Cloning the Fisher rat thyroid cell line (FRTL-5): variability in clonal growth and 3,'5'-cyclic adenosine...

To investigate the stability of FRTL-5 cells we cloned stock cells by limiting dilution in a 6-hormone medium with bovine TSH (bTSH) (10 mU/ml) (6H medium). One third of the wells with irradiated fibroblast feeder cells developed FRTL-5 colonies. One hundred and twenty clones were obtained, of which 29 (8%) developed sufficiently for functional analysis. Clones were tested for growth responses to bTSH stimulation as evidenced by 72-h [3H]thymidine uptake and TSH receptor activation by cAMP generation. Clonal growth responses to bTSH were of three types: dose-related growth increase, absence of growth stimulation, and stimulation by bTSH in concentrations up to 100 microU/ml and inhibition of growth above 100 microU/ml. All clones tested showed evidence of extracellular cAMP accumulation in response to bTSH. However, sensitivity to bTSH varied from 1 to 100 microU/ml and maximum cAMP secretion with 1 mU/ml bTSH varied from 2 to 13 pmol/ml. Whereas certain clones showed high sensitivity to bTSH with respect to both growth and cAMP responses (e.g. 1B-6), there were clones which showed disparity in this relationship, as evidenced by poor growth dependency but high cAMP responses or by growth stimulation yet insensitivity with respect to cAMP secretion. These data demonstrate that the FRTL-5 line contains cells with variable responsiveness to bTSH. Whereas the FRTL-5 line is heterogeneous and subject to developmental variation, cloning by limiting dilution allows the derivation of highly bTSH-sensitive cells, such as 1B-6, from the stock cultures.