ErbB/HER ligands in human breast cancer, and relationships with their receptors, the bio-pathological features and prognosis.

BACKGROUND: The aim of this study is to provide an expression profile of ErbB/HER ligands in breast cancer. We analysed the relationships with their receptors, the bio-pathological features and prognosis. PATIENTS AND METHODS: Epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), amphiregulin (AREG), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EREG) and neuregulins1-4 (NRG1-4) were quantified in 363 tumours by real-time reverse transcription-polymerase chain reaction using TaqMan probes. RESULTS: Ligands were detected in 80%-96% of the cases, except NRG3 (42%) and EREG (45.5%). At least one ligand was expressed in 304 cases (cut-off: upper quartile). Almost all combinations of receptor and ligand co-expressions were observed, but TGFalpha is preferentially expressed in tumours co-expressing EGFR/HER3, NRG3 in those co-expressing EGFR/HER4, AREG and EREG in those co-expressing HER2/HER4. EGF and AREG were associated with estradiol receptors, small tumour size, low histoprognostic grading, high HER4 levels. TGFalpha, HB-EGF and NRG2 were negatively related to these parameters. In Cox univariate analyses, EGF was a prognostic factor. CONCLUSION: Our study demonstrates that (i) ErbB/HER ligands, including BTC and EREG, are expressed in most breast cancers; and (ii) TGFalpha, HB-EGF and NRG2 high expressions are related to the biological aggressiveness of the tumours.

Real-time reverse-transcription PCR to quantify a panel of 19 genes in breast cancer: relationships with sentinel lymph node invasion.

At the Centre Oscar Lambret, the anticancer centre of the North of France, sentinel lymph node (SLN) procedures are routinely performed for localized (T0-T1, N0, M0) breast carcinoma without any previous treatment, in order to prevent the deleterious effects of axillary lymph node dissection. The present study was undertaken to assess if the expression in the tumor of a panel of 19 genes would allow to predict histological SLN involvement. We looked at cytokeratin 19 (CK19), mucin-1 (MUC1), mammaglobin (MGB1), cyclin D1 (CCND1), the four members of the HER/ErbB growth factor receptor family (EGFR, HER2-4), insulin-like growth factor-1 receptor (IGF-1R), estradiol receptors (ERalpha, ERbeta), progesterone receptor (PR), vascular endothelial growth factors (VEGF, VEGF-C), urokinase-like plasminogen activator (uPA), matrix metalloproteinases 2 and 9 (MMP2, MMP9), ets-related transcription factor ERM, and E-cadherin (CDH1). Their expression was quantified by real-time RT-PCR in 134 breast cancer samples and the relationships with SLN metastases were analyzed. A slight increase (35-40%) in CK19 and HER3 expression was observed in the tumors of patients with SLN metastases compared to those of patients without metastases, even if neither CK19 expression nor HER3 expression allowed to distinguish patients with micrometastases from patients with macrometastases. We conclude that the tumoral expression of biological parameters involved in cell proliferation or playing a critical role in the metastatic process, including tumor invasion and angiogenesis, is not strongly associated with SLN metastases.

Expression of nerve growth factor receptors and their prognostic value in human breast cancer.

Nerve growth factor (NGF) has been shown recently to be mitogenic for human breast cancer cells. In the present study, we have assayed the expression of NGF receptors (NGFRs: TrkA and p75) mRNAs in 363 human primary breast cancers, using real-time quantitative reverse transcription-PCR. NGFRs were found in all of the tumor biopsies. TrkA and p75 were positively correlated and were respectively associated with the histoprognostic grading and the tumor type. NGFRs were both related to progesterone receptors. In univariate analyses, TrkA (>upper quartile) was associated with longer overall survival. Histoprognostic grading, tumor size, node involvement, and steroid receptors were also prognostic factors. In Cox multivariate analyses, TrkA was not a prognostic parameter. This study demonstrates the expression of NGFRs in breast cancer and points out that patients with high levels of TrkA have a more favorable overall survival prognosis.

