Estrogen conjugates and serum factors mediating the estrogenic trophic effect on MCF-7 cell growth.

Estrogens stimulate growth of MCF-7 breast cancer cells in monolayer culture. Possible interference of serum factors leading to an estrogen-insensitive cell growth was analyzed in various experiments carried out on serum batches producing no estradiol stimulation. Out of five estrogen conjugates, only 3-glucurono-estradiol partly suppressed the inhibition of hydroxytamoxifen; the conjugate also reduced the estrogen receptor content of the cells, probably by a down regulation process ("processing"). Moreover, prolonged subcultures in dextran-coated charcoal-treated serum attempting to remove possible intracellular estrogens produced no growth stimulation. Interference by hormone carriers of the serum was ruled out by the fact that two strong synthetic estrogens, moxestrol and diethylstilbestrol with weak binding affinity for these carriers, were unstimulatory. Reduction of the carrier concentration also failed to confer any estrogen sensitivity. This lack of effect of most estrogen conjugates and serum carriers seems to contradict the hypothesis of their interference leading to an estrogen-insensitive growth. Presence in the serum of potential inhibitors towards estrogen action was also examined. Dilution of sera inducing an estrogenic stimulated growth failed to show any growth increase, either in the absence or presence of estradiol, thus excluding the possibility of a major influence of an antagonism on growth control. Moreover, clonogenic assays in soft agar eliminated the hypothesis that a difference between "active" (stimulatory with estradiol) and "inactive" serum batches may result from distinct adherence properties rather than from real growth stimulation. All of these data are consistent with the concept that serum factors which are not of estrogenic nature mediate the trophic effect of estradiol; their absence in some serum batches may lead to an estrogen-insensitive cell growth.

Hydroxy derivatives of tamoxifen.

In the exploration of the structural features that affect the RBA (binding affinity for the estrogen receptor of rat uterus relative to that of estradiol) in the tamoxifen [trans-(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl ]-1,2-diphenyl-1-butene] series, several derivatives variously substituted in the 1-phenyl group have been synthesized. [In the tamoxifen series, the descriptors E and Z, which define the configuration of the geometrical isomers and depend on the location and nature of substituents in the aromatic moieties and the ethyl group, may vary, although the relative configuration (cis or trans) does not. In order to avoid confusion the terms cis and trans will be used in this paper to refer to the relative positions of the 4-[2-(dimethylamino)ethoxy]phenyl and ethyl (or hydroxyethyl, hydroxypropyl, or bromo) substituents attached to the ethene moiety.] The final stage of each synthesis involved acid-catalyzed dehydration of a tertiary alcohol, and, in contrast to the known 3- and 4-hydroxy derivatives which were obtained as near-equimolar cis,trans mixtures, only the trans forms of the 2-hydroxy, 2-methyl, 2,4-dihydroxy, and 4-hydroxy-2-methyl derivatives were obtained. Also, in contrast to the trans forms of the 3- and 4-hydroxy derivatives, which are readily equilibrated to cis,trans mixtures, the trans 2-hydroxy derivative could not be isomerized. Tamoxifen and 2-methyltamoxifen had similar RBA's (approximately 1% of that of E2), but that of 2-hydroxytamoxifen was much lower (0.1%). Introduction of a second hydroxyl group (2,4-dihydroxy derivative) enhanced the RBA, and for the 4-hydroxy-2-methyl derivative, the RBA and growth inhibitory activity against the MCF-7 mammary tumor cell line in vitro were high and comparable to those of 4-hydroxytamoxifen, a metabolite of the parent drug. Tamoxifen derivatives hydroxylated at positions 3 or 4 of the 1-butene moiety and the 5-hydroxy-1-pentene analogue were also synthesized, but they had very low RBA values.

Phenotypic change of the transplantable MXT mammary adenocarcinoma into mixed bone producing sarcoma-like tumors.

