[CANCER RESEARCH 37, 1644-1648, June 1977] SUMMARY Some of the dynamics of neoplastic transformation in vitro have been studied with the use of benzo(a)pyrene as the carcinogen in the C3H/1OT1/2 morphological transfor mation assay. Experiments that involved the dispersion of cells into new culture dishes at various times after carcino gen treatment have shown that no change in the fraction of potentially transformed cells occurs while cultures grow to form a confluent monolayer, that little or no change in the fraction of potentially transformed cells occurs for approxi mately 3 weeks after confluence is attained, and that this fraction increases rapidly some 7 weeks after BP treatment. When confluent benzo(a)pyrene-treated cultures are dis persed in new culture dishes prior to the onset of growth toward focus formation , the formation of transformed foci is suppressed at high cell densities of seeding. This phenome non is independent of the total number of divisions under gone by cells after treatment. We suggest that phenotypic expression of morphological transformation is dependent on colony interactions in the C3H/1OT1/2 system, which we do not yet understand, but which are independent of time posttreatment expressed either in cell generations or abso lute time. INTRODUCTION C3H/1OT1/2 clone 8 is a line of mouse embryo fibroblasts, isolated by Reznikoff et a!. (12), that is subject to a high degree of density-dependent inhibition of growth. Carcino genic stimuli [polycyclic aromatic hydrocarbons (ii), X-rays (14), smoke condensate (3), etc.] are followed by the ap pearance of foci of multilayered cniss-cross colonies some 6 to 8 weeks after treatment. Transplantation of cells from such foci to C3H neonatal mice results in tumor formation (3, 9, 10, 11). Spontaneous appearance offoci is infrequent (<10@@).It has been shown, however, that normal untrans formed BALB/3T3 cells give rise to tumors, when implanted in host animals preattached to a solid substrate (5, 6, 16). Similar results have also been obtained with the C3H/1OT1/ 2 line (C. Heidelberger, personal communication). This I This work was supportedin part by GrantsNCI5ORO1and CA 15010- O2ETfrom the National Cancer Institute, Grant NIEHS 5P01 ES 00597 from The National Institute of Environmental Health Sciences, and by the Massa chusetts Institute of Technology Undergraduate Research Opportunities Pro gram. Received June 21, 1976; accepted February 25, 1977. raises questions as to the nature of the C3H line, which will be discussed later. For the purpose of clarity, however, the term “transformation,― as used in this paper, will refer to the morphological alteration in cellular growth patterns, from monolayered and ordered to multilayered and criss crossed. Independent of its potential as an assay for carcinogenic potential of a test agent, the reproducibility of the system and its adaptability for defined experimental study make it a useful tool for the characterization of the process of carci nogenesis in vitro. Reznikoff et a!. (11) found that increasing initial cell den sity above 1000 cells/60-mm dish at the time of carcinogen treatment suppressed the appearance of transformed foci. This observation suggested 2 possible explanations. First, close contact between normal and potentially transformed colonies, which is maximized at high inoculurn concentra tions, might exert an inhibitory effect on the production of transformed foci. Second, cells seeded at high density re quire fewer divisions to form a confluent monolayer relative to cells seeded at low density. Thus, the observed inhibition might reflect a requirement for a particular number of cell divisions before neoplastic expression would be possible. Cells treated with carcinogens (8, 9), oncogenic viruses (2), and X-rays (7, 14) have been reported to require a minimum number of cell generations before transformation could be observed. This requirement is now understood as 2 separate requirements, 1 for initial permanent fixation of the transformed state (usually 1 cell division), without which the capacity to express transformation is rapidly lost and, the 2nd, a growth requirement hypothetically needed for phenotypic expression (8, 9). The 1st cell division may be needed to fix a potential carcinogenic event. The additional generations could provide a means by which a key factor no longer synthesized is reduced to a low concentration through dilution. For example, expression of phage resist ance mutations in bacteria may require as many as 14 generations after treatment (13). Expression of the 6-thio guanine-resistant state in human lymphoblasts also re quires some 14 generations (15). The experiments de scnibed in this paper were designed primarily to determine whether the initial density-dependent inhibition of focus formation observed by Aeznikoff et a!. was due to the inter action of potentially transformed colonies with other cob nies or whether it was the result of a large number of cell generations required for the expression of carcinogen-in duced transformation, i.e., a long phenotypic lag. CANCERRESEARCHVOL. 37 1644 Cell Density Dependence of Focus Formation in the C3H/1OT1/2 Transformation Assay1 Daniel A. Haber, David A. Fox, William S. Dynan, and William G. Thilly Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 fD. A. H. , W. G. T.J; Harvard Medical School, Boston, Massachusetts 02115 (D. A. F.J;and McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 fW. S. 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