Multiplex reverse transcription polymerase chain reaction assessment of sialyltransferase expression in human breast cancer.

Increased sialylation, especially involving the Sialyl-Lewisa and Sialyl-Lewisx determinants, has been reported in breast cancer. A multiplex reverse transcription-PCR method was used here to determine the expression of five sialyltransferases (ST3Gal III, ST6Gal I, ST3Gal IV, ST3Gal I, and ST3Gal II) in 49 patients surgically treated for locoregional breast cancer. We assessed the relationship between these expressions and clinical, pathological, and biological features. The most expressed sialyltransferase was ST3Gal 1II, which is involved in Sialyl-Lewisa synthesis. ST3Gal III expression was positively correlated to ST6Gal I and ST3Gal IV expressions, to tumor size, and to the number of involved axillary nodes. Patients with high ST3Gal III expression had a shorter overall survival. High ST6Gal I expression was associated with histoprognostic grade III. ST6Gal I expression was negatively correlated to expression of progesterone receptor. In conclusion, high ST3Gal III and ST6Gal I expressions in human breast tumors are associated with poor prognosis markers.

Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using a 'real time' quantitative RT-PCR assay.

Quantitative competitive RT-PCR techniques have been developed to detect BCR-ABL fusion transcripts in CML but they are hardly reproducible. In this work, we have developed BCR-ABL quantification by real time RT-PCR using the ABI PRISM 7700 (Perkin Elmer), a new technique which allows simple and rapid quantification of a target sequence during the extension phase of PCR amplifications. A fluorogenic probe labeled with both a reporter dye at the 5' end and a quencher-dye at the 3' end hybridizes to the target sequence on the third exon of the ABL gene. The exonuclease activity of the Taq DNA polymerase cleaves the probe and releases the reporter dye, resulting in an increase in the fluorescence signal. The absolute copy number of the target sequence (BCR-ABL) or a control gene (ABL) in an unknown sample can then be calculated using a calibration curve prepared from a set of BCR-ABL RNA standards, and results are expressed as a BCR-ABL/ABL ratio. In our hands, the sensitivity of a serial dilution of total RNA from a positive cell line (K562) in a negative cell line (HL60) was 10(-4). Fifteen CML patients in cytogenetic CR, including 11 allografted patients, two autografted patients and two patients treated by IFN were studied sequentially by this new real time quantitative RT-PCR technique in parallel with conventional qualitative two round nested RT-PCR. The two autografted patients showed high BCR-ABL/ABL ratio in all samples. The two patients treated by IFN showed a progressive decrease in the ratio. In the 11 allografted patients, four were sequentially studied 2 years or more after allo-BMT, and all ratios were below 10(-4). The four patients remained in clinical and cytogenetic CR. The seven other allografted patients were studied immediately after the procedure. Three of them showed a progressive decrease in the BCR-ABL/ABL ratio which reached 10(-4) 7 months after allo-BMT. The three patients remained in hematologic and cytogenetic CR. The remaining four allografted patients had progressive increase of BCR-ABL ratio; three developed cytogenetic relapse 9, 11, 28 months after allo-BMT, and the last patient remained in cytogenetic CR in the bone marrow but developed granulocytic sarcoma. Results of real-time quantitative RT-PCR were in agreement with those of qualitative two round nested PCR. However, evolution changes in the results of real-time quantitative RT-PCR often preceded those of the conventional technique: a decrease of the BCR-ABL/ABL ratio preceded progression from first round to second round positivity and then negativity with the classical technique; conversely, an increase in the ratio preceded evolution with the classical technique. Thus, real-time quantitative RT-PCR may show better correlation with clinical and cytogenetic evolution than conventional qualitative techniques and may help in making early therapeutic decisions in CML, especially after molecular relapse.

Prognostic value of the type I growth factor receptors in a large series of human primary breast cancers quantified with a real-time reverse transcription-polymerase chain reaction assay.

