Phenotypic variability in anchorage-independent growth by a human breast tumor cell line

Possible mechanisms responsible for expression of anchorage independence were investigated with the use of the human breast carcinoma cell line Hs578T. This phenotype was not stable and most likely occurred via alterations in gene expression, causing secretion of growth factor (s). Colony-forming efficiency in methylcellulose was proportional to initial plating density at low passage number and increased with passage in culture. At high passages, there was less sensitivity to initial plating density, suggesting less dependence on surrounding cells for feeding effects. Clonal variants isolated in methylcellulose maintained a higher plating efficiency when kept in suspension, and removal of selective pressure resulted in the loss of the high level of expression upon subsequent challenge in suspension. In contrast to other systems, anchorage-dependent clones acquired the ability to grow in methylcellulose after only one passage in culture. This suggests that rapid variation at rates not typical of classical somatic mutation plays a role in expression of anchorage independence. Exposure to 5-azacytidine, which decreases methylation of DNA and may thereby activate gene transcription, increased the incidence of anchorage-independent growth 20 times; whereas treatment with 6-azacytidine had no effect. Unconcentrated medium conditioned by cells previously exposed to 5-azacytidine or by cells at high passage stimulated growth in suspension by as much as 10.9 times. The factor(s) present in this conditioned medium was stable to heat up to 63 degrees C for 30 minutes and was larger than 12,000-14,000 mol wt as determined by dialysis.