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 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)

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.

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.

Positive interference of merthiolate in the TDx digoxin assay in control samples used in the Belgian External Quality Assessment (EQA) Scheme.

We investigated the possible origin of the spuriously high results observed with the Abbott TDx Immunoassay in the 1991 Belgian external quality assessment scheme for digoxin. The present work ascribes this discrepancy to a matrix effect induced by the addition of merthiolate as preservative to the control samples. It consequently stresses the importance of avoiding the use of this compound for preparing such samples.

Factors influencing between-laboratory variability of C-reactive protein results as evidenced by the Belgian External Quality Assessment(EQA) Scheme.

Based on results from the Belgian External Quality Assessment (EQA) Scheme, we studied the main factors affecting the between-laboratory variation of C-reactive protein determination. Participants using homogeneous systems with several calibration points generally achieved better performance. Working temperatures influenced the results to a lesser extent. The present study stresses the importance for EQA organizers to collect more detailed information about CRP analytical methods used by the participants. It also suggests that manufacturers should be more involved in the management of quality, in particular by striving for standardization of the material (kit and calibrator) they produce for CRP assay.

Receptors for oestrogen, progesterone and epidermal growth factor in normal and tumorous canine mammary tissues.

Receptors for oestrogen (ER), progesterone (PR) and epidermal growth factor (EGF-R) are found in normal mammary tissue (NMT) and/or mammary tumours (MT) from all species studied, including dogs. The aims of this study were to define the possible influences of mammary histology, age, location in the mammary chain and of hormonal status and cycle stage in the expression of ER, PR and EGF-R in mammary tissues from healthy dogs and from dogs with MT. Carcinomas that had lost their glandular structure had significantly lower amounts of receptors. NMT either from healthy or affected dogs had significantly higher amounts of ER than MT. PR levels were significantly higher in benign lesions than in NMT. Steroid receptors in NMT from healthy dogs varied significantly with age (older dogs having more ER), location (posterior glands having higher ER concentrations) and cycle stage (the highest ER concentrations being found in the mid-luteal phase and the lowest PR concentrations in the early luteal phase). In NMT from affected dogs, higher steroid receptor concentrations were found in posterior glands; as in healthy dogs, ER concentrations were low in the follicular phase and high in the luteal phase and PR were high in anoestrus. Steroid receptor content in MT did not vary significantly with age, location or cycle stage or with hormonal status, but tended to vary with cycle stage in a manner similar to that found in NMT from the same dogs. In dogs affected with MT and treated with medroxyprogesterone acetate (MPA), NMT had low concentrations of PR but MT from the same dogs had high PR concentrations. EGF-R were found in the majority of the samples (+/- 65% of MT and +/- 85% of NMT) but there was no significant relation between the concentrations and the parameters studied. Nevertheless, EGF-R content was higher in NMT in the proliferative stages (oestrus, early and mid-luteal phase) than in the non-proliferative stages (early pro-oestrus and anoestrus). EGF-R and ER were significantly and positively correlated only in malignant tumours. There is no apparent difference between affected and healthy dogs in the regulation of ER, PR and EGF-R expression in NMT; on the other hand, some differences between NMT and MT are observed in the regulation of PR (for example under the influence of MPA) and in the correlation between EGF-R and ER expression.

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.

[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.

Hormone sensitivity in vitro and in vivo of v-ras-transfected MCF-7 cell derivatives.

Human mammary carcinoma cell lines (MCF-7) were analysed for their hormone sensitivity before and after transfection with a v-Ha-ras oncogene or with a neomycin-resistance gene followed by selection in vitro or in vivo. Our aim was to test how the expression of the ras oncogene would influence the estradiol sensitivity of MCF-7 cells. In culture, MCF-7 cells expressing the viral p21 oncogene product, as compared to parental MCF-7 cells and their control derivatives, showed lower levels of a 67-kDa estrogen receptor. Progesterone receptors, however, remained sensitive to up-regulation by estrogens. The oncogene-expressing cells were less sensitive than all controls to stimulation of proliferation by 10(-8)M estradiol or to inhibition of proliferation by 2-CH3-4-OH tamoxifen, and this was not dependent upon the type of culture medium used. After s.c. or i.p. injection into female athymic nude mice, ovariectomized or left intact, the growth of MCF-7 cells expressing the ras oncogene product and of all control cells was sensitive to stimulation by estrogen supplementation. Conversely, cell lines derived from tumors generated with long latency in untreated athymic nude mice by v-ras-expressing MCF-7 cells showed efficient formation of quickly growing tumors in the absence of estrogen supplementation. No differences were observed in invasion and metastasis of the different MCF-7 cell types injected into athymic nude mice that were supplemented with estrogens or not.

[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.

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.

Estrogen receptor status and estradiol sensitivity of MCF-7 cells in exponential growth phase.

