ABSTRACT
The polypeptide profiles of bovine vascular endothelial cells (from pulmonary artery and descending aorta), smooth muscle cells (from pulmonary artery) and fibroblast cells (from skin and lung) were examined by high-resolution two-dimensional polyacrylamide gel electrophoretic techniques. A set of polypeptides (molecular weights between 43 X 10(3) and 47 X 10(3) and pI values from 6.0-4.8, respectively) exhibited patterns that were unique to the three cell types. In the case of smooth muscle cells, these polypeptides exhibited cell-density-dependent expression. These results allow for identification of the three cell types on the basis of their highly specific polypeptide signatures.
Subject(s)
Fibroblasts/analysis , Muscle, Smooth, Vascular/analysis , Peptides/analysis , Animals , Arteries/analysis , Cattle , Cell Count , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Endothelium/analysis , Isoelectric Focusing , Lung/analysis , Pulmonary Artery/analysis , Skin/analysisABSTRACT
We have used high resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) to compare the polypeptides synthesized by normal human diploid fibroblasts cells which have significant proliferative capacity with those of cells which are terminally non-dividing. Normal cells that are terminally non-dividing (i.e., have no remaining proliferative potential) synthesize two polypeptides that are not detected in young cells which are either actively proliferating or growth arrested by incubation in medium containing a low concentration of serum (0.3%) for 72 h. Continued maintenance of young cells in the growth-arrested state ultimately leads to the detectable synthesis of one of the polypeptides synthesized by terminally non-dividing cells. Some preliminary biochemical properties of these two polypeptides are examined.
Subject(s)
Peptides/analysis , Cell Cycle , Cell Line , Clone Cells , Diploidy , Electrophoresis, Polyacrylamide Gel , Humans , Leucine/metabolism , Lung/cytology , Sulfur Radioisotopes , TritiumABSTRACT
We have isolated a diploid fibroblast culture from human fetal lung with an in vitro lifespan of about 100 population doublings. The culture grows very well at clonal densities and long-lived clones can be isolated for use in cellular aging studies. The longer in vitro lifespan of the culture has allowed us to isolate from it a clone, containing a dominant and recessive mutation, having significant remaining proliferative potential. The nature of the mutations will allow for hybrid selection, after fusion of the mutant clone with wild type human cells. The mass culture and clones derived from it provide a valuable resource for cell aging studies.
Subject(s)
Diploidy , Hybridization, Genetic , Lung/cytology , Aminopterin/pharmacology , Cell Division/drug effects , Cell Line , Cell Survival/drug effects , Clone Cells , Female , Fetus , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , Hypoxanthines/metabolism , Karyometry , Mutation , Ouabain/pharmacology , PregnancyABSTRACT
The percentage of cells capable of forming colonies of at least a given size has been found to provide a reliable estimate of the remaining doubling potential of several human diploid fibroblast cell lines and for chick embryo fibroblasts. In five independently derived cell lines of human diploid fibroblasts we have found the same linear relationship between population doublings remaining and the percentage of cells able to form colonies of at least 16 cells. For chick embryo fibroblasts there is a linear relationship between the percentage of cells forming colonies of 64 or more cells and remaining proliferative potential. When human diploid fibroblasts were grown in medium containing hydrocortisone at 5 microgram/ml the increased in vitro lifespan was reflected in the colony size distribution long before the end of the in vitro lifespan.
Subject(s)
Cell Survival , Mitosis , Cell Count , Cell Line , Cell Survival/drug effects , Cells, Cultured , Colony-Forming Units Assay , Fibroblasts/cytology , Humans , Hydrocortisone/pharmacology , Lung/cytology , Mitosis/drug effectsABSTRACT
In the past, it has been difficult to grow human diploid fibroblast cells at clonal densities. Newly devised cell culture media and rigorously controlled environmental conditions have greatly increased the ease with which such cells can be cloned. The present work was undertaken to determine whether, under appropriate conditions, diploid fibroblast cells from human embryonic lung, grow as well at clonal densities as in mass culture. The parameters studied were: (1) population doubling time, (2) in vitro proliferative capacity, (3) attachment, (4) percentage of non-dividing cells. In all cases essentially the same results were obtained for cultures at clonal densities and mass cultures. These results indicate that the behavior of these types of cells in clonal culture can be used to infer the behavior of individual cells and clones within a mass culture.