The B6D2F1 mouse mammary adenocarcinoma was adapted to grow in vitro as monolayer. After in vitro passaging of tumor cells, phenotypic changes occurred that were expressed in vivo. Following intraperitoneal inoculation of tumor cells, bone-forming tumors developed. These tumors consisted of undifferentiated adenocarcinoma mixed with large amount of cartilagenous and osseous tissue. The etiology of these phenotypic changes was not yet determined. However, hypothesis of the possible origin of the cartilage and bone forming tissue was formulated. The biologic characterization of the intraperitoneally bone-forming tumor was achieved and the experimental conditions to preserve and induce the reproducible sarcoma-like bone forming tumors were defined. Our data support the usefulness of this new original model for fundamental research as well as for screening of anticancer drugs.

Influence of inoculation sites and tumor cell culture techniques on the phenotype of a mixed cartilage- and bone-producing mouse mammary tumor.

We have previously reported the characterization of an intraperitoneally (IP) transplantable bone-forming MXT tumor. However, the question was unresolved as whether the bone-forming cells originated from either the host animal or from the neoplasm itself. The present work attempts to answer this question by studying the influences of inoculation sites (subcutaneously, SC; intraperitoneally, IP; in the brain, IB; intracranially, ICR) on both the cartilage- and bone-forming tumor phenotypes. Furthermore the influence of cell culture procedures (two- and three- dimensional cultures) on these phenotypes was investigated. SC administered MXT cancer cells never produce bone-forming tumors, suggesting the existence in the dermis of substance(s) inhibitory to the formation of cartilage or bone. On the contrary, our data clearly demonstrate that bone-forming tumors can be obtained by either IP route, in a way which mimics endochondral ossification, or in the brain (IB), a region usually devoid of connective tissue. This observation substantiates the hypothesis according to which the tumor itself is able to produce osseous tissue. Another main finding is the increasing occurrence of skeletal tissues produced by cells proceeding from three-dimensional culture. Finally, ICR and IB tumors exerted a bone-lytic action against the host skull suggesting that tumor cells either produce osteolytic substances (prostaglandins, enzymes) and/or that they contain various cell types exhibiting different properties toward osteogenesis. This model offers new perspectives for studying the mechanisms of both normal and pathologic osteogenesis.

Growth factor-like activity of phenol red preparations in the MCF-7 breast cancer cell line.

Hormonal responsiveness of the estrogen-sensitive MCF-7 human breast cancer cell line is known to vary between laboratories although the causes and implications of these variations remain unclear. Our findings lead us to conclude that the pH indicator phenol red (PHR) has growth factor-like effects in addition to its well known estrogen-like effects. To demonstrate this hypothesis, we have assessed the importance of PHR either in the absence or in the presence of the estrogens contained in the serum added to the culture medium. The basal growth rate of MCF-7 cells was significantly reduced by short-term or long-term withdrawal of PHR. The stimulatory effects of estradiol and the inhibitory effects of the antiestrogen 2-CH3,4-OH-tamoxifen (MHT) were not significantly affected by long-term withdrawal of the dye. Moreover, long-term cell maintenance without PHR alone or in complete estrogen-depleted medium did not change their basal steroid receptor content. The molecular structure of the estrogen receptor which is usually modified under estrogenic stimulation remained identical whether or not the cells were maintained in the presence of the dye. Maintaining cells without the dye in the presence of serum estrogens led to the death of the cell line after 50 transfers. Lastly, addition of PHR had clearcut growth stimulatory effects on the hormono-independent cell line Evsa-T.

Relationship between receptors for epidermal growth factor and steroid hormones in normal, dysplastic and neoplastic canine mammary tissues.

The concentrations of receptors for epidermal growth factor (EGF-R), oestrogen (ER) and progesterone (PR) were measured in 108 samples from canine mammary tumours and 132 samples of normal mammary tissue removed surgically from 84 bitches. The history and clinical signs were also recorded. Binding sites of high affinity were detected in 70 per cent of both types of tissue and no significant variations in EGF-R concentrations or positivity were observed with the histology, location, size or number of mammary tumours or the age of the animal. A significant direct correlation (P = 0.002) was observed between the concentrations of ER and EGF-R only in malignant tumours. The concentrations of EGF-R were significantly correlated (P = 0.04) in normal mammary tissues adjacent to and distant from the lesions, but not between normal tissue and tumour tissue. No significant differences were observed in the expression of EGF-R in normal and neoplastic tissues from the same bitches. The direct correlation between the concentrations of EGF-R and ER in malignant tumours could be related to an oestrogen-dependent expression of EGF-R or to a similar pattern of regulation of the receptors.

