Cell Line, Transformed ; Cell Transformation, Viral ; DNA, Viral ; genetics ; Epithelial Cells ; cytology ; virology ; Human papillomavirus 16 ; genetics ; Humans ; Plasmids

Animals ; Cell Transformation, Viral ; Hepatocytes ; cytology ; Humans ; Phenotype ; Simian virus 40 ; physiology

BACKGROUNDTo construct human papillomavirus type 18 (HPV18 E6E7) adeno-associated virus (AAV) for studying the role of HPV E6E7 in the development of human cancer.

METHODSHPV18 E6E7 genes were inserted into adeno-associated virus expression vector and then infected 293 cell line. The expression of HPV18 E6E7 genes were confirmed by using RT-PCR/Southern blot assay.

RESULTSThere was HPV18 E6E7 genes in the malignantly transformed cell line. The 293TL cells compared with the parent cells transformed cells grew more rapidly, lost their contact inhibition and formed more and large colonies in soft agar.

CONCLUSIONSHPV18 E6E7 AAV was successfully constructed and could induce malignant transformation. HPV18 E6E7 AAV can be use for studying the immortalization and malignant transformation of human normal epithelial cells.

Cell Line ; Cell Transformation, Neoplastic ; Cell Transformation, Viral ; DNA, Viral ; analysis ; DNA-Binding Proteins ; Dependovirus ; genetics ; Epithelial Cells ; cytology ; virology ; Fetus ; Humans ; Kidney ; cytology ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; Polymerase Chain Reaction

Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs. Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection. Colonies were isolated by puromycin selection and expanded by multiple passages. Immunohistochemistry, RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. The positive colonies were isolated and subcultured, designated immortalized precartilaginous stem cells (IPSCs), which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR. SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting, and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR. These findings suggested that IPSCs strain with SV40Tag was constructed successfully.
Cartilage/*cytology ; Cell Proliferation ; Cell Transformation, Viral ; Cells, Cultured ; Fetus ; Simian virus 40/*genetics ; Stem Cells/*cytology ; Transfection

OBJECTIVETo establish an immortalized oral epithelial cell line.

METHODSNormal human oral epithelial cells were transfected with HPV16E6/E7 open reading frames using recombinant retroviral system pLXSN. Expression of HPV16E6 and E7 protein were tested by Western blot in three kinds of cells. To define cellular biological characterization of HPV16E6/E7 transfected cells, a series analysis were performed, including protraction of growth curve, HE staining, immunocytochemical staining and scanning electron microscope observation. The tumorigenicity was assessed by colony formation and transplanting the cells into nude mice.

RESULTSHuman oral epithelial cells transfected with HPVE6/E7 has been in culture for over 18 months. The cell line was named HIO615. Western blot analysis showed HIO615 expressed HPV16 E6 and E7 protein. HIOC were positive for cytokeratin, tonofibril and desmosome as observed by scanning electron microscope. The number of large colonies of dense multilayer cells was low (0.77%). No tumor developed in nude mice injected subcutaneously with HIOEC.

CONCLUSIONA human immortalized oral epithelial cell line induced by HPV16E6 and E7 has been successfully established.

Animals ; Cell Line, Transformed ; Cell Transformation, Viral ; Humans ; Mice ; Mice, Inbred BALB C ; Mouth Mucosa ; cytology ; ultrastructure ; virology ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins

OBJECTIVETo study the effect of hepatitis C virus core protein (HCV-C) on human normal biliary epithelial cells (BEC) transformation and tumor development.

METHODSBEC cells were transfected with plasmid pcDNA HCV-C (expressing HCV-C) by lipofectamine and selected in G418. The expression of HCV-C gene and protein was determined by PCR and immunohistochemical staining, respectively. Biological effect of transfected cells was observed through cell proliferation assay, anchor independent growth, and tumor development in nude mice. The expression of HCV-C protein in the induced tumor was evaluated by immunohistochemistry.

RESULTSHCV-C was strongly expressed in BEC cells transfected with plasmid pcDNA HCV-C and the positive signal was located in cytoplasm. The HCV-C expression protein in the induced cytoplasm. Cell proliferation assay showed that the population doubling time in the pcDNA HCV-C transfected cells was much shorter than that in the pcDNA3 and non-transfected cells (14 h, 28 h, 30 h respectively). The cloning efficiencies of transfected cells with pcDNA HCV-C, pcDNA3 and non-transfected cells were 36%, 2.5% and 1.5%, respectively (P < 0.01). Tumor developed in nude mice inoculated with pcDNA HCV-C transfected cells after the inoculation. HE staining showed bile duct carcinoma character and immunohistochemistry confirmed HCV-C expression in the tumor tissue. The positive control group also showed tumor development, while no tumor mass obtained in the nude mice inoculated with pcDNA3 and non-transfected cells even 36 days after the injection.

