metallothionein and Cell-Transformation--Viral

Copper (Cu) is one of the essential metals and its homeostasis is strictly regulated. Metallothionein (MT) is induced by excess Cu to mask the Cu toxicity. Although the role of MT in Cu toxicity has been explained in terms of Cu sequestration, its role under Cu-deficient conditions is not known. This study was carried out to determine the role of MT in Cu depletion by a Cu(I)-specific chelator, bathocuproine sulfonate (BCS), in cultured cells established from MT-knockout mouse and its wild type. Viability was decreased more severely in MT-null cells than in wild-type cells by BCS treatment. The expression levels of both MT isoforms were increased by BCS treatment in wild-type cells. Thus, MT was shown to be induced under Cu-deficient conditions to maintain the activities of intracellular cuproenzymes such as cytochrome c oxidase and Cu,zinc-superoxide dismutase.

Topics: Animals; Cell Culture Techniques; Cell Line, Transformed; Cell Survival; Cell Transformation, Viral; Chelating Agents; Copper; Dose-Response Relationship, Drug; Electron Transport Complex IV; Fibroblasts; Metallothionein; Mice; Mice, Knockout; Phenanthrolines; Protein Isoforms; Simian virus 40; Superoxide Dismutase; Superoxide Dismutase-1

The UROtsa cell line was isolated from a primary culture of normal human urothelium through immortalization with a construct containing the SV40 large T antigen. It proliferates in serum-containing growth medium as a cell monolayer with little evidence of uroepithelial differentiation. The working hypothesis in the present study was that this cell line could be induced to differentiate and express known features of in situ urothelium if the original serum-containing growth medium was changed to a serum-free formulation. We demonstrated that the UROtsa cells could be successfully placed into a serum-free growth medium consisting of a 1:1 mixture of Dulbeco's modified Eagle's medium and Ham's F-12 supplemented with selenium (5 ng/mL), insulin (5 microg/mL), transferrin (5 microg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). Under serum-free growth conditions, confluent UROtsa cells were shown by light microscopy to produce raised, three-dimensional structures. Routine ultrastructural examination disclosed these three-dimensional areas to consist of a stratified layer of cells that strongly resembled in situ urothelium. The cells displayed numerous desmosomal connections, complex interactions of the lateral membranes, and abundant intermediate filaments within the cytoplasm. Freeze fracture analysis demonstrated that the cells possessed tight-junction sealing strands and gap junctions. The overall morphology was most consistent with that found in the intermediate layers of in situ urothelium. The basal expression patterns of the metallothionein (MT) and heat shock proteins 27, 60, and 70 were determined in these cells, and expression was in agreement with that known to occur for in situ urothelium. The cells were also successfully tested for their ability to be stably transfected using expression vectors containing the MT-3 or MT-2A genes. The findings suggest that the UROtsa cells grown with a serum-free medium could be a valuable adjunct for studying environmental insult to the human urothelium in general and for the stress response in particular.

Topics: Cell Culture Techniques; Cell Division; Cell Line; Cell Transformation, Viral; Culture Media; Gene Expression; Heat-Shock Proteins; Humans; Kidney Tubules, Proximal; Metallothionein; Models, Biological; Protein Isoforms; RNA, Antisense; RNA, Messenger; Transfection; Ureter; Urothelium

Epidemiology shows a clear correlation between chronic infection with the hepatitis B virus (HBV) and development of hepatocellular carcinoma (HCC). The potential role of the transactivating hepatitis B virus X protein (HBx) in transformation by HBV is controversial. Here we report that HBx suppresses transformation of primary rat embryo fibroblasts (REFs). Cooperating oncogenes like c-Ha-ras and c-myc transform REF very efficiently but cotransfection with HBx suppressed transformation of REFs down to 5%. Similarly, transfection of HBx together with the cooperating oncogenes Ha-ras and SV40 LTAg or c-Ha-ras and mutant p53 reduced the number of foci to 13%. Comparable results were obtained with HBx in the context of the whole HBV. Suppression of focus formation in REF could be partly relieved by cotransfection of apoptosis inhibitors Bcl-2 or E1B. However, cotransfection of apoptosis inhibitors crmA and p35 did not influence the proapoptotic functions of HBx. Thus, HBx may specifically activate the Bcl-2 sensitive pathway leading to apoptosis. Experiments with 13 HBx linker scanning mutants revealed that the domains necessary for HBx dependent transactivation overlap with the domains needed for the apoptotic/growth arrest functions of HBx.