Comparison of biochemical and immunoenzymatic macromethods and a new immunocytochemical micromethod for assaying estrogen receptors in human breast carcinomas.

Estrogen receptors (ERs) were assayed in 23 breast carcinomas by: (1) the conventional biochemical assay with dextran-coated charcoal (DCC); (2) the immunoenzymatic assay using a monoclonal antibody (MAb), ER-EIA (Abbott); and (3) an original cytochemical method using another MAb, ER-ICA (Abbott). The first two techniques were performed on biopsy samples, whereas the last was carried out on fine needle aspiration (FNA) samples. The ER contents in aspirates were evaluated by: (1) scaled proportions of colored neoplastic cells; (2) scaled coloration intensity; (3) total grading (= proportion plus intensity); (4) product grading (= proportion times intensity); and (5) a new index (NI) described in this paper. The ER-EIA assay correlated best, with a high statistical significance, with the NI (P less than .001); NI was also the only index that significantly correlated (P less than .05) with the DCC results. The results show that the ER-ICA assay offers the great advantages of being applicable to FNA specimens and of producing rapidly available results. This new technique enriches the panel of MAbs for the diagnosis of adenocarcinomas and offers a new tool for the therapeutic follow-up of breast cancer patients. Our preliminary results suggest that the anti-ER MAbs might be helpful for measuring the hormone dependence of small lesions not assayable by DCC, even under endocrine therapy, thus avoiding false-negative assays.

[Local growth factors for breast cancer: general review and potential value for clinician]. / Les facteurs de croissance locaux du cancer mammaire: revue générale et intérêt potentiel pour le clinicien.

The discovery of a panel of peptides produced by normal and cancerous cells and capable of stimulating cell growth in an autocrine and paracrine fashion has been witnessed in recent years. This article focusses on the local growth factors thought to play a role in the biology of breast cancer and reviews which growth factors' receptors have been associated with prognosis of primary breast cancer. The potential of those growth factors for the development of new anticancer treatment strategies is also briefly discussed.

[Immunocytochemical assay of estrogen receptors in puncture products of breast carcinomas]. / Dosage immunocytochimique du récepteur d'oestrogène dans les produits de ponctions de carcinomes mammaires.

In a pilot study, estrogen receptors (ER) were assayed on 42 surgically removed breast tumors by the following 3 methods: biochemical assay with dextran coated charcoal (DCC), Abbott immunoenzymatic (ER-EIA) and immunocytochemical (ER-ICA) technics. DCC and ER-EIA were performed on biopsy specimens while ER-ICA was run on cytocentrifugated cells obtained by fine needle aspiration (FNA). ER contents were expressed according to an index taking into account the proportion of colored neoplastic cells and the intensity of staining. Statistical correlation coefficient (Spearman and Kendall) concordance, sensitivity and specificity between the results were calculated (ER - ICA/ER - EIA: P less than 0.001, r = 0.38, concordance = 83%, sensitivity = 86%, specificity = 77%; ER - ICA/DCC: P less than 0.05, r = 0.22, concordance = 77%, sensitivity = 85%, specificity = 63%; ER - EIA/DCC: P less than 0.001, (r = 0.60). As previously reported, both immunoassays showed good agreement. The weaker but nevertheless significant correlation found with reference DCC may be due to the heterogeneity of tumoral ER content. This hypothesis is supported by the variability of ER - ICA assays on multiple FNA performed in 16 cases from our series. Use of multidirectional FNA slightly improved the results. Nevertheless, ER - ICA appear to be a good semi-quantitative method and might be helpful in the follow-up of metastasis treated with anti-estrogen, especially in small lesions not assayable by DCC.

