title: A melanoma cell line sensitive to expression of a fusion protein binding the retinoblastoma protein family pocket domain.
abstract: An established melanoma cell line (MM96L) was transfected with selectable plasmid constructs encoding either whole SV40 large T antigen, or beta-galactosidase fusions with the retinoblastoma protein (Rb)-binding region of SV40 large T antigen and a nonbinding mutant derivative of it. Both of the beta-galactosidase fusions also encoded the large T nuclear targeting signal. Transcription of inserted genes was regulated through a Zn+2-inducible metallothionein IA promoter, which provides tight but not absolute control of expression. Only the wild-type large T segment fusion was functionally active in the binding of Rb protein. Stable lines derived from primary transfectants with the expression plasmid encoding the mutant large T segment fusion showed a normal FACS scan profile, a normal growth rate, and (upon induction) high levels of nuclear staining for beta-galactosidase. However, cells transfected with the wild-type (Rb-binding) large T segment fusion grew slowly, with surviving clones assuming a predominantly tetraploid karyotype and relatively much lower levels of beta-galactosidase activity upon Zn+2 induction. The latter cells, but not those transfected with the corresponding non-Rb-binding fusion construct, also exhibited elevated cell death and apoptosis in response to the inducer Zn+2. These results implied that expression of an Rb-binding protein has deleterious effects on the melanoma cell line growth and may reflect a role for Rb of a related pocket protein in maintaining the differentiation state of these transformed cells.
authors.affiliation: Department of Pathology, Medical School, University of Queensland, Brisbane, Queensland, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Khan
authors.forename: R J
authors.initials: RJ
authors.lastname: Collins
authors.forename: I S
authors.initials: IS
authors.lastname: Dunn
mesh_terms: D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D000956:Antigens, Viral / Q000096:biosynthesis; D017209:Apoptosis; D001483:Base Sequence; D016923:Cell Death; D002999:Clone Cells; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D004790:Enzyme Induction / Q000187:drug effects; D019309:Epstein-Barr Virus Nuclear Antigens; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D008969:Molecular Sequence Data; D011123:Polyploidy; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013539:Simian virus 40 / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000494:pharmacology; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000952:Antigens, Polyomavirus Transforming; D000956:Antigens, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D001616:beta-Galactosidase; D015032:Zinc