Mesenchymal cells from primary BMS (baby mouse skin) cultures formed secondary monolayers subject to density-dependent inhibition. The monolayers remained quiescent but in good condition for 6-8 weeks if given weekly medium changes. Exposure of the primary cultures to fluorescent light and/or oxygen produced "altered" cells that gave rise in the secondary cultures to foci of up to 10(4) cells after 15 days. These foci overgrew the background BMS cells. The rate of growth, morphology, and arrangement of the altered cells varied greatly between foci but much less within a focus, which usually showed one or more characters of neoplastic cells. The initiation of foci was apparently not transmissible by an infectious agent.

Long-term cell lines were readily developed from a proportion of either baby mouse skin (BMS) cultures passing through colchicine-induced crisis or altered-cell foci selected from BMS cultures exposed to light and/or oxygen followed by colchicine. The developing cell lines behaved as though they passed through a continuing, profound genetic reshuffling process, which was usually lethal but which in some cases eventually yielded a gene set that favored long-term survival. Some cell lines have passed 120 cell doublings, and none has shown a second crisis or signs of senescence. As soon after isolation as measurement was possible (19-50 days) the cell lines were predominantly or entirely tetraploid or subtetraploid. Although BMS cells and all of the cell lines were density inhibited, the BMS, C14, C21 and C23 cells overgrew (formed colonies on) monolayers of the same cells in most combinations. The cell lines retained a variety of neoplastic morphological characters, although their morphology was more normal than in the original focus. No cell line, however, showed anchorage-independent growth or formed tumors in syngeneic hosts. The cell lines may all, therefore, be regarded as preneoplastic.

Mice (C57BL) succumbed to cultured B16 melanoma cells i.p. with reproducible kinetics and an MST2 of about 26 days. Serum from tumour-bearing or normal mice was treated at 0 degree C with fixed SAC cells and injected i.p. into fresh tumour-bearing mice. If serum was given 7 days or less after B16 inoculation, the MST of the mice was highly significantly increased by up to 32%. Similar activity has been generated in normal human, rabbit and guinea-pig serum, while untreated sera were ineffective. Apparently the sera contained an inactive native precursor that was activated by the SAC to produce an anti-tumour agent. Precursor and product were both relatively labile at 0 degree C. Anti-tumour activity was eluted at pH 2.5 from SAC or Sepharose-protein-A pretreated with serum, thus implicating the protein A component of SAC. The eluates contained haemolytically active C1, the first component of complement, and five crude C1 preparations made by standard methods showed good anti-tumour activity. However, seven other highly haemolytic C1 preparations had no anti-tumour effect. Similarly, two crude preparations of the subcomponent C1q had good anti-tumour activity, but eight other, more pure and highly haemolytic C1q preparations were inactive in mice. Thus the anti-tumour principle was not C1 or C1q alone, although it had some chemical properties in common with these substances. It remains unidentified, but has potential interest for cancer therapy.

When primary baby mouse skin (BMS) cultures were subcultured for 48 hours into media containing 10(-6) to 10(-7) M colchicine or demecolcine, the number of altered cell foci appearing after 3-4 weeks' maintenance at 36 degrees C was substantially enhanced over drug-free controls. This applied whether or not the primary cultures had been irradiated with white fluorescent light. The additional presence of cytochalasin D and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) sometimes improved and sometimes partly suppressed the enhancing effect of the antitubulin drugs, and these drugs were omitted for reproducible focus enhancement. The enhancement depended on passage through DNA synthesis in presence of colchicine, which did not prevent concurrent or subsequent DNA synthesis but induced a substantial proportion (greater than 33%) to replicate in the tetraploid (4n to 8n) chromosome configuration. Another effect was to induce widespread asymmetric nuclear division, allowing the potential for chromosome loss. All these effects occurred within the first one or few cell cycles after removal of the antitubulin drugs. The results suggest that the generation of tetraploidy perhaps followed by chromosome loss may be an important factor in the rapid induction of altered cell foci. Pre-existing DNA damage is another important factor.