We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into human intrahepatic biliary epithelial (IBE) cells from three different normal individuals. These IBE cell lines grow in a hormone-supplemented medium in the presence of NIH/3T3 fibroblast coculture. These cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratins 7 and 19 and gamma-glutamyl transpeptidase while being negative for the hepatocyte markers albumin and asialoglycoprotein receptor. To evaluate ion transport pathways in IBE cell lines, we utilized intracellular pH (pHi) measurements obtained using the intracellular fluorescent indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In the absence of HCO3(-)-CO2, an amiloride-sensitive Na(+)-H+ exchanger participated in the regulation of basal pHi. In the presence of HCO3(-)-CO2, a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive, Na-, Cl-, and HCO3(-)-dependent acid extrusion mechanism accounted for approximately 60% of pHi recovery from acidic pHi; this mechanism is most consistent with the presence of a Na-dependent Cl-HCO3- exchanger (Na+HCO3(-)-Cl-H+). Under basal conditions, Cl- depletion revealed a DIDS-sensitive alkalinization consistent with a Na-independent Cl(-)-HCO3- exchanger. These model systems will allow the opportunity to study the normal mechanisms of IBE function and to study the pathobiology of IBE processes in disease states.
To elucidate the role gap junctions play in the bystander transfected with the HSVtk gene failed to exert a bystander effect, we examined the cytotoxic effect of herpes simplex effect, whereas N2A transfectants coexpressing the convirus thymidine kinase (HSVtk)
We have constructed infectious retroviral vectors, derived from Moloney murine leukemia virus, that efficiently transduce the polyoma virus tumor (T) antigens individually. The parental vector we have chosen [pZIP-NeoSV(X)l] expresses a dominant selectable marker for neomycin resistance and is a shuttle vector capable of propagation in both eukaryotic and prokaryotic cells, thus facilitating its use in structure-function studies. To address the relationship between polyoma T-antigen tumorigenesis and the effects of individual T antigens on growth control and differentiation, we used these vectors to introduce and stably express large, middle-sized, or small T antigens into mouse fibroblasts and preadipocytes. All cDNAs introduced into the vector are expressed stably even in the absence of selective pressure. The stable expression of small T antigen is noted particularly because cell lines expressing small T antigen have not been readily available prior to the use of retroviral vectors. Small T antigen-induced increase in saturation density of NIH 3T3 cells can be scored on the basis of the morphology of drug-resistant colonies. Middle-sized T antigen eliminates the growth requirement of NIH 3T3 cells for epidermal growth factor in a defined medium and permits growth in platelet-poor plasma, indicating elimination of the platelet-derived growth factor requirement as well. Large T antigen suppresses mouse preadipocyte (3T3-F442A) differentiation. These vectors and these functional assays of T-antigen activity permit genetic analysis of the relationship between tumorigenesis by T antigens and the alteration of cellular growth and differentiation.The role of the individual polyoma tumor (T) antigens in tumorigenesis and in the polyoma virus replication cycle is currently the subject of intense study. Large T antigen (LTAg) is a nuclear protein with origin-specific DNA binding properties, and it is essential for viral DNA replication (1). In addition, this protein also negatively regulates early viral transcription (1) and is both necessary and sufficient to immortalize primary rodent fibroblasts (2). Middle-sized T antigen (MTAg) is sufficient to transform immortalized cell lines to form foci in monolayer cultures and colonies in soft agar and to become tumorigenic (3). MTAg immunoprecipitates contain tyrosine kinase activity (4), and recent results indicate that the tyrosine kinase activity in MTAg immunoprecipitates is pp60'csrc (5). An activity in MTAg immunoprecipitates will also phosphorylate the membrane phospholipid phosphatidylinositol (6), leading to the generation of two potent second messengers, diacylglycerol and inositol trisphosphate, which are important signals in normal cellular regulation (7) and may be important in MTAg transformation (8). Unlike LTAg and MTAg, small T antigen (STAg) has no known intrinsic or associated biochemical activity. Bastin and coworkers (9) have shown, however, that either STAg or LTAg must complement MTAg to cause tumor formation in newborn rats. In addition, Benj...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.