Angiogenic factor of a rat mammary tumor cell line (RMT-1) (I). Secretion of two distinct angiogenic factors into serum-free conditioned medium by RMT-1 cells.

Serum-free conditioned medium of a rat mammary tumor cell line RMT-1, established from a rat mammary carcinoma induced by 7,12-dimethylbenz[a]anthracene (DMBA), produced the complete angiogenic response in both rabbit cornea and chick embryo chorioallantoic membrane assays. The angiogenic activity in the RMT-1 conditioned medium was separated into two fractions on a column of heparin-Sepharose; one was eluted with 0.1 M NaCl and the other with 0.5 M NaCl, which are referred to hereafter as rAF-1 and rAF-2, respectively. These two angiogenic factors were further purified separately by FPLC on a Superose 12 column. The partially purified rAF-2 had an apparent Mr of 30,000-50,000 and seemed to exhibit mitogenic activity toward Balb/c 3T3 cells, while the partially purified rAF-1, with an apparent Mr of 10,000-30,000 did not have a mitogenic effect on these cells. Both rAF-1 and rAF-2 were resistant to heat and acid treatment, and exhibited trypsin sensitivity, suggesting that they are heat and acid stable peptides. The two angiogenic factors did not stimulate the proliferation of cultured vascular endothelial cells. These results suggest that RMT-1 secretes two distinct angiogenic factors into the medium and that these two secretable angiogenic factors participate cooperatively in the induction of the angiogenic response produced by a DMBA-induced rat mammary tumor in vivo.