Topics: Animals; Apoptosis; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; Clone Cells; Embryo, Mammalian; Fibroblasts; Genes, Tumor Suppressor; Genes, Viral; Genome, Viral; Hepatitis B virus; Metallothionein; Oncogenes; Promoter Regions, Genetic; Rats; Rats, Inbred F344; Trans-Activators; Transfection; Viral Regulatory and Accessory Proteins; Viral Structural Proteins

Metallothioneins (MTs) may modulate a variety of cellular processes by regulating the activity of zinc-binding proteins. These proteins have been implicated in cell growth regulation, and their expression is abnormal in some tumors. In particular, MT-IIA is expressed 27-fold less in human colorectal tumors and tumor cell lines compared with normal tissue (Zhang et al., 1997). Here we demonstrate that MT-IIA downregulation occurs when human cells become immortal, a key event in tumorigenesis. After immortalization MT-IIA expression remains inducible but the basal activity of the MT-IIA promoter is decreased. MT-IIA downregulation at immortalization is one of the most common immortalization-related changes identified to date, suggesting that MT-IIA has a role in this process.

Topics: Cadmium; Cell Transformation, Viral; Cellular Senescence; Chloramphenicol O-Acetyltransferase; Culture Media; Down-Regulation; Gene Deletion; HeLa Cells; Humans; Metallothionein; Neoplasm Proteins; RNA, Messenger; Simian virus 40; Transfection; Up-Regulation

Primary cultured embryonic cells derived from mice with disrupted metallothionein (MT) I and II genes and from control mice were transformed with a plasmid encoding the simian virus 40 (SV40) large T antigen. The resulting MT-/- and MT+/+ cell strains showed similar cell morphology, cell cycle and no significant differences in glutathione levels or in the activities of glutathione-related enzymes and antioxidant enzymes. The MT-/- cells were more sensitive to Cd than MT+/+ cells, though no increase in the sensitivity to Zn, Cu, Hg or Ni were observed in MT-/- cells. MT+/+ cells accumulated more Cd than MT-/- cells but showed less lesion, suggesting the role of MT induced by Cd in MT+/+ cells as a scavenger of toxic Cd ion. These results suggest a dominant protective role of MT against Cd compared with other metals. SV40-transformed MT-/- cells seem to be a useful tool for the investigation of cellular function of MT.

Topics: Animals; Cadmium; Cell Transformation, Viral; Cells, Cultured; Copper; Mercury; Metallothionein; Metals; Mice; Mice, Transgenic; Nickel; Simian virus 40; Zinc

We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony-forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.

Topics: Antigens, CD; Antigens, CD34; Antigens, Polyomavirus Transforming; Antigens, Surface; Bone Marrow; Bone Marrow Cells; Cell Division; Cell Transformation, Viral; Cells, Cultured; Gene Expression Regulation; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cells; Humans; In Vitro Techniques; Interleukin-6; Metallothionein; Platelet-Derived Growth Factor; Promoter Regions, Genetic; RNA, Messenger; Stem Cell Factor; Zinc

An inducible oncogene construct has been engineered by coupling the MC29 v-myc oncogene to the sheep metallothionein promoter. Transfection of this plasmid, which also contains the neomycin resistance gene, into Rat-1 cells, has resulted in the isolation of independent clones resistant to G418. Certain of these clones were found to exhibit inducible transformation in response to ZnSO4. Transformation was graded with increasing ZnSO4 levels and was reversible when ZnSO4 was removed from the media. By analyzing v-myc mRNA levels, the inducible alterations in cellular morphology and growth were found to be associated with increased v-myc expression. The metallothionein promoter exhibited negligible constitutive expression of v-myc and none of the clones isolated exhibited spontaneous transformation. Our results show that the use of a metallothionein promoter v-myc construct facilitates the study of inducible fibroblast transformation.