Effect of an homo-aza-steroidal ester on estrogen receptor.

A new modified steroid esterified with the cytotoxic moiety, p-[N,N-bis(2-chloroethyl)amino]phenyl-acetic acid, has been synthesized and tested for interaction with estrogen receptor and cytotoxic activity on the MCF-7 cell line.

Comparison of estrogen and progesterone receptor expression in normal and tumor mammary tissues from dogs.

Concentrations of estrogen (ER) and progesterone (PR) receptors were measured by radioreceptor assay in tumor (n = 319) and normal (n = 166) mammary tissue from 248 bitches. Correlations between ER and PR and between receptor expression in tumor and normal mammary tissue from the same bitches were evaluated. The influence of tumor, clinical, or hormonal variables on receptor expression also was studied. Approximately 80% of tumor and 95% of normal mammary tissue expressed detectable concentrations of ER, PR, or both. Direct correlation was found between ER and PR concentrations in normal and tumor tissues. Median ER concentrations were significantly higher (46 +/- 47 fmol/mg of cytosolic protein vs 27 +/- 24 fmol/mg of cytosolic protein; P = 0.0002) in normal than in tumor tissue. On the other hand, PR concentrations were significantly higher (57 +/- 52 fmol/mg vs 77 +/- 99 fmol/mg; P = 0.03) in tumors (especially benign tumors) than in normal tissue. Poorly differentiated malignant tumors expressed lower concentrations of receptors than did benign or well differentiated malignant tumors. The ER and PR concentrations decreased with increasing size of the lesion. Hormonal status of the bitch significantly (P < 0.05) influenced receptor expression in normal tissue: bitches in the luteal phase of the estrous cycle had higher concentrations of ER (69 +/- 62 fmol/mg) than did ovariectomized bitches (24 +/- 19 fmol/mg) or bitches in anestrus (38 +/- 45 fmol/mg) or the follicular phase (13 +/- 7 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in oestrogen, progesterone and epidermal growth factor receptor concentrations and affinities during the oestrous cycle in the normal mammary gland and uterus of dogs.

Changes in the concentrations and affinities of receptors for oestrogen (ER), progesterone (PR) and epidermal growth factor (EGF-R) were studied in mammary glands of healthy bitches with regard to age, the location in the mammary chain and the stage of the oestrous cycle. Uterus was used as the reference tissue for the evaluation of steroid receptors. Mammary and uterine samples from 7 healthy bitches were taken at five stages of the oestrous cycle in such a way that all the locations in the mammary chain were represented at each stage of the cycle (10 samples/dog). ER, PR and EGF-R were detected by biochemical assays using increasing concentrations of tritiated (steroids) or iodinated (EGF) ligands. A significant direct correlation was found between the ER and PR concentrations for mammary and uterine samples. No significant correlation was found between the steroid receptors and EGF-R concentrations. Mammary ER concentrations were significantly higher in bitches of 5 years of age or older than in younger ones; in posterior glands (4th and 5th pairs) than in anterior glands; and in the mid-luteal phase. Mammary PR did not vary significantly with age or location but was significantly lower in the early luteal phase than in other phases. A similar decrease in PR concentrations was observed in the uterus during the early luteal phase and uterine ER and PR concentrations were very low in the mid-luteal phase. Mammary EGF-R were not significantly higher in the early or mid-luteal phase than in pro-oestrus or anoestrus. The differences observed between the uterine and mammary steroid receptor concentrations during the oestrous cycle could be due to different mechanisms for regulating steroid receptor expression in the two tissues. Mammary EGF-R concentrations may be linked, as in other species, to cellular proliferation and/or to the serum progesterone concentrations.

Iodination of mouse EGF with chloramine T at 4 degrees C: characterization of the iodinated peptide and comparison with other labelling methods.

