To determine whether tumor necrosis factor-alpha (TNF alpha) plays a physiological role in normal mammary gland development, TNF alpha and TNF receptor expression were measured in epithelial cells (MEC) isolated from mammary glands of virgin, pregnant, lactating, and postlactational (day 7 of involution) rats. TNF alpha messenger RNA (mRNA) increased significantly during pregnancy, then decreased during lactation and involution. The 26-kDa transmembrane form of TNF alpha protein, undetectable in MEC from virgin rats, increased throughout pregnancy and lactation, and disappeared during involution. In contrast, p55 TNF receptor (TNFR) mRNA levels peaked in early lactation and declined thereafter, whereas p75 TNFR mRNA levels rose steadily through lactation. Using agonistic antibodies specific to either TNFR, the individual roles of each TNFR were investigated in MEC in primary culture. The p55 TNFR was found to be the sole mediator of TNF alpha-induced proliferation. Intriguingly, the two receptors were found to have opposing effects on functional differentiation (casein accumulation), with inhibition occurring through the p55 receptor and stimulation through the p75 receptor. Taken together, these results suggest that TNF alpha plays a role in the growth and development of the mammary gland. In addition, both TNF receptors are important for TNF alpha function and may mediate different effects.
In contrast to the cytotoxic or cytostatic effect of TNFalpha on many breast cancer cell lines, TNFalpha stimulates growth and morphogenesis of normal rat mammary epithelial cells (MEC). The present studies were carried out to determine whether there are intrinsic differences between normal and malignant MEC which may explain the differing responsiveness to TNFalpha. Freshly isolated rat MEC organoids from normal mammary gland or 1-methyl-1-nitrosourea-induced mammary tumors were treated with TNFalpha for 21 days. Unexpectedly, TNFalpha stimulated growth and morphogenesis of both normal and transformed MEC in primary culture, although in transformed cells its effects were delayed and the majority of the colonies were histologically abnormal, with multiple cell layers and no lumen. Since NFkappaB is a key mediator of TNFalpha action and has been implicated in carcinogenesis, the expression of the p50, p52, p65, and c-rel NFkappaB proteins in normal and transformed MEC was determined. Expression of p52 was significantly reduced in tumor cells, and p50 was absent, although its putative precursor, p105 was abundant. There were no changes in the levels of p65 or c-rel. TNFalpha induced a pronounced and sustained increase of a p50 homodimeric NFkappaB/DNA complex in both normal and transformed MEC. However, in transformed MEC, NFkappaB binding was initially undetectable but then increased in response to TNFalpha. Thus, NFkappaB expression and DNA binding activity are altered during mammary carcinogenesis. In addition, the significant increase in NFkappaB/p50 DNA-binding was temporally coincident with TNFalpha-induced growth and morphogenesis, suggesting that it may play a significant role in both normal development and carcinogenesis.
Our laboratory has shown that tumor necrosis factor-alpha (TNF alpha) can regulate normal mammary epithelial cell (MEC) growth, morphogenesis, and, under certain circumstances, functional differentiation in a manner similar to epidermal growth factor (EGF). As TNF alpha has been shown to up-regulate EGF receptor (EGFR) expression and function in other systems, the present studies were undertaken to determine whether TNF alpha action in MEC was indirect through stimulation of the EGFR. An inhibitor of EGFR tyrosine kinase activity, PD158780, failed to block proliferation induced by 40 ng/ml TNF alpha and only partially inhibited growth in response to 2 ng/ml TNF alpha. PD158780 was also unable to suppress the extensive morphological development induced by either TNF alpha concentration. In contrast, the effects of TNF alpha and PD158780 on functional differentiation (i.e. casein accumulation) were time dependent. When measured on day 7 after 48 h of treatment, casein accumulation was unaffected by either concentration of TNF alpha or by PD158780. When assessed on day 21 after 16 days of treatment, however, casein levels were decreased by 40 ng/ml TNF alpha and increased by PD158780. Significantly, this PD158780-induced increase in casein was not observed in MEC that had been treated with both PD158780 and TNF alpha. These results thus suggest that EGFR tyrosine kinase activity is not necessary for TNF alpha action in normal MEC.
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