Betty Badger

Role of transforming growth factor-α-related peptides in the autocrine/paracrine control of experimental breast cancer growth in vitro by estradiol, prolactin, and progesterone

SummaryWe have recently suggested that estradiol (E2), prolactin (oPrl), and progesterone (Pg) support the growth of the hormone-responsive N-nitrosomethylurea (NMU) rat mammary tumor in soft agar through autocrine/paracrine mechanisms. To gain insight into the nature of these hormonally regulated growth factors, we tested the ability of two monoclonal antibodies (MAb-425 and 528) directed against the epidermal growth factor receptor (EGF-R) to inhibit the colony-stimulating effects of conditioned media (CM) obtained from E2, oPrl, and Pg-treated NMU rat mammary tumors. Since both MAbs are specific for human EGF-R, MCF-7 breast cancer cells grown in soft agar in the absence of serum were used as our indicator system. Both MAb-425 and 528 totally abolished the colony-stimulating effect of genuine EGF, while having no agonistic/antagonistic action when added alone. Both MAb-425 and 528 markedly inhibited the colony-stimulating effect of rat mammary tumor E2-CM in a dose-dependent fashion. MAb-425 was also found to inhibit the growth-promoting action of Pg-CM, although this effect appeared to be somewhat less consistent and pronounced than that observed with E2-CM. In contrast, the colony-stimulating effect of Prl-CM was only rarely and, usually, modestly affected by the addition of either MAb-425 or 528. Our data suggest that in the NMU mammary tumor grown in soft agar, EGF/TGFα-related peptides are produced upon exposure to E2 and possibly Pg but only rarely following Prl administration. The possible role of these growth factors as mediators of hormonal effects in our experimental system remains to be established.

Role of transforming growth factor-α (TGF-α) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: Studies using TGF-α and EGF receptor antibodies

Abstract The biological role of transforming growth factor-α (TGF-α) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-α and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E 2 )-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control). E 2 -stimulated colony growth in 5 tumors (167% of control) and the antibody blocked E 2 -stimulation in 3 of the 5. Inhibition of E 2 -stimulated growth in 3 and basal growth in 4 other tumors by the EGF receptor antibody suggests that endogenously secreted TGF-α has a role as an autocrine/paracrine growth factor in constitutive and E 2 -stimulated tumor cell proliferation in a majority of human tumors. A polyclonal antibody against TGF-α was used to study the role of TGF-α in E 2 -, prolactin(Prl)- and progesterone(Prog)-stimulated proliferation of NMU(nitrosomethylurea)-induced rat mammary tumor cells under similar culture conditions. TGF-α, E 2 , Prl and Prog stimulated colony growth equally to 176, 187, 168 and 181% of control. The antibody produced significant and similar inhibition of TGF-α and E 2 -stimulated growth (95 and 83%). In contrast, inhibition of Prl- and Prog-stimulated growth by the antibody was only 24 and 37%. The TGF-α ligand antibody did not have an agonist or antagonist effect when added alone. Thus, TGF-α seems to be a major stimulatory growth factor mediating E 2 -induced tumor cell proliferation in rat mammary tumors. It is less important in Prl- and Prog-induced tumor growth and not essential for basal growth in these tumors. We conclude that TGF-α is a biologically important autocrine/paracrine growth factor in primary human breast cancer cell proliferation and in E 2 -induced rat mammary tumor growth.

Selectivity of polyamine involvement in hormone action on normal and neoplastic target tissues of the rat.

The present experiments were designed to evaluate the polyamine involvement in hormonal actions on proliferation and receptor content of neoplastic tissue (hormone-responsive breast cancer) as well as on growth of normal endocrine target tissue (uterus) in the same animals. Administration of estradiol and perphenazine (to stimulate endogenous prolactin release) stimulated N-nitrosomethyl-urea (NMU)-induced rat mammary tumor growth following ovariectomy-induced tumor regression. Such hormonal activation of breast cancer growth was completely abolished by treatment with α-difluoromethyl-ornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, which lowered tumor content of polyamines. The growth inhibitory effect of DFMO was partially reversible by exogenous putrescine administration. In contrast, the rise in cytosolic content of progesterone receptors induced by hormonal treatment was not affected by suppression of tumor polyamine levels by DFMO. Similarly, DFMO administration failed to influence the hormone-induced increase in uterine weight in the same animals. Thus, our data suggest selectivity of polyamine involvement in hormone actions, which, in our experimental system, seems to be restricted to the endocrine control of neoplastic cell proliferation.

