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.

Measurement of mRNA of 11 biomarkers by RT-PCR to detect lymph node involvement in cervical cancer.

Lymph node metastases are a major prognostic factor in cervical carcinomas. The aim of this study was to characterize the expression of 11 markers in cervical tumors and negative lymph nodes and to determine which ones could be helpful for improving the specificity of molecular diagnosis of nodal involvement. Using TaqMan RT-PCR, we studied the expression of CK19, MUC1, HER1-HER4, VEGF, VEGF-C, uPA, MMP9, and PRAD1 in uterine cervical tumors and in histologically nonmetastatic lymph nodes of 8 patients diagnosed with locally advanced cervical cancer. We observed that CK19, MUC1, HER1-HER3, uPA, and VEGF had a significantly higher expression in cervical tumors than in the negative nodes, whereas VEGF-C expression level was higher in the negative nodes than in the tumors. PRAD1 harbored similar expression levels in the tumors and in the negative nodes. Interestingly, 1 of the 4 patients who presented a clinical recurrence, showed elevated HER1, HER2, uPA, and VEGF in the histologically negative nodes. Our results suggest that CK19, MUC1, HER1-3, uPA, and VEGF are biomarkers that have a higher expression in tumoral cervical tissues compared with the negative lymph nodes and could be useful to diagnose nodal involvement in uterine cervical carcinoma. Our results should encourage us in continue to investigate a greater number of patients, including patients with histologically involved nodes.

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.

Proteomic analysis reveals that 14-3-3sigma is down-regulated in human breast cancer cells.

The class of molecular chaperones known as 14-3-3 is involved in the control of cellular growth by virtue of its apparent regulation of various signaling pathways, including the Raf/mitogen-activated protein kinase pathway. In breast cancer cells, the sigma form of 14-3-3 has been shown to interact with cyclin-dependent kinases and to control the rate of entry into mitosis. To test for a direct role for 14-3-3 in breast epithelial cell neoplasia, we have quantitated 14-3-3 protein levels using a proteomic approach based on two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF). We show here that 14-3-3sigma protein is strongly down-regulated in the prototypic breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with normal breast epithelial cells. In contrast, levels of the alpha, beta, delta, or zeta isoforms of 14-3-3 were the same in both normal and transformed cells. The data support the idea that 14-3-3sigma is involved in the neoplastic transition of breast epithelial cells by virtue of its role as a tumor suppressor; as such, it may constitute a robust marker with clinical efficacy for this pathology.

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.

Prognostic value of circulating soluble E-selectin concentrations in node-negative breast cancer patients.

Several studies have suggested that endothelial cells participate in tumor development. Soluble E-selectin (sE-selectin) is specifically released by activated endothelial cells, and its serum concentration can be considered a marker of endothelial activation. In this study, we assessed the prognostic value of sE-selectin concentrations in node-negative breast cancer patients. Serum sE-selectin concentrations were measured by an ELISA method prior to surgery in 456 node-negative breast cancer patients. We analyzed also tumor size (TS), histoprognostic grading, and steroid hormone receptor status. The mean sE-selectin concentration was 24.9 +/- 15.0 ng/ml. The sE-selectin concentrations were mildly correlated with the TS but not with the other factors. For prognostic analyses, the median follow-up duration was 7.5 years. The cutoff sE-selectin concentration used was 40 ng/ml. In overall survival studies, univariate analyses demonstrated a prognostic value of sE-selectin, TS, and histoprognostic grading, and multivariate analyses demonstrated a prognostic value of sE-selectin and TS. For disease-free survival, univariate and multivariate analyses demonstrated a prognostic value of sE-selectin and TS. sE-selectin concentration is an easily measurable and strong prognostic factor in node-negative breast cancer patients. These results provide further evidence for the role of adhesion molecules expression by endothelial cells in tumor progression.

Prognostic value of p53 and urokinase-type plasminogen activator in node-negative human breast cancers.