We measured the expression of the type I growth factor receptor gene family [epidermal growth factor receptor (EGFR), c-erbB-2, c-erbB-3 and c-erbB-4] in a series of 365 unselected primary breast cancers. The expression was quantified with a real-time one-step reverse transcriptase-PCR (RT-PCR) assay, based upon the 5' nuclease activity of the Taq polymerase and using an Abi Prism 7700 Sequence Detector System (Perkin-Elmer, Courtaboeuf, France). c-erbB-3 and c-erbB-4 were positively correlated to each other (Spearman test) and negatively correlated to EGFR. EGFR and c-erbB-2 were inversely correlated to the presence of estradiol receptors (ER) and progesterone receptors (PgR), and positively correlated to the histoprognostic grading (HPG). Conversely, c-erbB-3 and c-erbB-4 were positively correlated to the presence of ER and PgR, and inversely correlated to the grading HPG. EGFR was inversely related (chi2 test) to the presence of ER and PgR, and positively associated with HPG. In contrast, both c-erbB-3 and c-erbB-4 were inversely related to HPG, and positively associated with the presence of ER and PgR. The expression level of EGFR and c-erbB-2 was significantly higher in ER- and PgR-negative tumors compared with ER- and PgR-positive tumors (Student's t test), and in tumors with higher grade compared with tumors with lower grade. The expression level of c-erbB-3 and c-erbB-4 was significantly higher in ER- and PgR-positive tumors compared with ER- and PgR-negative tumors and in tumors with lower grade compared with tumors with higher grade. In overall survival studies, Cox univariate analyses showed prognostic values of EGFR [> or = median; P = 0.026; risk ratio (RR), 1.6], c-erbB-3 (> or = median; P = 0.0093; RR, 0.58), c-erbB-4 (> or = median; P = 0.0024; RR, 0.52), HPG, node involvement, tumor diameter, ER, and PgR. In Cox multivariate analyses, tumor diameter, ER, and PgR had a prognostic value. In relapse-free survival studies, univariate analyses demonstrated prognostic values of tumor diameter, node involvement, and c-erbB-4 (P = 0.015; RR, 0.65). These three parameters maintained their prognostic value in multivariate analyses (c-erbB-4, P = 0.035; RR, 0.67). This study confirms that EGFR expression and c-erbB-2 expression are markers of tumor aggressiveness in breast cancer. Conversely, we demonstrate that c-erbB-3 and c-erbB-4 elevated expressions are associated with a better prognosis.

Glyceraldehyde-3-phosphate dehydrogenase gene expression in human breast cancer.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been widely used as a control RNA in Northern blotting and in reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. We investigated the expression of GAPDH in a large series of primary breast cancers and in MCF7 human mammary epithelial breast cancer cells treated with oestradiol. The expression of GAPDH was quantified by a real-time one-step RT-PCR assay, based upon the 5' nuclease activity of Taq polymerase using an Abi Prism 7700 Sequence Detector System (Perkin Elmer, France). Using the Spearman test, GAPDH expression was found to correlate inversely with the age of the patients at diagnosis (P = 0.003; r = -0.147), oestradiol receptors (ER) (P<0.0001; r = -0.327) and progesterone receptors (PgR) (P < 0.0001; r = -0.206). A positive correlation was observed between GAPDH expression and the histo-prognostic grading (HPG) (P < 0.0001; r = 0.344). Moreover, the overall survival (OS) and the relapse-free survival (RFS) were significantly reduced in patients whose tumours showed an enhanced level of GAPDH expression (OS, P = 0.046; RFS, P = 0.021). Multivariate analyses demonstrated that GAPDH was not an independent prognostic factor. Finally, in MCF7 cells treated with oestradiol. a statistically significant dose-dependent increase in GAPDH expression was observed. These results show that GAPDH expression is associated with breast cancer cell proliferation and with the aggressiveness of tumours. The present study demonstrates that, in cancer, the use of GAPDH gene expression should not be used as a control RNA.

Further characterization of the light breast cyst fluid protein, GCDFP-15.

