During repeated passage of BALB/3T3 cells and testing for anchorage-independent growth, a single transformed clone was isolated from agar, and five subclones were derived from it. These subclones differed from one another in morphology on a solid substratum, efficiency and size of colony formation in agar, and rate of tumor formation in nude mide. With weekly passage over a period of 6 months, the differences in morphology and growth in agar gradually decreased. The subclone which produced the fastest-growing tumors in nude mice after 4 weeks of culture produced the slowest-growing tumors after 18 weeks, and a change in the opposite direction was made by another subclone. There was no difference among the subclones in growth rate on plastic. The distribution of chromosome numbers was heterogeneous but overlapping in all the primary subclones at 16 and 24 weeks, with no statistically significant difference in the mean number of chromosomes per subclone. An extremely high degree of variation must have occurred to produce the multiple differences between the subclones, and the same type of variation could have been responsible for the subsequent changes with repeated passage. The high frequency and graded nature of the changes and the concurrent involvement of several traits suggest an epigenetic basis for the variation.
This work was supported in part by USPHS Grant CA 15744 and in part by a grant from the United States Department of Energy, Office of Environment, under Contract AT03-79EV10277.