Comparison of ovariectomy and retinyl acetate on the growth of established 7,12-dimethylbenz[a]anthracene-induced mammary tumors in the rat.

Prolonged exposure to retinyl acetate (RA) in the diet inhibits the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancers in rats. The effectiveness of RA was examined when given 6 months after the administration of DMBA. Non-inbred female Sprague-Dawley rats with DMBA-induced mammary tumors were divided into 3 groups and treated for 4 weeks as follows: Group 1 served as controls, group 2 was ovariectomized, and group 3 received 328 mg RA/kg diet. Ovariectomy (OVX) markedly reduced both the number and size of the tumors. RA administration failed to induce any significant regression in tumor number but significantly retarded tumor growth when compared to tumor growth in group 1 controls. The levels of estradiol, progestin, and prolactin (PRL) receptors were significantly reduced after OVX, whereas only the levels of PRL receptors declined significantly after RA administration. Circulating progesterone concentrations were not affected in the RA-treated group but the plasma PRL level was significantly increased. The present studies show that if treatment with RA is delayed until 6 months after carcinogen administration, the protective effect of RA can still be observed although its effectiveness is less dramatic than when it is administered earlier.

N-nitroso-N-methylurea-induced mammary tumors in the rat: role of prolactin and a prolactin-lowering drug.

Female outbred Sprague-Dawley rats bearing N-nitroso-N-methylurea (NMU)-induced mammary tumors received various endocrine therapies 3 months after the first NMU injection. Rats were divided into 5 groups (15-20 rats/group) and received a 4-week treatment as follows: group 1, controls; group 2, ovariectomized; group 3, 0.5 mg 2-bromoergocryptine (CB-154) injected so twice daily; group 4a, pituitary implant under the kidney capsule; and group 4b, CB-154 injected during the last 2 weeks of the experiment in rats bearing a pituitary implant. Castration of rats with established NMU-induced tumors resulted in a decrease in both tumor number and size, but these parameters again started to increase 3 weeks post castration. CB-154 failed to reduce the tumor number but did arrest the increase in tumor size. In the animals with a pituitary implant, both tumor number and tumor size increased progressively at a greater rate than in control animals, whereas the addition of CB-154 (group 4b) stabilized the tumor growth. Ovariectomy (OVX) resulted in a significant decline of steroid receptor levels. Prolactin (PRL) receptor levels were significantly stimulated by the pituitary implant, and CB-154 prevented this increase. The present studies confirmed that NMU-induced mammary tumors are less hormone-dependent (response to OVX) than 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumors. The role of PRL also appears to be less important, at least for established tumors, for NMU-induced mammary tumors than for DMBA-induced mammary tumors.

Early alterations at the plasma membrane of breast cancer cell lines in response to estradiol and hydroxytamoxifen.

The time course of the early stage of estradiol-17 beta (E2) and hydroxytamoxifen (OHTAM) action at the plasma membrane of hormone-responsive MCF-7 and non-responsive MDA-MB-231 (MDA) breast cancer cell lines was investigated using scanning electron microscopy (SEM), electron probe X-ray microanalysis and microelectrophysiology analysis. SEM showed a marked increase in the density and the length of microvilli (MV) on MCF-7 cells treated with 1 nM estradiol for 1 min. This membrane response disappeared at 5 min. No early effect was obtained with OHTAM, but both compounds produced a similar surge of heterogeneous MV at 15 min of treatment. The morphological change induced by E2 subsided at 60 min, whereas that of OHTAM persisted. X-ray microanalysis and computer determination of peak/background ratios permitted the demonstration that these morphological alterations were concomitant with a rise in the intracellular level of potassium. Microelectrophysiology analysis showed a sharp transitory decrease in the membrane potential of MCF-7 cells in response to estradiol. In the estrogen-insensitive MDA cells, the hormone did not modify the membrane potential and K levels decreased at 1 and 5 min before rising again to control levels at minute 15 when MV appeared. With OHTAM, potassium decreased significantly at 60 min of treatment. These initial and transitory changes in surface morphology paralleled by alterations in potassium level may be consistent with the occurrence of estrogen membrane receptors on target cells, a new aspect of steroid hormone action.

Prolactin receptors in N-nitroso-N-methylurea-induced rat mammary tumors: relationship to tumor age and down-regulation in short-term explant culture.