We measured the levels of p53 and urokinase-type plasminogen activator (uPA) in 634 tumor tissues from 634 different node-negative primary breast cancer patients who underwent locoregional surgery in the Center Oscar Lambret between July 1989 and September 1994. p53 and uPA were assayed using commercially available kits in cytosols prepared for estradiol receptor (ER) and progesterone receptor (PgR) assays. The optimum clinical thresholds were chosen for prognostic studies: 4 ng/ml for p53 and 0.5 ng/ml for uPA. p53 was elevated in 13.7% of the tumors, and uPA was elevated in 27.5% of the tumors; they were negatively related (chi 2 test) to ER and PgR and positively related to histoprognostic grading (HPG) and tumor diameter. uPA was negatively correlated to ER and PgR, and p53 and uPA were positively correlated to each other (P = 0.0001; Spearman test). In the prognostic studies, the 316 patients who did not receive adjuvant chemotherapy were included to avoid treatment interference; this number corresponds to all of the patients operated on between 1989 and 1992. The mean duration of follow-up of living patients was 4 years. In overall survival studies, Cox univariate analyses demonstrated a prognostic value of p53 (P = 0.011; risk ratio, 1.59), uPA (P = 0.038; risk ratio, 2.32), PgR, HPG, and tumor diameter. In Cox multivariate analyses, only HPG had a statistically significant prognostic value. In relapse-free survival studies, univariate analyses demonstrated prognostic values of uPA (P = 0.0011) and of age, and both parameters retained their prognostic value in multivariate analyses (uPA: P = 0.0004). This study demonstrates not only that p53 and uPA have prognostic value but also that these two parameters are linked to other classical clinical, histological, or biological prognostic parameters, as well as to each other. Moreover, because uPA is of prognostic value in multivariate relapse-free survival studies, uPA is an important prognostic factor in node-negative breast cancer patients.

The relationship between concentrations of circulating soluble E-selectin and clinical, pathological, and biological features in patients with breast cancer.

Increasing evidence suggests that E-selectin contributes to tumor growth and metastasis. E-selectin may increase tumoral angiogenesis and the adhesion of tumoral cells to endothelial cells at distant sites. The aim of this study was to assess the relationship between concentrations of circulating soluble E-selectin (sE-selectin) and clinical, pathological, and biological features in patients with breast cancer (BC). Concentrations of sE-selectin were analyzed by an ELISA method in sera from 113 patients with metastatic BC, 30 patients with primary inflammatory BC, 105 patients with primary noninflammatory BC, and 42 healthy controls. These concentrations were analyzed in terms of the clinical and pathological features of the tumors as well as in terms of the concentrations of serum inflammatory parameters (erythrocyte sedimentation rate, C reactive protein, interleukin 1 beta, and tumor necrosis factor alpha), the response to chemotherapy or hormone therapy, and the survival duration. Tumoral angiogenesis was also assessed in 68 patients with primary noninflammatory BC who had had primary surgery. The mean concentration of sE-selectin in the metastatic BC group was significantly higher than the mean concentration found in the healthy control group (33.5 versus 21.8 ng/ml; P < 0.01). In metastatic BC, the mean concentration of sE-selectin was significantly higher in patients with liver metastasis than in patients without liver metastasis (55.3 versus 26.0 ng/ml; P < 10(-5). The univariate analysis showed that high concentrations of sE-selectin were associated with reduced overall survival (P < 0.05), but this probably reflected the association between high concentrations of sE-selectin and liver metastasis. In patients with primary noninflammatory BC, a negative correlation was found between sE-selectin concentrations and the tumoral microvessel count (r = -0.47; P = 10(-4). In patients with primary inflammatory or noninflammatory BC, no correlation was found between concentrations of sE-selectin and tumor size, lymph node involvement, response to chemotherapy or hormone therapy, and survival. No correlation was found between the concentrations of sE-selectin and serum inflammatory parameters in any of the patient groups. This study suggests that in patients with metastatic BC, levels of sE-selectin are higher in the presence of liver metastasis. In patients with primary BC, high concentrations seem to be associated with reduced tumoral angiogenesis. Although several studies have previously demonstrated that the expression of cell surface E-selectin enhances the metastatic process, the shedding of sE-selectin in circulation may be considered a mechanism of inhibition of tumor progression.

Milk from dams fed an obesogenic diet combined with a high-fat/high-sugar diet induces long-term abnormal mammary gland development in the rabbit.

