The frequency of immortalization of human fibroblasts and mammary epithelial cells transfected with SV40 large T-antigen.

SV40 T-antigen-expressing human cells generally have an extension of lifespan until a period called "crisis" begins. On rare occasions a clone of cells emerges from the population in crisis and gives rise to an immortalized cell line. The present study compares the frequency of immortalization of cells from two different human lineages, lung fibroblasts and mammary epithelial cells.

E6 of human papillomavirus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts.

Immortalization is the consequence of the inactivation or bypass of two mortality stage mechanisms, M1 and M2, which are controlled by several genes including Rb and p53 in human fibroblasts. Abrogation of the M1 controls can be obtained through the activity of DNA tumor virus genes such as E6 and E7 of human papillomavirus 16 (HPV16). Fibroblasts expressing both E6 (which binds p53) and E7 (which binds Rb) bypass M1 and continue replicating (exhibit an extended lifespan) until an independent mechanism, M2, is activated.

Growth of rat mammary tumor line 64-24 in liposome-supplemented defined medium. II. Effect of liposome B and prolactin on colony forming efficiency.

During studies on serum-free clonal growth of rat mammary tumor line 64-24, we observed that liposome B causes a major increase in colony-forming efficiency. This phenomenon was studied with a short-term assay based on phase contrast microscopic observation of the effects of liposome B on recently plated, serum-depleted cells. In the serum-free medium, colony forming efficiency is not determined primarily by viability (measured by dye exclusion) or cell attachment.

Growth of rat mammary tumor line 64-24 in liposome-supplemented defined medium. I. Effect of liposome B components on colony growth.

An improved serum-free medium has been developed that supports growth of rat mammary tumor line 64-24 with far less protein supplementation and with a much smaller inoculum than previously possible. An initial survey showed that MCDB 202 supported clonal growth with 1% dialyzed serum. The remaining serum was then replaced with 5 micrograms/ml insulin, 10 ng/ml epidermal growth factor (EGF), 1 micrograms/ml hydrocortisone, 50 ng/ml ovine prolactin, and 5 micrograms/ml liposome B (a mixture of soy lecithin, sphingomyelin, cholesterol, vitamin E, and vitamin E acetate in liposome form).