A light protein of breast cyst fluid from women with gross cystic disease, termed GCDFP-15 in the literature, has been investigated. This light protein was purified by preparative electrophoresis on sodium dodecyl sulfate polyacrylamide gel. Its isoelectric point has been determined as 3.75 and its molecular weight has been estimated at 17 400. The light protein was a glycoprotein containing about 163 amino acid residues; the glucidic fraction corresponded to 11% of the molecular weight. The N-terminal amino acid was blocked and the C-terminal amino acid was determined as valine. Antisera raised against this light protein have proved to be specific. In the literature, there is evidence suggesting that apocrine secretion is of prime importance in conditioning the biochemical composition of breast cyst fluid. Further information is needed to substantiate the hypothesis that in gross cystic disease the apocrine epithelium itself or some of its functional aspects are associated with the risk of neoplasia. The physicochemical characterization of the breast cyst fluid protein can contribute to the study of its biosynthesis and provide a better understanding of the physiopathology of gross cystic disease and its relationship to breast carcinoma.

Effect of extracellular ATP on breast tumor cell growth, implication of intracellular calcium.

We studied the effects of purine nucleotides and particularly adenosine triphosphate (ATP) in two (one hormonosensitive, MCF7 and one hormonoinsensitive, MDA-MB 231) human breast tumor cell lines. As described in other cells, we observed that purine nucleotides produced transient elevations in intracellular calcium ions, [Ca2+]i, in both types of cells as determined from Indo-1 fluorescence of loaded cells. In the absence of external calcium the [Ca2+]i transients consisted of single narrow peaks while an extension of peak duration along with a biphasic appearance were observed in the presence of external calcium. The potency of different purine nucleotides in elevating [Ca2+]i was ATP > ADP >> AMP > adenosine (which was inefficient) proving the presence of P2 purinergic receptor subtypes. Suramin, a compound known to compete with ATP for its binding sites, nearly abolished the effect of ATP on [Ca2+]i increase. while verapamil, a calcium channel blocker, was unable to abolish such an an ATP-induced [Ca2+]i increase. The concentrations of ATP required to increase [Ca2%bdi ranged from 10(-7) M to 10(-3) M, the maximal effect being obtained with 10(-4) M ATP. At this latter concentration, ATP induced cell growth inhibition which was dose-independent as triggered only when maximal elevation of [Ca2+]i was attained. This ATP concentration also induced maximal apoptotic features in both types of cells. Together, our results highlighted an 'all or none' effect of ATP on breast tumor cell growth mediated by its effect on [Ca2+]i liberation from intracellular stores, the first rise of [Ca2+]i being further amplified by an influx of calcium from extracellular space. The attainment of sufficient [Ca2+]i level then triggers cellular events.

1,25-dihydroxyvitamin D3 receptors in normal and malignant human colorectal tissues.

1 alpha,25-Dihydroxyvitamin D3, (1,25(OH)2D3), the active metabolite of vitamin D3, has important physiological effects on growth and differentiation in a variety of malignant and non-malignant cell types. In order to better understand the significance of 1,25(OH)2D3 receptors (VDR) in human colorectal cancer, we determined the levels of VDR in paired samples (malignant and adjacent normal tissues) of 24 colorectal cancer bearing patients. Our results demonstrated differences in the relative abundance of VDR between normal and transformed tissues according to the localization of the tumor. While colonic tumors exhibited significantly higher VDR values than their normal counterparts, the contrary seemed to occur in the rectal tumors. In colonic tumors, we found significant correlations between VDR levels and the absence of node involvement or a low Astler-Coller stage. The increased VDR values in colonic tumors as compared with the normal adjacent tissues, may warrant, at least in this type of cancer, the action of 1,25(OH)2D3 or its non-calcemic analogs, to help induce cell differentiation and growth inhibition.

Comparative study of intracellular calcium and adenosine 3',5'-cyclic monophosphate levels in human breast carcinoma cells sensitive or resistant to Adriamycin: contribution to reversion of chemoresistance.