A modified Chloramine T labelling procedure was used to iodinate mEGF in order to perform radio-receptor assays. The reaction was conducted at 4 degrees C with 1 mu g Chloramine T only. The tracer obtained was characterized by its maximal binding, specific activity and binding properties compared with the native peptide. Fast Liquid Protein Chromatography was performed to analyse the homogeneity of the preparation and membrane extracts from A431 cells were used to purify the tracer. The modified Chloramine T procedure was compared with two other methods: the classical Chloramine T iodination and the labelling procedure using Enzymobeads. The modified Chloramine T procedure is reproducible, provides labelled mEGF with high binding capacity (65 to 80% with canine placental membrane extracts) and high specific activity (351 +/- 107 mu Ci/mu g mEGF) and seems to preserve the binding properties of the native peptide.

Guide-lines in the design of new antiestrogens and cytotoxic-linked estrogens for the treatment of breast cancer.

Antiestrogens (Tamoxifen) are used for the treatment of breast cancer. However these compounds are also weak estrogens that may stimulate tumor growth. Cytotoxic-linked estrogens (Estradiol Mustard, Estracyt) are devoid of major therapeutic activity. This led to search for new antiestrogens devoid of estrogenicity and for active cytotoxic-estrogens. Hydroxylation of C-4 of triphenylethylene antiestrogens (tamoxifen, CI 628 and U 23,469) largely increases their binding affinity for the estrogen receptor (ER). Hydroxylation also increases the in vitro antitumor activity of the drugs as shown by their higher ability to inhibit the growth of the ER-positive cell line MCF-7. Triphenylethylene antiestrogens contain an aminoethoxy side chain which appears essential for their physiological activity. Removal of the chain of tamoxifen suppresses its antiestrogenicity and antitumor activity. The grafting of side chains on a weak estrogen of the gem-diphenylethylene category produces "symmetrical" antiestrogens devoid of estrogenic activity. This observation raises the question of the role played by the third phenyl ring of the triphenylethylenes since the trans-isomers of the latter display antiestrogenicity and the cis-isomers estrogenicity. Comparison of the binding affinity for ER and antitumor activity of di- and triphenylethylene antiestrogens suggests that this third phenyl ring increases the interaction with ER of the 4-phenolic group of the drugs and/or their aminoethoxy side chain. An analogue of this chain is without any biological activity suggesting that the di-(tri)pheny-lalkene structure is required for promoting the interaction of the chain with ER. New chemical structures yielding antiestrogens with antitumor activity are also reviewed. New cytotoxic estrogens designed for producing lethal damage of DNA show a low binding affinity for ER. Moreover, there is no evidence suggesting specific antitumor activity. Such activity may be more easily obtained with estrogens bearing reagents for proteins rather than DNA. The biological properties of a 2-mesylate derivative of estrone irreversibly interacting with ER supports the concept. On MCF-7 cells, the drug displays a strong antitumor activity which can only be suppressed by high, equimolar, concentrations of estradiol. It is devoid of cytotoxic activity on the ER-negative cell line Evsa-T suggesting that ER is involved in its action.

Tritiated actinomycin-D staining method: a valuable tool to study oestrogen receptor-induced modifications of transcriptional activity in normal and neoplastic cells.

Exposure at 4 degrees C of purified cell nuclei from uterus and hormone-sensitive mammary cancer to their own cytosol preincubated with oestradiol produced a significant increase in their ability to bind [3H] actinomycin D (3H-AMD). This increase did not occur under conditions preventing the transfer of the oestrogen-receptor into the cell nucleus, nor in cancer cell nuclei devoid of oestrogen-receptors. Uterine cytosol preincubated with the strong antioestrogen 4-hydroxytamoxifen did not modify the 3H-AMD binding capacity of the uterine nuclei but significantly suppressed the increase induced by oestradiol. Moreover, ultrastructural study of tumour nuclei revealed that the oestradiol receptor-induced increase was associated with a marked chromatin dispersion. These results strongly suggest that the 3H-AMD staining method is a valuable tool for the assessment of oestrogen receptor-induced modifications of transcriptional activity in normal and pathological tissue as well.