Adrenergic compounds (epinephrine and norepinephrine) are the most important hormones released during stress. Several different receptors are associated with their action in different tissues. However, alpha(2)-adrenoceptors have not yet been described in either normal or tumour human breast tissue. The aim of this work was to describe and characterize these receptors in several tumour and non-tumour human cell lines. The expression of alpha(2)-adrenoceptors was analyzed at the RNA (RT-PCR) and protein ([(3)H]-rauwolscine binding and immunocytochemistry) levels in different human breast cell lines, and the biological activity assessed by [(3)H]-thymidine incorporation. The cancer IBH-6, IBH-7 and MCF-7 and the non-tumour HBL-100 cells line, expressed both alpha(2B)- and alpha(2C)-adrenoceptor-subtypes. A single subtype was expressed in malignant HS-578T (alpha(2A)) and MDA-MB-231 and non-tumour MCF-10A cells (alpha(2B)). All cell lines exhibited significant binding for the specific antagonist [(3)H]-rauwolscine. The alpha-, alpha(2)-, and the alpha(1)-compounds with known affinity for alpha(2)-adrenoceptors, including epinephrine, norepinephrine, yohimbine, clonidine, rauwolscine and prazosin, competed significantly with binding in MCF-7 cells. In addition, IBH-6, IBH-7 and MCF-7 cells showed significant staining with specific antibodies against alpha(2B)- and alpha(2C)-adrenoceptor-subtypes, when tested by immunocytochemistry. In all cell lines, the specific agonist clonidine or oxymetazoline stimulated [(3)H]-thymidine incorporation. EC(50) values were in the range of 20-50 fM for IBH-6, IBH-7, and HS-578T; 0.14 pM for MCF-7; 2-82 pM for HBL-100 and MCF-10A cells, and a biphasic behaviour with a maximum value at 38.0 pM, was observed for MDA-MB-231 cells. The specific alpha(2)-adrenergic antagonist rauwolscine always reversed this stimulation at 0.1 nM. In conclusion, this study describes for the first time, the presence of alpha(2)-adrenoceptors in human epithelial breast cell lines. Moreover, activation of these receptors was associated with an enhancement of cell proliferation.
The histamine H2 receptor (H2r) belongs to the heptahelical receptor family; upon agonist binding, members of this family activate a G protein and the downstream effector adenylyl cyclase. Like other G protein-coupled receptors, exposure of H2r to agonists produces a desensitization of the response. The present study focused on the desensitization mechanism of this receptor. Using transiently transfected COS-7 cells expressing tagged-H2r, the desensitization induced by amthamine, characterized by decreased cAMP production, was studied. Results show that the receptor was rapidly desensitized with a t(1/2) = 0.49 +/- 0.01 min. Because of the rapid nature of H2r desensitization, receptor phosphorylation was examined as a likely mechanism for signal attenuation. H2r desensitization was not affected by protein kinases A and C (PKA and PKC) inhibitors but was remarkably reduced by Zn(2+), an inhibitor of G protein-coupled receptor kinases (GRKs). Cotransfection experiments using tagged H2r and different GRKs (2, 3, 5, or 6), demonstrated that GRK2 and GRK3 were the most potent in augmenting desensitization, causing a reduction in the maximal response to amthamine and a decrease of the t(1/2) for desensitization, whereas GRK5 and GRK6 did not affect the signaling. Receptor phosphorylation correlates with desensitization for each GRK studied, whereas phosphorylation that is dependent on protein kinases A and C seemed irrelevant in receptor signal termination. These results indicate that in H2r-transfected COS-7 cells, exposure to an agonist caused desensitization controlled by H2r phosphorylation via GRK2 and GRK3.
We examined the effects of histamine and its agonists on the expression of the c-fos and c-myc proto-oncogenes at the transcriptional and translational levels in the human promonocytic U937 cell line. Histamine transiently increased cAMP and c-fos expression through H2 receptors. Dibutyryl cAMP also increased c-fos mRNA and protein, and levels remained elevated even after 12 hr of treatment. Dose-dependence studies using histamine and dimaprit showed that the EC50 values for cAMP production and c-fos increase were similar, suggesting that cAMP might be involved in c-fos induction via H2 receptors. Furthermore, studies carried out using H7, a protein kinase A/protein kinase C inhibitor, blocked c-fos induction, whereas no effect was observed with bisindolylmaleimide, a specific protein kinase C inhibitor. No modification of c-myc expression could be detected on treatment with histamine or its analogues. Nevertheless, dibutyryl cAMP induced a down-regulation of the levels of this proto-oncogene. In addition, dibutyryl cAMP inhibited cell growth in a dose-dependent manner, whereas histamine failed to affect proliferation and differentiation of U937 cells. Cells pretreated with dimaprit showed a decrease in the cAMP response to subsequent addition of H2 agonists, whereas the cAMP response to prostaglandin E2 remained unaltered. This homologous mechanism of H2 receptor desensitization was time dependent. These results indicate that histamine activates several mechanisms involved in the induction of differentiation, such as cAMP and c-fos production, but fails to promote differentiation of U937 cells, apparently due to the rapid desensitization of H2 receptors.
Human breast cancer primary cultures are useful tools for the study of several aspects of cancer biology, including the effects of chemotherapy and acute gene expression in response to different hormonal/chemotherapy treatments. The present study reports the conditions for primary culture of breast cancer samples from untreated patients and the most effective collagenization method to dissociate human samples consisting in an overnight incubation with 1 mg/ml types II or IV collagenase and further incubation in DMEM:F12 (1:1) medium supplemented with glutamine, bovine insulin, penicillin-streptomycin, HEPES, estradiol, cortisol (F), tri-iodothyronine (T(3)), transferrine (TR), and 10% fetal calf serum (FCS). These conditions proved to be appropriate for both primary culture and the development of stable cell lines. Of the seven cell lines obtained, three fast growing and estrogen receptor (ER)+/progesterone receptor (PgR)+/EGF receptor (EGFR)+ have been characterized. The cells are able to grow both in soft agar and in nude mice, and express cytokeratins, all parameters characteristic of malignant epithelial cell lines. The cells also exhibit an increased proliferation rate in the presence of estradiol, progesterone, and EGF, suggesting the presence of the corresponding receptors. The mRNA expression of type alpha- and beta-ER as well as EGFR, was confirmed by RT-PCR. In conclusion, the novel cell lines described, arose from primary tumors and are sensitive to estradiol, progesterone, and EGF. This not only expands the repertoire of breast cancer cells available as potentially useful tools for examining most parameters in breast cancer "in vitro", but also provides unique new models to explore the complex regulation by steroids as well as growth factors in such cells.
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