Multidrug resistance (MDR) corresponds to the cross-over resistance of tumour cells to structurally unrelated cytotoxic chemotherapeutic drugs. One of the mechanisms causing this resistance is the enhanced expression of a transmembrane drug efflux pump P-glycoprotein (P-170). Reversal of P-glycoprotein-associated MDR has received much attention in recent years. In experimental cell lines, P-170 and the glutathione redox cycle seem to contribute to this phenomenon; P-170 may be inactivated by calcium and calmodulin antagonists and the glutathione redox cycle altered by buthionine sulphoximine (BSO). Treatment of human MCF-7 breast cancer cells with chemosensitizers (CS), such as verapamil, trifluoperazine or BSO, for 72 hr resulted in an enhanced sensitization of cells to Adriamycin, trifluoperazine being the most potent compound in the reversion of chemoresistance. In these Adriamycin sensitive or resistant cells, treated or not by the CS, the possible role of calcium and cyclic adenosine monophosphate (cAMP) in mediating the reversion of chemoresistance to Adriamycin was investigated. It was found that intracellular calcium was approximately 2-fold higher in resistant than in sensitive cells, the opposite was true for cAMP. Modifications in calcium and cAMP levels were observed in MCF-7 resistant cells after treatment with verapamil and BSO; trifluoperazine had no effect on these two parameters. These results seemed to rule out any implication of calcium and cAMP levels in the contribution of these three chemosensitizers in the mechanisms of reversion of chemoresistance to Adriamycin.

Binding of transferrin to membrane-associated proteoglycans in breast-cancer cells.

Cell surface associated proteoglycans were isolated from MCF-7 breast tumor cells by mild trypsin digestion, extraction by guanidine-hydrochloride and purification by ion-exchange chromatography. Immunological studies showed that transferrin can bind to membrane associated proteoglycans (MAPG). However, these MAPG are not recognized by anti-transferrin receptor antibodies either in an intact, or in a form stripped of glycosaminoglycan chains. Considering the possible involvement of secreted transferrin in proliferation or differentiation of breast tumor cells, we suggest that transferrin binding to MAPG may be related to a specific function of transferrin.

Intracellular calcium and breast-cancer cell-growth and differentiation.

The growth of normal human breast epithelial cells in vitro, as well as those of other cell types is strongly influenced by the concentration of calcium in the culture medium [Ca++]e. The aim of this study was to ascertain if calcium also affects breast tumor cell growth in vitro. To address this question, the metastatic breast cancer cells MCF-7 were grown at low (0.04 mM, L-Ca) and high (2.5 mM, H-Ca) [Ca++]e. In each culture condition, we estimated intracellular calcium levels (Ca++]i from Indo-1 fluorescence by the ratio method. We showed that [Ca++]i increased with [Ca++]e, the [Ca++]i values ranging from approximately 50 to 250 nM. Changes of [Ca++]i ware accompanied by changes of cell shape and cell kinetic parameters. In H-Ca, cells were flat and 3 times larger than in L-Ca and the percentage of cells in the S+G2+M phases as well as the percentage of Ki-67 positive cells rapidly dropped on days 3-4 of culture in contrast to cells grown in L-Ca. In H-Ca, the cell growth arrest corresponded to maximal [Ca++]i which was stable during the stationary phase; at that time, a switch from H-Ca to L-Ca resulted in a drop of [Ca++]i and a resumption of cell growth.. In H-Ca, modifications in cell differentiation parameters such as diminution of ER expression and increases of lipid content and EMA expression were observed as compared to cells grown m L-Ca. Our results suggest that MCF-7 cells have retained some calcium dependency and that agents that can increase [Ca++]i in breast tumor cells may limit their proliferation and trigger at least a partial differentiation.

Expression of BRCA1 gene in ewe mammary epithelial cells during pregnancy: regulation by growth hormone and steroid hormones.

