Hiroyoshi Takamizawa

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.

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.

Neuron-specific enolase as a serum marker for immature teratoma and dysgerminoma

Neuron-specific enolase (NSE) was measured with an enzyme-immunoassay in sera from 54 patients with malignant (24 cases) and benign (30 cases) germ cell tumors of ovarian origin. Serum NSE contents were clearly raised above control value (greater than 10 ng/mg) in 4 of 8 patients with immature teratomas and 5 of 6 with dysgerminomas. NSE was also measured in nine cell lines of germ cell tumors. Among these cell lines, high NSE contents were detected in the cell extracts and culture supernatants from PA-1 and Tera-II lines. In immunohistochemical study, widespread positive staining for NSE was shown in dysgerminomas, whereas the immunostaining was confined to neural elements in both an immature teratoma and xenograft tumors derived from PA-1 and Tera-II lines in nude mice. These findings suggest that serum NSE measurements are of diagnostic value not only for immature teratomas but also for dysgerminomas.

Effect of methotrexate on the growth and human chorionic gonadotropin secretion of human choriocarcinoma cell lines in vitro

The effect of methotrexate (MTX) on the growth and human chorionic gonadotropin (hCG) secretion of five gestational and two nongestational human choriocarcinoma cell lines was studied in vitro. A striking heterogeneity in hCG secretion was noted among the cell lines. The growth of cells which secreted large quantities of hCG, such as HCCM-5, BeWo, and IMa, was inhibited by continuous exposure to 2 X 10(-8)M MTX. In contrast, cells which secreted little hCG, such as SCH, ENAMI-1, and GCH-1, showed no response to the growth inhibitory action of 2 X 10(-8)M MTX and the 3H-thymidine uptake was not reduced by treatment with MTX at doses of up to 10(-4)M for 48 hours. The common morphologic alterations observed in the cells which responded to MTX were an increase in the number of multinucleated giant cells, the appearance of vacuoles and granules in the cytoplasm, and enlargement of the nuclei. Increased hCG secretion was observed in accordance with the appearance of such morphologically altered cells. Part of the mechanisms of resistance to MTX appeared to involve both impairment of MTX uptake by the cells and an increase in the level of intracellular dihydrofolate reductase.

Invasion potential of human choriocarcinoma cell lines and the role of lytic enzymes

The chick chorioallantoic membrane (CAM) was used as an assay system to examine the invasive potential of human choriocarcinoma cell lines. When 5 X 10(6) cells were inoculated into the CAM at the 10th day of postfertilization, three of eight cell lines formed extensively invasive tumors within the CAM. A tendency to correlation between the tumorigenic potential of cell lines in hamster cheek pouches and their invasive potential in the CAM was noted. In addition, the invasive capacity of cell lines correlated well with the amount of collagenase but did not correlate with the amount of plasminogen activator or cathepsin B secreted by them. It is concluded that a heterogeneity of invasive potential in the CAM exists among human choriocarcinoma cell lines and the role of the collagenase secreted by them is suggested.

The immunocytochemical localization of new soluble placental tissue proteins (PP14, 16, 17, 19, 20 and PP21) in human andCynomolgus monkey placentae

SummaryApparently Placenta-specific placental tissue proteins (PP14 and PP17) and solitary tissue proteins (PP16, 19, 20 and PP21) were investigated by avidin-biotin immunoperoxidase technique in the human andcynomolgus monkey placentae, membranes, decidua and umbilical cords. In human early placentae, PP14, 16, 17, 19 and PP21 were localized mainly in the cytoplasm of villous syncytiotrophoblast. PP20 was localized in the cytoplasm of basal chorionic trophoblasts. In human term placentae, positive stainings for PP16, 19 and PP21 were observed mainly in all kinds of trophoblastic cells, while positive stainings for PP14, 17 and PP20 were weakened in the trophoblastic cells. PP20 was clearly localized in the cytoplasm of Hofbauer-like cells in the villous stroma. The membrane of villous syncytiotrophoblast showed strongly positive stainings for PP21. PP21 was also localized in the membrane of amniotic and umbilical epithelium. The umbilical epithelium was cytoplasmically positive for PP14, 16 and PP20. Clear positive stainings for PP14 and PP21 were found in the cytoplasm of fetal polymorphonuclear neutrophils. All of the placental proteins were immunocytochemically positive in the decidual large cells. In thecynomolgus monkey placentae, similar immunostaining results were obtained. The monkey could, thus, serve as a model for the investigation of the placental proteins.

Serum levels of six tumor markers in patients with benign and malignant gynecological disease

SummaryWe studied the pretreatment serum levels of 6 tumor markers in gynecological patients with and without malignant disease. The tumor markers were carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), ferritin, Schwangerschaftsprotein 1 (SP1), Schwangerschaftsprotein 3 (SP3) and cancer antigen 125 (CA125). The results were as follows: (1) Serum CA125 and TPA levels were raised in 81% and 57% of patients with ovarian serous cystadenocarcinoma: CEA and SP3, in 52% and 43% respectively of patients with ovarian mucinous cystadenocarcinoma; CA125, TPA and SP3, in 76%, 48% and 48% respectively of patients with other ovarian malignancies; and TPA and SP3, in 56% and 40% respectively of patients with endometrial carcinoma. (2) Serum levels of TPA, ferritin and CA125 were more often raised with advancing stages of malignant disease. (3) Serum TPA levels were elevated in 55% of patients with stage I endometrial carcinoma, and serum SP3 levels were elevated in 35% of patients with a stage I malignant ovarian neoplasm and in 45% of patients with endometrial carcinoma. (4) One of the 6 tumor markers showed a raised level in 84% of patients with gynecologic malignancy as against 56% in those with benign gynecologic diseases.

Human cell clones, RSa and UVr-1, differing in their capability for UV-induced virus reactivation and phenotypic mutation.

UVr-1 is a human cell clone established as a variant with increased resistance to cell killing by ultraviolet light (UV, principally 254 nm wavelength) from a UV-sensitive cell clone, RSa. Both cells have been characterized to have much the same capacity of UV-induced DNA repair synthesis in whole cells, and the parent RSa cells were recently found to be hypermutable. In the present study UVr-1 cells were characterized in comparison RSa cells with respect to UV-induced virus reactivation and phenotypic mutation. Survival levels of UV-irradiated vaccinia virus and herpes simplex virus type 1 (HSV-1) were much the same in logarithmically proliferating UVr-1 and RSa cells. Correlated with these host cell reactivation levels, the same extent of UV-induced DNA repair replication synthesis was observed in isolated nuclei of the two cell clones. Enhancement of survival levels of UV-irradiated HSV-1 was detected when proliferating RSa cells were irradiated with UV prior to the virus infection. In contrast, this enhanced virus reactivation (EVR) was not detected in similarly irradiated and infected UVr-1 cells. As for phenotypic mutation frequencies assessed by the cloning efficiency of cells with increased resistance to ouabain cell killing (OuaR), OuaR mutants were not obtained from UVr-1 cells either with or without UV irradiation. When the proliferation of cells was synchronized, both EVR and OuaR mutations were detected in RSa cells irradiated with UV at any cell cycle phase, being greatest in the later half of the G1 phase. However, there was no detectable EVR or mutation in any phase of synchronous UVr-1 cells. The hypomutability of UVr-1 cells and hypermutability of RSa cells in a G1 cell cycle phase was also found even if 4-nitroquinoline 1-oxide was used as a mutagen or mutant cells with increased resistance to 6-thioguanine cell killing were estimated.