Mammary tumors were induced by N-nitrosomethylurea (NMU) in young female Sprague-Dawley rats and time of appearance of the tumors was recorded daily. A total of 41 tumors of different ages (7 to 110 days old) were removed and prolactin (PRL) receptors were measured using [125I] human growth hormone (hGH) (equipotent lactogenic activity to oPRL). All but one tumor contained high PRL receptor levels. However, there was a slight decline of both free and total PRL receptor levels with increasing tumor age, with total prolactin receptor levels in tumors 7-20 days old being 32.0 +/- 1.4% compared to 26.0 +/- 2.0% in tumors 66-110 days old (p less than 0.05). In addition, for 24 of these tumors which were of sufficient size, explant cultures were carried out up to 24 h in medium-199 containing insulin and cortisol in the presence or absence of 20 microgram/ml oPRL. At this concentration, an occupation of free and a reduction of total receptor levels was observed in all explant cultures. This down-regulation also appeared to vary as a function of tumor age. Total PRL receptors declined 24.9 +/- 2.3% in young tumors (7-35 days old) compared to 41.7 +/- 4.4% in tumors 50-110 days old (p less than 0.01). The present data are important in that they demonstrate that PRL receptor levels are relatively high regardless of the age of the tumor with, however, a slight decline with increased tumor age. In contrast, the responsiveness of the tumors measured by the ability of prolactin to down-regulate its own receptors is greater in older tumors.

[Breast tissue: estrogen receptors. Immunohistochemical and biochemical analysis of normal and hyperplastic tissue and carcinoma in situ. Apropos of 86 cases]. / Tissu mammaire: récepteurs estrogéniques. Analyse immunohistochimique et biochimique du tissu normal, hyperplasique et du carcinome in situ. A propos de 86 cas.

Estrogen receptors (ER) were investigated using immunohistochemical techniques in 54 cases and biochemical techniques in 38 cases on a series of biopsy specimens of normal, hyperplasic and malignant mammary tissue (intraductal carcinoma). Immunohistochemical data were submitted to quantitative and semi-quantitative analysis (SAMBA 200, TITN). On normal tissue, immunostaining is bright and evenly distributed in galactophores and ductulo-lobular junctions; it is unevenly distributed but consistently present in lobules and increasing after menopause. On hyperplasic tissue, immunostaining of papillary cystadenomas and epitheliosis and fibroadenosis zones was similar to normal tissue with respect to the intensity and heterogeneity. Conversely, their immunohistochemical features are close to blunt duct adenosis and atypical lobular hyperplasia of ductulo-lobular junctions. On malignant tissue, immunostaining is unpredictable, it can be evenly distributed, unevenly distributed in patches or gradients, or totally absent. Immunohistochemical techniques can only be used to assess the intensity and distribution of ER in function of cell type in normal tissue and during a cancerous process. Thus strong receptivity of ductulo-lobular junctions may reinforce the claim that these structures are the targets for estrogen co-carcinogenic substances. The heterogeneity of ER at the onset of carcinoma suggests the possibility of several pathway of development. The fact that myoepithelial cell never seem to display estrogen type receptivity makes it logical to seek a special sensitivity for them.

Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems.

A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000-16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.

Prolactin receptors in explant cultures of carcinogen-induced rat mammary tumors.

The turnover, down-regulation and role of intracellular organelles in the down-regulation of prolactin (PRL) receptors have been investigated in N-nitrosomethylurea (NMU)-induced rat mammary tumors cultured in short-term explants. Tumor explants are capable of maintaining PRL receptors for 24-48 hr. This maintenance reflects a dynamic phenomenon involving receptor synthesis, since addition of cycloheximide (1 microgram/ml) in the culture medium results within 12 hr in a marked decline of PRL receptor levels. A down-regulation of total PRL receptors (measured after exposure of membranes to 3M MgCl2) is observed in cultures containing concentrations of 20 micrograms/ml or greater of ovine PRL (oPRL). Lysosomotropic agents, such as chloroquine (100 microM) are ineffective in either increasing basal PRL receptor levels or in preventing the PRL-induced down-regulation in NMU-induced mammary tumor explants. Cytochalasin B (20 microM), without effect on basal PRL binding, prevents the down-regulation of PRL receptors, whereas colchicine (10 microM) results in a decline of PRL receptor levels both in the absence and in the presence of oPRL. The present data suggest a different pattern of PRL receptor regulation in vitro for tumors compared to normal rabbit mammary explants.

Toward a new approach in tumor cell heterogeneity studies using the concept of order.

A new methodology was developed to study dynamic processes topographically in biological systems by means of a graph-theoretical method. It is based upon order parameters obtained from a minimal spanning tree analysis coupled with computer simulations. The method was used to analyse the heterogeneous behavior of two neoplastic cell lines after treatment with laminin. The laminin-induced cell detachment was quantitated and shown to be inversely related to cell population density and thus to cellular interactions. Our statistical analysis is a very powerful tool to obtain information from seemingly disorderly heterogeneous biological models.