Clonal analysis of the stepwise appearance of anchorage independence and tumorigenicity in CAK, a permanent line of mouse cells.

To provide information on the number of steps involved in preneoplastic progression in vitro, the induced and spontaneous appearances of anchorage-independent and tumorigenic variants were studied with clones of a permanent cell line of morphologically transformed, anchorage-dependent, non-tumorigenic pseudodiploid mouse cells (CAK). Tumorigenicity was assayed in the nude mouse by s.c. coinoculation of CAK cell derivatives with 2 x 10(6) human fibroblasts. Under the assay conditions, as few as 10 tumorigenic cells formed tumors. The assay permitted detection of tumorigenic variants soon after their origin and reduced the spontaneous development of new variants that result from extensive proliferation of clonal cell populations prior to testing for tumorigenicity. Anchorage-independent, nontumorigenic variants of CAK cells originated spontaneously at an estimated rate of about 10(-4)/cell/generation. Tumorigenic variants appeared spontaneously during proliferation of an anchorage-independent cell clone at an estimated rate of about 10(-7)/cell/generation but were undetectable among anchorage-dependent CAK cells. In contrast, N-methyl-N'-nitro-N-nitrosoguanidine treatment induced the appearance of tumorigenic variants in both anchorage-dependent and -independent clones with an estimated frequency of about 10(-4)/surviving clone former, which was similar to the induced frequency of ouabain-resistant variants in the same cells. Anchorage independence was expressed without tumorigenicity in new anchorage-independent variants but tumorigenic cells were always anchorage independent. We propose that CAK cells can become tumorigenic by a three-step pathway that includes changes causing morphological transformation, anchorage independence, and tumorigenicity. Our evidence is also consistent with an alternative two-step pathway where anchorage independence and tumorigenicity are acquired in a single step, since anchorage-independent, tumorigenic clones were derived from anchorage-dependent cells soon after a single mutagenic treatment.