OBJECTIVE: Steroid hormones (estradiol and progesterone) in association with prolactin and growth hormone are involved in lobulo alveolar development of the mammary gland during pregnancy. We hypothesized that the BRCA1 gene may be induced by these different hormones. METHODS AND RESULTS: In this study, we have demonstrated by Northern blot and in situ hybridization, that the expression of ovine (o) BRCA1 mRNA in mammary epithelial cells increased dramatically during a short period in the second half of pregnancy (days 70 to 112) and decreased at the end of pregnancy. The increase in oBRCA1 mRNA expression is concomitant with rapid lobulo alveolar growth. Using an in vivo protocol to artificially induce mammary gland development, we demonstrated by the real-time RT-PCR method that growth hormone in association with estrogen, progesterone and hydrocortisone induces an increase of BRCA1 mRNA expression in the ewe mammary gland. Moreover, we showed that estradiol and progesterone induce oBRCA1 expression in primary cultures of ewe mammary gland. CONCLUSIONS: These results suggest that BRCA1 is a potential regulator of the effects of steroid hormones and growth hormone in the induction of mammary epithelial cell proliferation.

Calcium regulation of heparan sulfate proteoglycans in breast cancer cells.

Breast tumor cells have been shown to be responsive to calcium in that external calcium modifies cell calcium, shape and growth. In order to highlight some of the numerous mechanisms by which calcium is operating, we investigated its influence on the cell microenvironment and particularly its effect on membrane-associated heparan sulfate proteoglycans. The breast cancer cells MCF-7 were grown either at low (0.04 mM) or high (2.5 mM) calcium concentration. After 3 days of culture, cells were labeled with Na2(35)SO4 for 24 h and cell-associated proteoglycans extracted and purified. We showed that calcium enhances approximately twofold the synthesis of sulfated proteoglycans and, among these sulfated proteoglycans, chemical treatments indicated a specific two- to threefold increase of heparan sulfate proteoglycans. In view of the increasing implication of heparan sulfate proteoglycans in numerous mechanisms such as cell-cell contact, cell-matrix interactions and cell growth control, it appears that calcium may be a target for modulating metastatic and growth processes in breast tumor cells.

Germline BRCA1 mutations in patients from 84 families with breast and/or ovarian cancers in northern France.

The BRCA1 gene modification is responsible for an autosomal dominant syndrome of inherited early onset breast and/or ovarian cancer. This gene is estimated to account for almost half of inherited breast cancers and three quarters of inherited breast/ovarian cancers. This suggests that about 1 in every 500 women may carry the BRCA1 mutation. The BRCA1 was isolated by positional cloning in 1994. More than 100 different mutations have been found in the germline of affected individuals. Using systematic sequencing, we looked at BRCA1 germline mutations in 84 patients treated at the Centre Oscar Lambret for breast and/or ovarian cancer who belonged to high-risk families. We found 39 mutations: 22 true mutations inducing modifications of the BRCA1 protein (BRCA1+), six mutations with unknown consequences on the BRCA1 protein, and eleven mutations corresponding to polymorphisms that had been described previously. All the BRCA1+ cases had a HPG3 tumour. The median age of discovery and the receptor positivity percentage are lower in hereditary breast cancer than in the standard population of the breast cancers treated in our centre. Conversely, most of the BRCA1+ patients are without node involvement. This shows that BRCA1 mutations are not always related to parameters thought to indicate a bad prognosis.

Influence of cAMP on E-cadherin expression and cell surface heparan sulfate proteoglycan synthesis in human breast cancer cells.

The growth of MCF-7 and MDA-MB-231 human breast cancer cells was inhibited by treatment with dibutyryl cAMP (dBcAMP, 10(-4) M). The effects on E-cadherin expression and cell surface associated heparan sulfate proteoglycans (HSPG) synthesis, both implicated in cell adhesion, were investigated. dBcAMP was demonstrated to increase E-cadherin expression in the E-cadherin positive MCF-7 cells. However, in the E-cadherin negative MDA-MB-231 cells, the treatment did not induce expression of this cell adhesion molecule. Furthermore, in the two cell lines, an increase of the [35S] Na2SO4 incorporation into the cell surface sulfated PG was observed subsequently to dBcAMP treatment. Interestingly, the proportion of cell surface HSPG was also enhanced by this treatment. Taken together, these results demonstrate that the decrease of the proliferation observed in the human breast cancer cells after dBcAMP treatment is associated with an increase in the cell-cell and cell-matrix interactions. This suggests that the metastatic process which involves lack of cohesiveness and migration of the cells may probably be counteracted by cAMP in the human breast cancer cells.