Topics: Animals; Cell Transformation, Viral; Fibroblasts; Genes, myc; Metallothionein; Plasmids; Promoter Regions, Genetic; Rats; RNA, Messenger; Sulfates; Transfection; Zinc; Zinc Sulfate

Cd2+-binding proteins of peripheral blood lymphocytes and monocytes have not well been characterized so far, although they are expected to be a clue for understanding Cd2+ toxicity in those immune competent cells. We separated a family of Cd2+-binding proteins from Cd2+-exposed human peripheral blood lymphocytes by gel filtration chromatography, and characterized them by SDS-gel electrophoresis. The proteins showed electrophoretic behaviours closely similar to metallothioneins (MTs) of HeLa cells derived from human cervical carcinoma. The proteins were also found in Cd2+-exposed monocytes, and were inducible by Cd2+ in both lymphocytes and monocytes. Anti-MT serum specifically precipitated these proteins, which were thus identified as MTs. These results suggest that the two classes of the cells involved in the immune system possess a protective mechanism against Cd2+ through MTs. A variety of human lymphoid cell lines derived from both T and B cells were also found to have capacity to synthesize MTs in response to Cd2+.

Topics: Adult; Burkitt Lymphoma; Cadmium; Cell Line; Cell Transformation, Viral; Humans; Leukemia; Lymphocytes; Male; Metallothionein; Monocytes; Multiple Myeloma

Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2-negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction.

Topics: Animals; Animals, Newborn; Antigens, Polyomavirus Transforming; Cell Transformation, Viral; Cells, Cultured; Ganglia, Spinal; Genes; Genes, Viral; Metallothionein; Microscopy, Electron; Promoter Regions, Genetic; Rats; Rats, Inbred Strains; Schwann Cells; Simian virus 40

To determine the potential role of pp60c-src in polyomavirus-transformed cells, we constructed a recombinant plasmid with the mouse metallothionein-I promoter upstream of a src gene in an anti-sense orientation. We cotransfected this plasmid into middle tumor antigen-transformed FR3T3 cells with a plasmid containing the neomycin resistance gene, and G418 resistant colonies were selected. Analysis of these cells for pp60c-src expression revealed that 50 of the 200 cellular clones screened were found to have decreased levels of c-src expression when compared with the parental middle tumor antigen-transformed cells. Three independent clones which transcribed the expected 3.6-kilobase src complementary RNA and had levels of pp60c-src kinase activity comparable to that of normal FR3T3 cells were further analyzed. In the presence of Cd2+, these clones grew significantly slower in monolayer cultures than either the parental transformed cells (FR18-1) or FR18-1 cells transfected with the neomycin resistance gene alone. The morphology of these clones in the presence of Cd2+ was distinct from that of either the parental FR18-1 cells or normal FR3T3 cells. The clones expressing the complementary src RNA were found to form fewer colonies in soft agar, form fewer foci on monolayers of normal rat cells, and form tumors more slowly following injection into syngenic rats when compared with parental FR18-1 cells. The results of these studies suggest that the level of pp60c-src kinase activity affects the growth characteristics and transformation properties of polyoma virus-transformed rat cells.

Topics: Animals; Cadmium; Cell Transformation, Viral; Drug Resistance; Metallothionein; Mice; Neomycin; Oncogene Protein pp60(v-src); Polyomavirus; Promoter Regions, Genetic; Protein Kinases; Rats; Recombinant Proteins; Retroviridae Proteins; RNA; RNA, Messenger

Cultured lymphoblasts derived from infants with Menkes' disease exhibit the same increased avidity for copper as do fibroblasts and most extrahepatic tissues from these patients. The Menkes' cells preferentially take up not only copper but also, on exposure to elevated metal concentrations, the other metallothionein-binding metals, zinc and cadmium. Menkes' lymphoblasts contain larger amounts of metallothionein than normal cells following exposure to each of these metals; the amount bound to this protein quantitatively accounted for the total cellular increment in metal in Menkes' cells. Induction of metallothionein synthesis caused both normal and Menkes' cells to subsequently take up increased amounts of 67Cu. These observations suggest that an enhanced capacity of Menkes' cells to accumulate metallothionein may be responsible for their increased uptake and retention of copper.

Topics: Biological Transport; Brain Diseases, Metabolic; Cadmium; Cell Line; Cell Transformation, Viral; Cells, Cultured; Copper; Herpesvirus 4, Human; Humans; Infant; Kinetics; Lymphocytes; Menkes Kinky Hair Syndrome; Metalloproteins; Metallothionein; Reference Values; Zinc