hygromycin-a and Cell-Transformation--Viral

To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.. We successfully isolated primary human hepatocytes from surgically resected liver tissue taken from a patient with liver hemangiomas. The freshly isolated cells were then immortalized with retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40T) and hygromycin-resistance genes flanked by paired loxP recombination targets.. The freshly isolated hepatocytes with high viability (85%) were successfully immortalized using retroviral gene transfer of SV40T. SV40T in the immortalized cells was then excised by Cre/loxP site-specific recombination. This cell population exhibited the characteristics of differentiated hepatocytes.. We successfully established reversibly immortalized human hepatocytes, which will provide an unlimited supply of cells for practical applications.

Topics: Anti-Bacterial Agents; Antigens, Polyomavirus Transforming; Cell Differentiation; Cell Line; Cell Proliferation; Cell Separation; Cell Survival; Cell Transformation, Viral; Cinnamates; Drug Resistance, Bacterial; Genetic Vectors; Hepatocytes; Humans; Hygromycin B; Integrases; Recombination, Genetic; Retroviridae; Serum Albumin; Serum Albumin, Human; Time Factors; Transduction, Genetic

Herpesvirus saimiri is a lymphotropic herpesvirus capable of immortalizing and transforming T cells both in vitro and in vivo. Immortalized and transformed T cells harbor several copies of the viral genome as a persisting genome. The mapping of the cis-acting genetic cis-acting segment (oriP) required for viral episomal maintenance is reported here. Viral DNA fragments that potentially contain oriP were cloned into a plasmid that contains the hygromycin resistance gene. After several round of subcloning followed by transfection, oriP was mapped to a 1.955-kb viral segment. This viral fragment permits stable plasmid replication without deletion or rearrangement as well as episomal maintenance without integration or recombination. The function of oriP depends on a trans-acting factor(s) encoded by the viral genome. The 1.955-kb viral segment includes a dyad symmetry region located between two small nuclear RNA genes and is located upstream of the dihydrofolate reductase gene homolog. Therefore, this oriP contains novel elements distinct from those of other DNA viruses.

Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Viral; Cinnamates; Cloning, Molecular; DNA Replication; DNA, Viral; Drug Resistance; Herpesvirus 2, Saimiriine; Humans; Hygromycin B; Molecular Sequence Data; Plasmids; Rabbits; Replication Origin; T-Lymphocytes; Trans-Activators; Transfection; Virus Replication

The dependence on human papillomavirus (HPV) oncoproteins of the growth of cervical cancer cell lines [C4-1, HeLa (both containing HPV 18 DNA), CaSki and SiHa (both containing HPV 16 DNA)], HPV 16-transformed human embryonic kidney cells, and HPV 16-transformed rat brain and 3Y1 cells was examined by using antisense RNA approaches. The cells were transfected with plasmids expressing RNA antisense to the HPV 16 or 18 open reading frames E6E7, together with plasmids expressing the hygromycin B resistance gene, and drug-resistant colonies were scored three weeks later. In all the human cell lines, the efficiency of colony formation was lowered by RNA antisense to the resident HPV type. Some of the rat cell lines responded to the antisense plasmids, but some did not. From a nonresponding rat tumor line (3Y1HP-1T), cell clones with various levels of E7 protein were isolated after transfection with the antisense plasmid, and were examined for anchorage-independent growth in soft agar. The colonies formed by the clones with lower E7 levels tended to be smaller and fewer than those formed by the clones with higher E7 levels. These findings strongly suggest that some of the transformed or cancer phenotypes of cells in vitro are dependent, even after extensive passages and malignant changes, on expression of the oncoproteins of the resident HPV.

Topics: Animals; Blotting, Western; Cell Division; Cell Transformation, Viral; Cinnamates; DNA, Viral; Drug Resistance; Female; Humans; Hygromycin B; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Rats; RNA, Antisense; Transfection; Tumor Cells, Cultured; Uterine Cervical Neoplasms