Alterations to the metabolic endocrine environment during early life are crucial to mammary gland development. Among these environmental parameters, the initial nutritional event after birth is the consumption of milk, which represents the first maternal support provided to mammalian newborns. Milk is a complex fluid that exerts effects far beyond its immediate nutritional value. The present study, therefore, aimed to determine the effect of the nutritional changes during the neonatal and prepubertal periods on the adult mammary phenotype. Newborn rabbits were suckled by dams fed a high-fat/high-sugar obesogenic (OD) or a control (CON) diet and then subsequently fed either the OD or CON diets from the onset of puberty and throughout early pregnancy. Mammary glands were collected during early pregnancy (Day 8 of pregnancy). Rabbits fed with OD milk and then subjected to an OD diet displayed an abnormal development of the mammary gland: the mammary ducts were markedly enlarged (P < 0.05) and filled with abundant secretory products. Moreover, the alveolar secretory structures were disorganized, with an abnormal aspect characterized by large lumina. Mammary epithelial cells contained numerous large lipid droplets and exhibited fingering of the apical membrane and abnormally enlarged intercellular spaces filled with casein micelles. Leptin has been shown to be involved in modulating several developmental processes. We therefore analyzed its expression in the mammary gland. Mammary leptin mRNA was strongly expressed in rabbits fed with OD milk and subjected to an OD diet by comparison with the CON rabbits. Leptin transcripts and protein were localized in the epithelial cells, indicating that the increase in leptin synthesis occurs in this compartment. Taken together, these findings suggest that early-life nutritional history, in particular through the milking period, can determine subsequent mammary gland development. Moreover, they highlight the potentially important regulatory role that leptin may play during critical early-life nutritional windows with respect to long-term growth and mammary function.

cAMP effect on extracellular matrix synthesis in human breast cancer cells.

The effect of a cAMP derivative (N6, O2-dibutyryl cyclic adenosine 3',5'-monophosphate: dBcAMP) on the cell cycle and on the synthesis of typical extracellular matrix (ECM) components, i.e. collagen and glycosaminoglycans (GAG), was studied in two hormone-responsive human breast cancer cell lines VHB-1 and MCF-7. The data showed that dBcAMP induced a decrease in the proportion of cells in S + G2 + M phases due to an increase of the non-cycling (G0 phase) cell number as revealed by the Ki-67 antigen immunocytochemical study. The collagen synthesis, estimated by [3H] proline incorporation into the cellular proteins followed by an enzymatic digestion with highly purified bacterial collagenase, was not modified by dBcAMP. In contrast, the GAG synthesis, analysed by [3H] glucosamine incorporation, was increased two-fold in the dBcAMP treated cells. As a comparison we also tested 4-hydroxy-Tamoxifen (4-OH-Tam) since it induces similar cell cycle perturbations as dBcAMP. However, we did not observe a stimulation of the GAG synthesis following 4-OH-Tam treatment. These data demonstrated that the increased GAG synthesis is due to cAMP and is not a consequence of perturbations in the cell cycle. We can therefore assume that the ECM modifications induced by dBcAMP may contribute to the growth inhibition of the hormone-responsive human breast cancer cells.

ERBB2 oncogene in human breast cancer and its clinical significance.

We reveiwed the relationships between ERBB2 amplification and/or overexpression in human breast cancer and the clinicopathological parameters described in the literature (97 studies involving 22,616 patients) in order to draw conclusions regarding its clinical interest. The mean of ERBB2 positivity (26%, ranging from 5 to 55%) is not dependent on the method used to evaluate ERBB2 amplification or overexpression. Despite the discrepancies observed between the different studies, several associations between ERBB2 positivity and the classical clinicopathological parameters were noted. There are clear relationships between ERBB2 positivity and the lack of steroid receptors, the histological subtypes of mammary tumours (ductal invasive and in situ), worse histological and nuclear grades, aneuploidy and high rate of proliferation. In univariate analyses, ERBB2 is strongly associated with poor prognosis. All these data indicate that ERBB2 is a marker of aggressiveness of the tumour. However, ERBB2 does not retain a clinical prognostic significance in multivariate analyses, since it is associated with several strong prognostic parameters. When considering the prognostic value of ERBB2 in relation to treatment, a significantly worse survival of the treated patients is noted in ERBB2 positive patients. This suggest that ERBB2 could be a marker of reduced response to chemotherapy and hormonal treatment. With respect to the tumour response to treatment, the results, provided as yet by pilot studies, remain controversial and further investigations are necessary to evaluate the predictive value of ERBB2. Finally, new therapeutic approaches targeting the cells overexpressing ERBB2 have been developed.

