ABSTRACT
We report the partial purification of growth inhibitors extracted from human and mouse multicellular tumor spheroids with extensive necrosis. Sephadex G-100 column chromatography of spheroid extracts separated inhibitory fractions which eluted just after the void volume of the column. Identical chromatography of monolayer cell extracts showed no inhibitory activity. High-performance liquid chromatography of spheroid extracts separated single active peaks at apparent molecular weights of 80-89 kD. These extracts were extremely heat labile, and activity was destroyed by moderate trypsin treatment. The isolation of similar growth inhibitors from spheroids of two cell lines suggests that inhibition is important in tumor cell growth control in a three-dimensional situation.
Subject(s)
Growth Inhibitors/isolation & purification , Neoplasm Proteins/isolation & purification , Adenocarcinoma/pathology , Animals , Cell Division/drug effects , Cell Line , Female , Growth Inhibitors/pharmacology , Humans , Mammary Neoplasms, Experimental/pathology , Mice , Neoplasm Proteins/pharmacologyABSTRACT
The RNA of an established line of Chinese hamster cells growing in cell culture contains a small number of uridines substituted at the 3-position with a gamma-linked alpha-aminobutyrate residue. Structure has been ascertained by: (a) examination of incorporation of isotopic labels from precursors; (b) degradation with anhydrous hydrazine and comparison of the products with synthetic material or with hydrazinolysis products of known uridines; and (c) comparison of the unknowns as their hydantoin derivatives with the 5-beta-(bromoethyl)hydantoin alkylation products of uridine and of 1- and 3-methylpseudouridine. In this manner it is shown that 18-S RNA of ribosomes contains a single residue of a nucleoside which we tentatively identify as 1-methyl-3-gamma-(alpha-amino-alpha-carboxypropyl)pseudouridine per molecule. RNA isolated from the supernatant fraction, sedimenting at 4 S and co-electrophoresing with transfer RNA on polyacrylamide gels, contains several similar bases, one of which is identified as 3-gamma-(alpha-amino-alpha-carboxypropyl)uridine. Each of the above nucleosides derives its alpha-aminobutyrate residue from methionine.
Subject(s)
Aminobutyrates/chemistry , Methionine/chemistry , Pseudouridine/analogs & derivatives , RNA/chemistry , Uridine/isolation & purification , Animals , CHO Cells , Centrifugation, Density Gradient , Chromatography, Paper , Cricetinae , Cricetulus , Hydralazine , Hydrogen-Ion Concentration , Models, Chemical , Pseudouridine/chemistry , Spectrophotometry , Tritium , Uridine/analogs & derivativesSubject(s)
Cells, Cultured/analysis , RNA, Transfer , RNA , Subcellular Fractions , Adenine Nucleotides , Adenosine Triphosphate , Amino Acyl-tRNA Synthetases , Animals , Benzoates , Carbon Isotopes , Cells, Cultured/cytology , Cells, Cultured/metabolism , Centrifugation, Density Gradient , Chromatography, DEAE-Cellulose , Chromatography, Ion Exchange , Cricetinae , Cytoplasm , Cytosine Nucleotides , Escherichia coli/enzymology , Methionine/metabolism , Methylation , RNA/metabolism , RNA Nucleotidyltransferases/isolation & purification , TritiumSubject(s)
RNA , Adsorption , Aluminum Silicates , Animals , Carbon Isotopes , Cell Nucleolus , Cell Nucleus , Centrifugation, Zonal , Chemical Phenomena , Chemistry , Cricetinae , Culture Techniques , Cytoplasm , Electrophoresis , Female , Gels , Methionine , Ovary , RNA, TransferSubject(s)
Nucleosides/isolation & purification , RNA/analysis , Animals , Cell Line , Cricetinae , Culture Techniques , Female , Guanine , Ovary , Spectrum AnalysisSubject(s)
Ovary/cytology , RNA , Animals , Cell Nucleus , Centrifugation, Zonal , Chromatography, Ion Exchange , Cricetinae , Culture Techniques , Electrophoresis, Disc , Female , Hydrogen-Ion Concentration , Methylation , Molecular Weight , Nucleotides , RNA/classification , RNA/isolation & purification , RNA, Transfer , Ribosomes , TritiumSubject(s)
Methionine/metabolism , RNA/metabolism , Ribosomes , Animals , Carbon Isotopes , Centrifugation, Zonal , Chromatography, Paper , Cricetinae , Culture Techniques , RNA/analysis , TritiumABSTRACT
The incorporation of methionine-methyl-(14)C into 18S ribosomal RNA of cultured Chinese hamster ovary cells in early and late interphase has been determined by zone-sedimentation analysis of phenol-extracted RNA preparations. Synchronized cell cultures were prepared for these studies by thymidine treatment and by mechanical selection of mitotic cells. The specific activity of 18S RNA labeled in late interphase was found to be 1.1-1.2 times that of 18S RNA labeled in early interphase. Upon correction for increase in RNA mass, the rate of methylation of 18S RNA in late interphase is about 1.9 times that in early interphase.
