Culture conditions for a human lymphoma cell line (Raji) and their application to mutation research

Experimental systems for the detection of somatic cell mutation to hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency in human lymphoma cells were developed. The cell line used was the Raji line established from Burkitt's lymphoma. The cloning efficiency (CE) in soft agar of the cells was enhanced approximately 5-fold by addition of 2 x 10(-4) to 4 x 10(-4)M dithiothreitol (DDT) and 1% human serum, as compared with the CE in control medium. Other thiols such a mercaptoethanol and thioglycerin were also able to increase the CE but not as efficiently as DTT. Under these culture conditions, 10(6) cells per 55-mm dish could be seeded into soft agar medium containing 5 micrograms/ml 6-thioguanine (6-TG) to detect a forward mutation without any metabolic cooperation. The optimum expression time was determined by treating the cells with 125 micrograms/ml ethyl methanesulfonate (EMS) for 20 hr at 37 degrees. A significant increase of mutation frequency was observed approximately 10 days after the 6-TG treatment, by which time the cells had gone through about 10 population doublings. After determining these experimental conditions, a comparison of mutation frequency among three mutagens, EMS, N-methyl-N'-nitro-N-nitrosoguanidine and ICR-170, was carried out. ICr-170 was the most efficient mutagen among these chemicals.