Beta-adrenergic receptors in human breast cancer: identification, characterization and correlation with progesterone and estradiol receptors.

Besides the variety of other cellular processes in which beta-adrenergic compounds have been involved, their effect on the second messenger cAMP is thought to be related to the growth and differentiation of malignant cells. Because beta-adrenergic receptor could be an easy target for a new combination treatment, we attempted to identify and characterize adrenergic receptor in breast tumor and sought for correlations with estradiol and progesterone receptors. Using L[3H]-dihydroalprenolol, a potent beta-adrenergic antagonist, we demonstrated in human breast tumor the presence of beta-adrenergic receptors with high affinity (Kd 1-3 nM) as shown by Scatchard analyses. Natural and synthetic agonists and antagonists inhibited the [3H] DHA binding, mainly in the order of beta 2-subtype potency. We verified that these receptors were normally coupled with G-protein. A slight correlation was shown between adrenergic and progesterone receptors, and no correlation between adrenergic and estrogen receptors. The presence of specific beta-adrenergic receptors functionally coupled with G protein incited us to study their physiological regulation, since it is tempting to speculate that circulating or locally released catecholamines or other natural compounds may participate in the process of growth and differentiation of the mammary gland through adrenergic receptors.

Distribution and prognostic value of the fibroblast growth factor-2 low-affinity binding sites in human breast cancer.

We performed a competitive binding study with 125I-labelled FGF (fibroblast growth factor)-2 and unlabelled FGF-2 in an unselected series of two hundred and thirty human primary breast cancers. One hundred and ninety-two breast cancer biopsies possessed FGF-2 low-affinity binding sites (FGF-2 LABS). The median dissociation constant was 2.4 nM (range, 1.03-18) and the median concentration of membrane protein was 6187.5 fmol/mg (range, 831-90,000). FGF-2 LABS concentrations were positively correlated to the progesterone receptor level. Cox univariate analyses showed that the FGF-2 LABS (> or = upper quartile) was associated to a longer overall survival (p = 0.05; RR = 0.042); node involvement, estrogen receptor progesterone receptor and histoprognostic grading were also prognostic. In Cox multivariate analyses, only the progesterone receptor, estrogen receptor, node involvement and FGF-2 LABS were prognostic factors; the FGF-2 LABS were associated with a longer overall survival (p = 0.033; RR = 0.068). The present study showed that FGF-2 LABS have only a limited role as a prognostic factor in breast cancer.

C-myc overexpression, c-mil, c-myb expression in a breast tumor cell line. Effects of estrogen and antiestrogen.

In breast tumor cell lines, c-myc amplification is frequently associated with estrogen unresponsiveness. We, however, succeeded in characterizing an estrogen-responsive cell line VHB1 derived from a duct cell carcinoma, which exhibits c-myc amplification and overexpression. We therefore studied the effects of estrogen and antiestrogen on c-myc expression in this particular cell line. We also investigated these effects on the expression of c-mil and c-myb oncogenes, also expressed but not amplified in VHB1 cells. Short-(1 h) and long-(72 h) term stimulations were performed. Our experiments showed that estradiol (E2 10(-8) M) was still able to stimulate c-myc expression equally either after short or long-term treatment. In the same way, the antiestrogen 4-hydroxytamoxifen equally decreased c-myc expression but the reversal effect of E2 after long-term antiestrogen treatment was more pronounced than after short-term treatment. The effects of E2 and 4-OH Tam on the expression of the not-amplified c-mil and c-myb oncogenes were stronger than those observed on c-myc expression; however, the E2 reversal effect was identical either after short or long-term antiestrogen treatment. Our results may enlighten some aspects of the complex action of some of the early- and late-growth regulated genes in breast cancer.