Proliferative patterns of MCF-7 human breast cancer cells have been reported to influence their estrogen receptor (ER) contents. However, the experimental conditions under which these variations in ER contents were described differed from those commonly used for maintaining exponential growth. We, therefore, investigated whether or not MCF-7 receptor status also fluctuated under normal growth conditions. MCF-7 cells were cultured up to 4 days in 96-multiwell dishes. On each day, cell number was spectrophotometrically assessed after fixation and coloration of the cells with hematoxylin; corresponding ER content was measured by the Abbott enzyme immunoassay in KCl extracts. At the three plating densities tested (5, 10 and 20 x 10(3) cells/ml), an obvious parallel was found between the cell number and the ER content suggesting an unchanged receptor status throughout the culture period. Regression analysis confirmed this impression. Additional fractionation by SDS-PAGE of total MCF-7 proteins extracted at various times of the culture (up to 7 days in 35 mm Petri dishes) gave identical patterns suggesting that ER synthesis is regulated as the majority of proteins. Growth experiments indicated that this situation conferred a constant estrogenic sensitivity to the cells: 24 h exposure to 10(-8) M estradiol on either the 1st, 2nd, 3rd or 4th day after plating resulted in the same increase in cell number. All these data indicated that ER contents of MCF-7 cells were maintained at a constant level under exponential growth which resulted in a constant estrogenic sensitivity.

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.

Endocrine effects of Trilostane: in vitro and in vivo studies.

Trilostane (4-alpha-5-epoxy-17 beta-hydroxy-3-oxo-5-alpha-androstan-2-carbonitrile) is a modified steroidal molecule. In vitro and in vivo studies in rats have shown that it inhibits adrenal, ovarian and placental steroid synthesis. It seems to act by exerting a selective blockade on 3 beta-hydroxysteroid dehydrogenase. In this study, we investigated whether this molecule interacts with hormone receptors for estrogen, androgen or progesterone. We also tried to demonstrate the effect which Trilostane may have on cellular cultures of human mammary carcinoma (MCF-7 Evsa-T). We also studied hormonal modifications in a series of 12 patients treated with different doses of Trilostane, since this drug is supposed to inhibit the production by the adrenal glands of mineralocorticoids, of glucocorticoids and of the precursors of estrogens. Our results indicate that Trilostane does not react with any of the main hormonal sex steroid receptors, nor does it interfere with cultures of human mammary cancer cells either containing estrogen receptors and therefore allegedly hormone-dependent (MCF-7 line), or estrogen receptor-negative cells, presumably independent of hormonal manipulations (Evsa-T cell line). Finally, endocrine studies on postmenopausal women with advanced breast cancer show that Trilostane significantly reduces the plasma levels of estrone and of its major androgen precursor (androstenedione). However, the latter inhibition is no different from that exerted by hydrocortisone acetate administered alone at a dose of 40 mg/day. The results of clinical trials comparing hydrocortisone alone with hydrocortisone plus Trilostane are awaited.

In vitro studies of canine mammary tumors: influence of 17-beta-estradiol and progesterone on cell kinetics parameters.

Using an in vitro tritiated thymidine nuclear labeling followed by autoradiography, the effects of 17-beta-estradiol (E2) or progesterone (Pg) were studied in 30 canine mammary tumors that were incubated and hormonally stimulated in vitro. In 10 of these tumors, the synthetic (S) phase duration was also measured in absence or in presence of E2, by using a double labeling with tritiated thymidine. Our results demonstrate that E2, and, to a lesser degree, Pg can induce cell replication in both estrogen receptor-positive (ER+ PgR+) and estrogen receptor-negative (ER- PgR-) canine mammary tumors. The mitogenic effect of E2 may involve a shortening of the DNA S cell cycle phase. We have also found a significantly positive relationship between the estrogen and the progesterone receptor concentrations and the basal proliferation rate in these tumors, whereas no correlation was found between steroid receptor contents and the maximal level of stimulation achieved after E2 or Pg exposure.

Analogues of tamoxifen: the role of the basic side-chain. Applications of a whole-cell oestrogen-receptor binding assay to N-oxides and quaternary salts.

Derivatives of tamoxifen (1) and 4-hydroxy-2-methyltamoxifen (2) in which the basic side chain has been modified by N-oxidation or by quaternization have been investigated with respect to the effects on affinity for the oestrogen receptor and on cytostatic activity towards the MCF-7 cell line in vitro. In addition to the conventional cytosol assay for receptor binding affinity (RBA) a recently developed whole-cell assay was employed. N-oxidation (e.g. 2----3) produced no significant alteration in RBA value either in cytosol or in whole cells, nor in activity towards the MCF-7 line. Quaternization with methyl iodide (1----4, 2----6) or ethyl bromide (1----5, 2----7b: the cis isomer 7a also formed) did not alter receptor binding in the cytosol assay but almost abolished binding in the whole cell and cytostatic activity. The whole-cell RBA values for 2 (0.45) and 3 (0.5) were lower than those for 4-hydroxytamoxifen (2.9), suggested to be due to the lower oestrogenicity of the 2-methyl derivatives since activity against MCF-7 cells was unimpaired. The even lower values of whole-cell RBA (0.01-0.02) for the quaternary ethyl bromide derivatives 7a and 7b were ascribed to poor penetration into the cell since these compounds had minimal cytostatic activity.

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.

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.