Uniqueness of each spontaneous transformant from a clone of BALB/c 3T3 cells

Seeding 2 X 10(4) cells from a clone of BALB/c 3T3 cells in agar led to the formation of about 100 small colonies (approximate diameter, 0.2 mm) and two large colonies (1-mm diameter). Seven of the former and both of the latter were isolated, and the morphology and growth properties of their cells were observed in repeated weekly passages. The seven subclones derived from the smaller agar colonies spread out on the dish and multiplied more slowly than the parental clone on plastic, but six of them produced more colonies in agar than the parental clone. The two subclones derived from the larger agar colonies had a fully transformed morphology, multiplied much faster than the parental clone on plastic, and produced a high percentage of large colonies in agar. Each of the subclones could be distinguished morphologically from the others and from the parental clone, and most of them could be distinguished on the basis of their colony-forming efficiency in agar. Most of the secondary subclones derived from an early passage on plastic of one of the two large agar clones, died out on the second passage after their isolation. Secondary subclones derived from the same subclone five weeks later had a wide range of fluctuating growth rates, but did not die out on passage. The two rapidly growing subclones derived from large agar colonies initiated fast-growing tumors in nude mice. None of the other subclones produced any visible growth in nude mice over a 3-month period. Large agar colonies of fast growing, morphologically transformed cells appeared once during further passage of the parental clone and two of the subclones. The results reveal a surprising degree of heritable diversity in morphology and growth characteristics of the progeny from a clonal line of nontransformed cells. They also indicate that, in this cell line, only those cells that have a high efficiency (greater than 20%) of large colony formation in agar have the capacity to form tumors in nude mice.