Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma.

We have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1,284 and 4,471 (numbering system from K. Seedorf, G. Kr?mmer, M. Dürst, S. Suhai, and W. G. R?wekamp, Virology 145:181-185, 1985), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1,137 and 1,138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene described by Seedorf et al. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene.     

Human papillomavirus type 18 DNA is integrated at a single chromosome site in cervical carcinoma cell line SW756.

SW756, a cervical carcinoma cell line, has multiple copies of human papillomavirus type 18 DNA sequences. The integration site of human papillomavirus type 18 DNA was localized by in situ hybridization to chromosome 12 at band q13. This single integration site corresponds to a heritable fragile site, which may have facilitated the integration of the viral DNA.     

Characterization of the WIDR: a human colon carcinoma cell line.

We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.     

Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.     

Calcium-dependent morphogenesis in a cell line derived from human uterine cervical carcinoma

Cells of the cervical carcinoma line C-4I replicate in culture media with 0.8–1.8 mM calcium and concommitantly form multilayered colonies with tonofilament bundles, desmosomes and an intercellular organization resembling stratified squamous epithelium. In medium with 0.02 mM calcium, the cells grow as monolayers of loosely cohesive cells, without desmosomes and with an altered tonofilament distribution. Upon return to 1.8 mM calcium, the morphological characteristics of stratified squamous epithelium are restored. The proliferative and morphogenetic response of this cervical cell line to extracellular calcium levels closely resembles the response of epidermal carcinoma cells and suggests that calcium may play a similar role in the regulation of differentiation in these two stratified squamous epithelia.     

Heterografts of human cervical cancer. Characterization of papillomavirus genomes

Three new human cervical carcinoma xenografts was established from clinical tumor specimens of patients with I-II stages of disease. Growth characteristics of models are writing. Xenografts designated CC 9, 24 and 25 contain integrated DNA HPV, complemented DNA HPV-16. DNA CC 5 contain neither DNA HPV-16, nor DNA HPV-18.     

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.     

Presence of human papillomavirus DNA sequences in cervical intraepithelial neoplasia.

Twenty two patients referred to a district colposcopy clinic because of an abnormal cervical cytology report or a suspicious cervix and found to have a cervical epithelial abnormality were studied. The techniques of cytology, histology, immunohistochemistry, and DNA-DNA hybridisation were used to detect infection by human papillomavirus. Using an indirect immunoalkaline phosphatase technique human papillomavirus antigen was found in biopsy specimens from six of the 22 patients and DNA of papillomavirus type 6 in biopsy specimens from 13 of these women, including four out of six whose histological diagnosis was cervical intraepithelial neoplasia grade 3. In eight cases where cytological, colposcopical, and histological investigations all indicated the presence of wart virus infection, papillomavirus type 6 DNA was found in seven. Papillomavirus type 6 DNA was found in more than half of the proved cases of cervical intraepithelial neoplasia. The presence of this viral DNA in women with no cervical abnormality is to be studied.     

Characterization of LHY-821, a novel moderately differentiated endometrial carcinoma cell line

Endometrial cancer is a major problem for women but only a small number of comprehensively characterized cell models are available for studies. Here, we established a new cell line derived from a Stage IIIc(1) Grade 2 endometrial adenocarcinoma. The cell line, designated LHY-821, was characterized using growth curve, karyotyping, immunohistochemical staining, immunoblotting, drug sensitivity assay, invasion assay, and xenografting in nude mice. LHY-821 has a doubling time of about 46?h and a colony-forming efficiency of approximately 71?%. These cells expresse high levels of progesterone receptor but not estrogen receptor and are sensitive to medroxyprogesterone acetate (MPA). LHY-821 also expresses pan-cytokeratin, PTEN, p53, β-catenin, IGF-1, and IGF-2. In addition, karyotype analysis revealed that LHY-821 possessed a near diploid karyotype including 6q-, 10p-, Xq-, 13q+, 17p+, and Triplo-12. LHY-821 showed highly tumorigenicity in nude mice (100?%) and weak invasiveness. Chemosensitivity tests showed that LHY-821 was sensitive to both carboplatin and paclitaxel. LHY-821 is an immortalized cell line which had survived more than 80 serial passages; it may provide a novel tool to study the molecular mechanism and potential treatment for endometrial cancer.     

Status of the human DNA papillomavirus in cervical tumors]

Cervical carcinoma is etiologically associated with the human papilloma virus (HPV), HPV 16 and HPV 18 being the most common. Viral DNA is thought to persist mostly in the episomal form in early tumor development, and in the integrated form in carcinomas. This assumption was checked with a new method that discriminated between RNAs transcribed from episomal and integrated HPV DNAs. Both forms were detected in carcinomas of Russian patients regardless of the disease stage. The data were verified by two other methods. RNA with sequences of the HPV transforming gene E7 proved to be transcribed from either DNA form. The results suggest that HPV integration is not crucial for carcinoma progression.     

The DNA binding domain of a papillomavirus E2 protein programs a chimeric nuclease to cleave integrated human papillomavirus DNA in HeLa cervical carcinoma cells

Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells.     