Role of polyamines in the growth of hormone-responsive experimental breast cancerin vivo

SummaryWe have provided evidence for a critical role of polyamines in the growth of the hormone-responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumorin vitro. The present experiments were designed to test whether polyamines are involved in the growth of this experimental tumorin vivo. To test this hypothesis, groups of rats bearing NMU-induced mammary cancers were randomly allocated to receive no treatment or escalating doses of the polyamine biosynthesis inhibitor α-difluoromethyl-ornithine (DFMO) (0.5%, 1%, 2%, 3% in drinking water). DFMO inhibited tumor growth in a dose-dependent fashion and consistently reduced tumor putrescine level. To evaluate the time dependency of this effect, additional groups of rats received either no treatment or 2% DFMO for 3, 7, 14, and 21 days. At all times DFMO suppressed tumor putrescine level as well as spermidine to spermine ratio. Finally, exogenous administration of putrescine (200 mg/kg/i.p./day × 21 days) given concomitantly with DFMO restored tumor growth, partially repleted tumor putrescine level, and raised the spermidine to spermine ratio to control levels. Putrescine, given alone, had no significant effect on either tumor polyamine levels or tumor growth. Except for modest weight loss, no major toxicity was encountered. These results indicate that polyamines play an important role in the growth of the NMU rat mammary tumorin vivo. The interaction between polyamines and hormones in supporting NMU mammary tumor growthin vivo remains to be elucidated.

Role of polyamines in the growth of hormone-responsive and - resistant human breast cancer cells in nude mice

Recent in vitro data suggest that at least some hormone-independent breast cancer cells exhibit increased polyamine biosynthesis and resistance to antipolyamine therapy. To address this issue under conditions of in vivo growth, we tested the antiproliferative effect of the polyamine synthetic inhibitor alpha-difluoromethyl-ornithine (DFMO) on hormone-dependent (MCF-7) and -independent (MDA-MB-231, BT-20) breast cancer cell lines growing in nude mice. We observed that DFMO significantly inhibited the growth of established tumors to a similar extent in all cell lines, even though tumor regression was only observed with MCF-7 cells. DFMO, while inhibiting E2-supported MCF-7 breast cancer growth, did not inhibit E2-stimulated progesterone receptor synthesis. Cellular levels of polyamines were highest in MCF-7 cells and lowest in the BT-20 cell line. Tumor content of spermidine was similarly suppressed by DFMO treatment in the 3 cell lines, while the spermine level was unaffected. Cellular putrescine levels were suppressed in MCF-7 and BT-20 cells. Administration of DFMO prior to implantation of fragments of MCF-7 or MDA-MB-231 tumors in nude mice significantly inhibited tumor development to a similar extent. The action of DFMO seemed to be predominantly tumoristatic since new tumors develop in some mice upon discontinuation of the drug. We conclude that the hormone-independent breast cancer cell lines tested do not exhibit increased polyamine biosynthesis or resistance to antipolyamine therapy when grown in vivo in nude mice.

Role of polyamines in the synthesis of prolactin-regulated growth factors by experimental breast cancer in culture

SummaryThe present experiments were designed to test whether polyamines play an essential role in the synthesis of growth factors induced by ovine prolactin (oPRL), using the N-nitrosomethylurea (NMU)-induced rat mammary tumor cultured in the soft agar clonogenic assay. Conditioned media (CM) obtained from tumors treated with oPRL and the polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) (1 mM) no longer exerted the colony-stimulating effect which was observed with oPRL-CM. Such growth-promoting activity was restored with conditioned media obtained from tumors treated with oPRL, DFMO, and increasing concentrations of spermidine from 1 to 500µM. The colony-stimulating effect of the CM employed could not be accounted for by the contaminating presence in the media of oPRL, DFMO, and polyamines. These results indicate that in our system polyamines play an important role in the synthesis of oPRL-regulated growth factors.

