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Human breast cancer MCF‐7 cells containing estrogen receptor are killed by transforming growth factor‐beta (TGF‐β). We isolated variants of MCF‐7 highly resistant to TGF‐β. Variants ES‐1 and ES‐4 were cloned, and the growth of ES‐1 and ES‐4 was found to be inhibited by estradiol, whereas estradiol stimulated the growth of the parental MCF‐7 cells. ES‐1 cells contained about 2‐fold higher level of estradiol receptor than MCF‐7 cells. Addition of estradiol to the culture medium for MCF‐7 and the variant changed the expression of several secreted proteins. The repertoire of secreted proteins was markedly altered in the variant. Polypeptides of molecular weight 52,000 (52 K), 65 K and 160 K were increased about 10‐ to 50‐fold in both estradiol‐treated MCF‐7 and ES‐1 cells. Polypeptide of 130 K was decreased in estradiol‐treated ES‐1 cells while this polypeptide was increased about 4‐fold in estradiol‐treated MCF‐7, as compared with untreated MCF‐7. Polypeptide of 100 K was specifically secreted in ES‐1 whether or not estradiol was present, but there appeared to be no significant amount of the 100 K protein in MCF‐7. The estradiol‐hypersensitive phenotype is discussed in relation to its aberrant expression of secreting proteins.

A mutant clone was originally isolated as a clone resistant to Na+/K+ ionophoric antibiotic monensin from mouse Balbk3T3 cells. was found to show low receptor-endocytosis activity for epidermal growth factor (ECF): binding activity for ECF in was less than one tenth of that in Balbk3T3. Anchorage-independent growth of MO-5 was compared to that of BaIbk313 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-@ efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of or BaIbk3T3 was significantly enhanced by TCF-@ while EGF did not further enhance that of or BaIbk3T3. Colonies of BaIbk3T3 formed in soft agar in the presence of TCF-@ showed low colony formation capacity in soft agar in the absence of TCF-@. Colonies of formed by TGF-@ in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-@. Pretreatment of with TGF-@ induced secretion of TCF-@-like activity from the cells, while the treatment of Balb/ c3T3 did not induce the secretion of a significant amount of TGF-@-like activity. The loss of ECF-receptor activity in the stable expression and maintenance of the "transformed" phenotype in MO-5 is discussed.

The effect of human TNF on cultured human microvascular endothelial (HME) cells was examined. Incubation with TNF alone transformed the morphology of HME cells from a cobblestone-like appearance into a disordered array of criss-crossed, elongated, spindle-shaped cells. Coadministration of epidermal growth factor (EGF) and TNF caused even more dramatic morphologic changes than TNF alone. Addition of basic fibroblast growth factor or insulin-like growth factor-I showed rather weak effects on cell morphology than EGF. Cell growth of HME cells was stimulated up to two-fold by TNF whereas addition of EGF additively enhanced the growth rate. Treatment of HME cells with 10 ng/ml EGF increased the binding of 125I-TNF, and Scatchard analysis showed increased TNF-R number by EGF treatment. Cellular response to TNF in the absence or presence of EGF was assessed by analyzing SDS-PAGE patterns of secreted proteins from HME cells. TNF enhanced the secretion of a protein of molecular weight 25,000 Da (25 kDa) which was found to be IL-6. In contrast, secretion of a polypeptide of 29 kDa was significantly increased when HME cells were treated with EGF, but not with TNF. Coadministration of TNF and EGF synergistically increased the secretion of the 29-kDa protein. This 29-kDa protein was found to be tissue inhibitor of metalloproteinases when assayed with antitissue inhibitor of metalloproteinases antibody. TNF and EGF also enhanced secretion of collagenase with Mr of approximately 55 kDa. Increased steady state levels of the inhibitor mRNA were observed when HME cells were treated with EGF, and coadministration of TNF further increased the levels. The morphologic transformation of HME cells by TNF and/or EGF is discussed in relation to their expression of the secreted proteins.