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.

Plasma c-erbB2 concentrations in relation to chemotherapy in breast cancer patients.

Amplification and overexpression of the C-ERBB2 oncogene have been associated with a poor prognosis and a lower response to chemotherapy in human breast cancer. In this study, plasma c-erbB2 concentrations were determined using an enzyme immunoassay in patients with breast cancer. The links between c-erbB2 concentration and tumoral response to chemotherapy were established. The patients with a c-erbB2 concentration higher than the cut-off value (27 U/ml) were considered as c-erbB2+. Ten of the 33 metastatic breast cancers were c-erbB2+. No statistically significant difference in response to chemotherapy was noted between c-erbB2+ and c-erbB2- patients (4/10 objective responses versus 10/23). Variations in c-erbB2 concentrations during treatment were not related to response to treatment.

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.

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.

1,25-dihydroxyvitamin-d-3 and breast-cancer - growth-inhibition by 2 analogs (ro23-4319 and ro23-7553) and detection of receptors in routinely formalin-fixed paraffin-embedded material.

In addition to the regulation of calcium absorption and bone mineralization, the active form of vitamin D-3, 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3), has been shown to inhibit the proliferation and induce the differentiation of a wide range of normal and malignant cells via binding to a specific intracellular receptor. In this study, we demonstrated that the growth of estrogen receptor positive (MCF-7 and T47D) and negative (MDA-MB-231 and BT-20) human breast cancer cells was inhibited in a dose-dependent manner by 22-ene-1,25(OH)(2)D-3 (Ro23-4319) and 16-ene-23-yne-1,25(OH)(2)D-3 (Ro23-7553), two noncalcemic analogues of 1,25(OH)(2)D-3. Moreover, we showed that the antitumor effect exerted by the antiestrogen 4-hydroxytamoxifen was enhanced by Ro23-7553 in MCF-7 cells. Taken together, these results provide further evidence for the clinical interest of the noncalcemic analogues of 1,25(OH)(2)D-3 both for patients with estrogen receptor positive and negative breast tumors. These observations combined with the potential pronostic Value of the 1,25(OH)(2)D-3 receptor (VDR) status in breast cancer led us to test an immunohistochemical method performed on 32 routinely formalin-fixed paraffin-embedded breast tumor samples which were until now unusable for VDR detection. We compared this method, involving a pretreatment of the tissue sections in a microwave oven, with the conventional biochemical assay. Our results showed that breast tumors were massively stained (80% to 98% of the tumor cell nuclei) and that the parallelism observed between the staining intensity and the VDR concentration, could be proposed to either select a responsive population of patients or to carry out retrospective studies intended to specify the pronostic interest of VDR in breast cancer. We also demonstrated, as others, that no relationship existed between the presence of VDR and the age of the patients, the presence of estrogen and progesterone receptors and the lymph node involvement.

Effect of calcium on e-cadherin expression in breast-tumor cells.

E-cadherins are homophilic adhesion molecules the expression of which is tightly linked to the invasiveness and the differentiated state of the cells. E-cadherin expression seems also inversely related to the expression of vimentin, an intermediate filament implicated in the metastatic potential of some cells. In breast tumor cells MCF-7, we have previously shown that calcium influences cell growth and promotes cell differentiation. In view of the importance of cell adhesion mechanisms in cell growth and invasion, we sought to determine whether calcium affects the regulation of E-cadherin expression and modifies the relationship between E-cadherin and vimentin expression. To address this question, cells were grown at low (0.04 mM) or high (2.5 mM) concentration of calcium and cadherin and vimentin expression was assessed by flow cytometry analyses. Our results show that calcium enhances cadherin expression in cadherin positive cells and decreases vimentin expression in these cells; in cadherin negative cells, calcium only decreases the expression of vimentin. The modifications of E-cadherin and/or vimentin expression suggests that drugs that can modify intracellular calcium may contribute to overcoming the progression of breast tumor cells toward increasingly malignant phenotypes.