Kinetic and morphometric responses of heterogeneous populations of NMU-induced rat mammary tumor cells to hormone and antipolyamine therapyin vivo

The present experiments were designed to evaluatein vivo the differential sensitivity of tumor cell subpopulations to hormone and polyamine manipulations using the hormone-responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumor. NMU tumor bearing rats were randomly assigned to control, ovariectomy, α-difluoromethyl-ornithine (DFMO) administration (an inhibitor of polyamine biosynthesis), or combination treatment, and were sacrificed on day 2, 4, or 7. The proportion of different cells was estimated by morphometric analysis and their replicative activities by [3H]-thymidine autoradiography.In tumors of intact rats, the fractions of glandular, myoepithelial, and non-epithelial cells were 85.3 ± 2.2%, 4.7 ± 0.7%, and 9.9 ± 1.9%, respectively. Ovariectomy induced a similar time-dependent decline in the labelling indices of each cell type (from 5% to 1%). It also decreased the fraction of glandular cells (74.9 ± 4.5%), while increasing the fraction of myoepithelial (8.6 ± 1.9%) and non-epithelial (16.3 ± 3.2%) cells. DFMO exerted similar but more modest effects. DFMO-induced tumor regression was also inferior to that observed with ovariectomy. Combined ovariectomy and DFMO induced a faster and greater suppression of all labelling indices than the individual treatments, even though tumor regression was not superior to that produced by ovariectomy alone. Combination treatment also produced more profound morphologic changes, reducing the fraction of glandular cells to 64.4 ± 3.9% and increasing that of non-epithelial cells to 26.6 ± 4.4%. Ovariectomy and DFMO reduced height but not width of glandular cells, resulting in a modest decrease in cell volume. The combination treatment, however, significantly suppressed all three parameters.Cellular levels of polyamines were only modestly affected by the treatment used, thus raising doubts on their role as mediators, at least of ovariectomy-induced effects. Nevertheless, these results emphasize the sensitivity of different cell components of NMU tumors to combined hormone and anti-polyamine therapy with regard to kinetic and morphometric changes.

Hormonal control of polyamine pools in experimental breast cancer in vivo: correlation with estrogen and progesterone receptor levels.

SummaryThe present experiments were designed to test whether, in the hormone responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumor, polyamine levels are under hormonal control and whether they correlate with estrogen (ER) and progesterone (PgR) receptor content. We observed that tumor regression induced by ovariectomy was associated with a decline in putrescine (Pu), spermidine (Sd) and spermine (Sm). Administration of estradiol and perphenazine (to stimulate endogenous prolactin release) to castrated rats restored tumor growth and contents of Pu and Sd to control values in a time dependent fashion while Sm levels were only modestly raised. The hormonal modulation of tumor polyamine levels was particularly obvious when the treatment effects on total pools (i.e. Pu+Sd+Sm) were analyzed. No significant correlation was observed between ER and PgR and polyamines in the tumors of intact rats as well as most of the treated groups. In contrast, a highly significant correlation was observed between ER and PgR levels. We conclude that in this experimental system cellular polyamine levels are hormonally regulated but are not correlated with the ER and PgR content of the tumor.

Species-specificity of estradiol regulated growth factors in breast cancer.

Recent evidence indicates that autocrine/paracrine mechanisms may mediate the mitogenic effect of estradiol (E2) both in human and experimental breast cancer. However, the species-specificity of E2-regulated growth factors with regard to their biologic action has not been evaluated. To test this issue, we examined, in the soft agar clonogenic assay, the colony-stimulating activity in human breast cancers of conditioned media obtained from rat mammary carcinomas exposed to E2 (rat E2-CM). Of 22 primary human breast cancers plated in soft agar in the absence of serum, 18 (82%) successfully grew with a mean colony number of 62.4 +/- 9.8 (S.E.M.) (range 14-193). Rat E2-CM significantly stimulated colony formation in 10/18 (56%) human breast cancers to 155 +/- 11% (S.E.M.) of control. E2 administration (10(-9) M) in these tumors had a virtually identical overall effect (154 +/- 13% of control colony number). In the remaining eight tumors (44%), neither rat E2-CM nor E2 had, in general, a significant colony-stimulating effect. The growth-promoting action of rat E2-CM and E2 was not influenced by the hormone receptor status of the tumor. These results suggest that E2-regulated growth factors may not be species-specific, at least with regard to their colony-stimulating effects in soft agar.