CONCLUSIONHCV-C protein showed human normal biliary epithelial cells transformation and tumorigenic features.

Animals ; Bile Duct Neoplasms ; etiology ; Bile Ducts ; cytology ; Cell Transformation, Neoplastic ; Cell Transformation, Viral ; Cells, Cultured ; Epithelial Cells ; pathology ; Female ; Hepacivirus ; Humans ; Mice ; Mice, Nude ; Plasmids ; Transfection ; Viral Core Proteins ; physiology

The immortalization and transformation of cultured human cells has far-reaching implications for both cell and cancer biology. Human cell transformation studies will increase our understanding of the mechanisms underlying carcinogenesis and differentiation. The neoplastic process can now be studied in a model human cell culture system. The accompanying biochemical and genetic changes, once identified, will help define the relationship between malignancy and differentiation. The present studies indeed demonstrate that the neoplastic process can now be studied in a human cell model system. Primary human cells treated with various carcinogens became immortalized in culture but were not tumorigenic. Additional exposure to either retroviruses, chemical carcinogenes or X-ray irradiation to these cells induced morphological alterations associated with the acquisition of neoplastic properties. These findings demonstrate the malignant transformation of human primary cells in culture by the combined action of either a DNA transforming virus and a retrovirus or a DNA virus and a chemical or X-ray irradiation, and support an multistep process for neoplastic conversion. It has been known that normal human cells in culture are remarkably resistant to experimentally induced tumorigenicity. However, as shown above, normal human cells could now be transformed into tumorigenic cells.
*Cell Transformation, Neoplastic/genetics ; Cells, Cultured ; DNA, Viral ; Fibroblasts/pathology ; Human ; Keratinocytes/cytology/pathology ; Osteosarcoma/genetics/pathology

OBJECTIVETo study the genetic stability of an immortalized cell line transformed by Epstein-Barr virus (EBV) after long subculture process.

METHODSIn the present study, the genetic stability including chromosome diploidy, karyotypes and microsatellite DNA were evaluated with chromosome banding techniques and microsatellite DNA detection. The telomerase activity of the immortalized cell line was detected by using the telomerase assay kit.

RESULTSFrom passage 1 to 30, there were no change of the diploidy, karyotypes of chromosome and microsatellite DNA, and the telomerase activity is negative.

CONCLUSIONThis study indicates that the immortalized cell line remains stable genetically within limited passages.

Cell Transformation, Viral ; genetics ; Herpesvirus 4, Human ; genetics ; Humans ; Lymphocytes ; cytology ; metabolism ; virology ; Microsatellite Repeats ; genetics ; Polymerase Chain Reaction

Amino Acid Sequence ; Animals ; Birds ; Cell Transformation, Viral ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; virology ; Models, Biological ; Molecular Sequence Data ; Oncogene Proteins, Viral ; genetics ; physiology

OBJECTIVETo establish immortalized B-lymphoblastoid cell lines of keloid pedigree transformed with Epstein-Barr (EB) virus and conduct karyotype analysis of the cells.

METHODSImmortalized B-lymphoblastoid cell lines were established by EB virus transformation of the peripheral blood B lymphocytes from the members of keloid pedigree. Karyotype analysis was performed for the cultured cells of passages 10, 20, 30, and 35 to evaluate their genetic stability.

RESULTSAltogether 27 immortalized lymphoblastoid cell lines with stable chromosome were obtained successfully from the keloid pedigree. No chromosomal abnormalities were found in the cultured cells until passages 30 and 35, in which variation in chromosome number and structure are detected.

CONCLUSIONThe cell lines of the keloid pedigree established in this study can be useful in future studies, and genetic analysis is conducted preferably with cells of early passages.

B-Lymphocytes ; cytology ; metabolism ; virology ; Cell Line, Transformed ; Cell Lineage ; Cell Transformation, Viral ; Female ; Herpesvirus 4, Human ; physiology ; Humans ; Karyotyping ; Keloid ; genetics ; pathology ; Male