Clonal variation in growth factor production by human carcinoma cells

Recent research has indicated that growth factors play an important role in normal and malignant cell growth. Previous work in this laboratory established that RPMI-2650, a cell line derived from a human carcinoma, produces growth factors including TGF-a, TGF- 3 and an autocrine factor. The purpose of this thesis was to investigate whether growth factor production is a property of all cells, or of a sub-population of cells. If high-producer clones were available it would greatly facilitate production of material for purification and characterisation. In order to answer this question it was necessary to isolate clones of RPMI-2650. A number of experimental variables were examined, with the objective of improving cloning efficiency. Initially, basic parameters related to the environment of the cells were examined : The use of a basal medium consisting of DMEM:HamsF12 (1:1) gave a higher cloning efficiency then MEM or DME. Choice of a suitable batch of serum was very important, and serum concentration was important in determining the size of the colonies. It was also found that trypsinizing cells at 4°C gave better cloning efficiencies than trypsinization at 37°C. On the other hand, use of gas atmosphere with reduced oxygen tension caused a decrease in cloning efficiency. Feeder cells proved to be of considerable significance. The use of mitomycin C-treated feeder cells consistently improved the cloning efficiency of RPMI-2650 cells; Mouse 3T3 or human RPMI-2650 cells were both effective as feeders. Use of RPMI-2650 conditioned medium and pre-coating of culture dishes with DEAE dextran gave a marginal improvement in cloning efficiency. Transfer of cells from the initial clone to a larger growth area proved difficult. This problem was largely overcome by a procedure involving transfer of cells to a small volume of medium (in 96-well microplates) following trypsinization using a cloning ring. Using this improved procedure, twenty independent clones of RPMI-2650 were isolated and frozen in liquid nitrogen. Many of the clones appeared morphologically distinct from one another. Fourteen of the clones were examined for cloning efficiency in monolayer and agar, and for production of TGF-a, TGF-3 and autocrine factors. Considerable variation between clones was observed in all of these properties. All clones examined, however, produced significant amounts of growth factors. It was concluded that growth factor production is not a property confined to a small sub-population of RPMI-2650 cells in culture. The techniques developed in this thesis should facilitate future work in preparing clonal populations of human cancer cells. The finding that TGF-a and TGF-S production is a general property of the carcinoma cells studied, rather than a property of a sub-population, suggests that production of these growth factors may be related in some way to the process of carcinogenesis