structural proteins. To examine antigenic properties of a L2 gene product, we constructed two plasmids which contain N-terminal (L2-N) and internal (L2-I) regions of the HPV6b L2 ORF and then each region was expressed in Escherichia coli as a fusion protein with E. coli beta-galactosidase (beta-Gal). Both L2-N/beta-Gal fusion proteins reacted with anti-beta-Gal antibody, but did not react with the antibody prepared against bovine papillomavirus type 1 (BPV1), in contrast with a high reactivity of HPV6b L1-beta-Gal fusion protein with the anti-BPV1 antibody. Antibody raised against the L2-I/beta-Gal protein in a rabbit reacted with viral antigens in the nuclei of cells in superficial epithelium of the condyloma acuminatum tissue, but did not react with the antigens in the bovine papilloma tissue. This antibody recognized a protein from condyloma acuminata which migrates to the position of mol wt 70K-76K on an electrophoresed SDS-polyacrylamide gel. These results suggested that the L2 ORF of HPV6b codes for a capsid protein which is less cross-reactive than the L1 antigen with anti-BPV1 antibody.

Suppressive Effects of Interferon on Cell Fusion by Sendai Virus

Suppressive effects of human IFN-alpha, IFN-beta and mouse-IFN on syncytium formation in human and mouse transformed cells have been studied using u.v.-irradiated Sendai virus (UV-Sendai virus). After treatment of human RSa cells with 500 units/ml human IFN-alpha for 16 h, the syncytium formation induced by UV-Sendai virus was reduced to less than 10% of controls. The suppressive effect was dependent on the amount of interferon added, and it was blocked by the addition of cycloheximide. Suppression of syncytium formation was also observed on human IFr cells, which are partially resistant to the anticellular effect of interferon but are sensitive to the antiviral effect of interferon. Human IFN-beta also had a suppressive effect on syncytium formation in human transformed cells, and human IFN-alpha, IFN-beta and mouse IFN showed species specificity in their effect on fusion. This anti-cell fusion activity was developed in IFr cells from about 5 h after addition, of IFN-alpha and when the cells were treated with IFN-alpha for 16 h, the resistant state of cell continued for over 20 h after removal of IFN-alpha.

Amplification and overexpression of the c-erbB-2 protooncogene in human gastric cancer.

SummaryThe c-erbB-2 protooncogene encodes a possible growth factor receptor. This gene has been studied as to whether it can be regarded as a prognostic indicator in human breast carcinoma. As amplification and overexpression of the gene have been reported in several adenocarcinomas, 24 specimens of human gastric cancers were examined by immunohistochemical staining (24 cases), by Southern blotting (23/24) and by Northern blotting (16/24). Amplification of the gene was detected in two moderately differentiated tubular adenocarcinomas (8.7%), and overexpression of c-erbB-2 m-RNA was detected in three moderately differentiated tubular adenocarcinomas (18.8%). By immunohistochemical staining of paraffin-embedded tissues using a polyclonal antibody to c-erbB-2 gene products, the cell membrane was stained positively in three cases of gastric cancers which overexpressed c-erbB-2 mRNA. Peritoneal metastases were found in six gastric cancers, including two moderately differentiated tubular adenocarcinomas in which amplification of c-erbB-2 occurred. These results suggest that amplification and overexpression of c-erbB-2 may be correlated with metastases in differentiated adenocarcinoma of the stomach.

Multiple effects of interferon on myogenesis in chicken myoblast cultures

Effects of chicken interferon on the differentiation of chicken skeletal muscle in vitro were examined. Continuous treatment of chicken myoblast culture with 200 IU/ml of interferon (10 IU/mg protein) resulted in significant inhibition of cell fusion and subsequent myotube formation. However, treatment of myoblast culture with 2 to 200 IU/ml of interferon increased activities of creatine kinase and myokinase in 4- or 6-day cultured muscle cells in a dose-dependent fashion. The effect of interferon on myokinase was less than on creatine kinase. Three-fold increase in creatine kinase activity induced by interferon was not accompanied by the accelerated transition of creatine kinase isozyme from BB- to MM-type. On the other hand, accumulation of acetylcholinesterase in interferon-treated cells at day 6 was suppressed to nearly half the level of control cells. Rates of actin and myosin synthesis in 4-day cultures estimated by pulse-labelling with [35S]methionine were also suppressed to 85% of control cultures. However, a proportion of 35S-labelled actin and myosin in labelled proteins associated with glycerinated cells was not changed by interferon treatment. These results indicate that partially purified interferon has multiple effects on the process of the myogenic differentiation of chicken myoblast in vitro.

Suppression of Murine Leukaemia Virus Production by Ouabain and Interferon in Mouse Cells

Ouabain markedly inhibited the growth of the mouse cell lines K3b and JLS-V9 and the production of murine leukaemia virus (MuLV) in them. The inhibition of MuLV production was abolished by exposing the cells to normal medium or by adding a high concentration (43 mM) of K+ ions to the ouabain-containing medium. MuLV production was reduced by ouabain more rapidly than host cell directed protein synthesis. After treatment of cells with ouabain (0.5 mM) for 7 h, extracellular reverse transcriptase reverse transcriptase activities were reduced by 87 to 92%. However, the intracellular level of polymerase activities remained almost unchanged (77 to 98% relative to the control). Mouse interferon inhibited the production of MuLV in K3b cells and this antiviral action was not blocked by 0.5 mM-ouabain.