Characterization of a new human cell line derived from a xenografted embryonal carcinoma

Summary A human cell line has been established from a transplantable xenografted human testicular tumor, which, both in the original tumor and in the xenograft, exhibited the histological characteristics of an undifferentiated malignant teratoma (embryonal cell carcinoma). The cells in culture were undifferentiated by biochemical, morphological, and ultrastructural criteria, growing as small islands of cells that tended to form aggregates at high density. The cells showed some variation in chromosome number with 30 to 40% of the cells having a normal human karyotype. The cells expressed high levels of alkaline phosphatase, which by heat inactivation and inhibition studies was 40 to 50% placental type alkaline phosphatase. None of the cultures produced human chorionic gonadotrophin, alphafetoprotein, carcinoembryonic antigen, or fibronectin, although at high cell densities plasminogen activator could be detected at low levels. Cell surface studies showed that the cells shared antigens with the murine embryonal carcinoma cell line F9, expressedβ ₂-microglobulin at very low and variable levels, and bound the lectin peanut agglutinin. These studies suggest that this cell line has some of the characteristics described for murine embryonal carcinoma cell lines.     

Production of human papillomavirus type 16 virions in a keratinocyte cell line.

Human papillomavirus type 16 (HPV-16) is strongly associated with carcinoma of the cervix, but the complete life cycle of the virus cannot be studied because no experimental system is available in which HPV-16 progeny are produced, and there is currently no source of HPV-16 virus particles. Most cell lines that harbor HPV-16 DNA contain the viral genome as integrated or concatenated DNA in which open reading frames are disrupted or deleted, but a human cervical keratinocyte cell line has been described which maintains HPV-16 DNA in monomeric episomal form (M.A. Stanley, H.M. Brown, M.W. Appleby, and A.C. Minson, Int. J. Cancer 43:672-676, 1989). This cell line was induced to form a stratified differentiating epithelium by grafting onto nude mice. Long-term grafts displayed the histological features of a low-grade cervical dysplasia, and terminally differentiated cells contained amplified levels of HPV-16 DNA, virus capsid antigen, and virus particles. This experimental system appears to permit the completion of the HPV-16 life cycle in virus-containing keratinocytes.     

Characterization of an unusual isoenzyme of N-acetyl-beta-D-hexosaminidase from a human colonic carcinoma cell line.

A sub-line with increased metastatic ability was previously isolated from an established human colonic carcinoma cell line [Kimball & Brattain (1980) Cancer Res. 40, 1574-1579]. The separation and characterization of the isoenzymes of N-acetyl-beta-D-hexosaminidase from each cell line are reported. The parental cell line contained A and B isoenzymes. The sub-line lacked the A-isoenzyme activity and contained an atypical B isoenzyme that was thermolabile, susceptible to alkylation and of lower molecular weight.     

Establishment and characterization of human uterine cervical epidermoid carcinoma cell line HHUS containing HPV 59 DNA

The cell line designated HHUS was established from human uterine cervical keratinizing squamous cell carcinoma. The HHUS cell line was subcultivated more than 70 times within 3 years. The cultured cells, polygonal or spindle, with neoplastic and pleomorphic features, appeared epithelial in shape, with a pavement-like arrangement and grew in multi-layers without contact inhibition. The chromosome number was varied from 40 to 88, and the modal number was stable in diploid range. The cultured cells produced keratinizing squamous cell carcinomas by heterotransplantation into the subcutis of nude mice. The HHUS cells were characterized as producing large amounts of SCC, in vitro and possessing HPV-59 DNA genomes.     

Characterization of a novel cutaneous human papillomavirus genotype HPV-125

The DNA genome of a novel HPV genotype, HPV-125, isolated from a hand wart of an immuno-competent 19-year old male was fully cloned, sequenced and characterized. The full genome of HPV-125 is 7,809-bp in length with a GC content of 46.4%. By comparing the nucleotide sequence of the complete L1 gene, HPV-125 is phylogenetically placed within cutaneotrophic species 2 of Alphapapillomaviruses, and is most closely related to HPV-3 and HPV-28. HPV-125 has a typical genomic organization of Alphapapillomaviruses and contains genes coding for five early proteins, E6, E7, E1, E2 and E4 and two late capsid proteins, L1 and L2. The genome contains two non-coding regions: the first located between the L1 and E6 genes (nucleotide positions 7,137-7,809, length 673-bp) and the second between genes E2 and L2 (nucleotide positions 3,757-4,216, length 460-bp). The E6 protein of HPV-125 contains two regular zinc-binding domains at amino acid positions 29 and 102, whereas the E7 protein exhibits one such domain at position 50. HPV-125 lacks the regular pRb-binding core sequence within its E7 protein. In order to assess the tissue predilection and clinical significance of HPV-125, a quantitative type-specific real-time PCR was developed. The 95% limit-of-detection of the assay was 2.5 copies per reaction (range 1.7-5.7) and the intra- and inter-assay coefficients of variation were 0.47 and 2.00 for 100 copies per reaction, and 1.15 and 2.15 for 10 copies per reaction, respectively. Testing of a representative collection of HPV-associated mucosal and cutaneous benign and malignant neoplasms and hair follicles (a total of 601 samples) showed that HPV-125 is a relatively rare HPV genotype, with cutaneous tropism etiologically linked with sporadic cases of common warts.