Isabel Alicia Luthy

Effect of medroxyprogesterone acetate (MPA) and serum factors on cell proliferation in primary cultures of an MPA-induced mammary adenocarcinoma.

SummaryThe effect of progesterone (Pg), medroxyprogesterone acetate (MPA), estradiol (E2), dihydrotestosterone (DHT) and dexamethasone (DEXA) was studied on thein vitro growth rate of a progestin-dependent (PD), estrogen-sensitive mammary tumor line originated in an MPA-treated BALB/c mouse (C4-HD), and on its estrogen-resistant variant (C4-HDR). The specificity of hormone action was further investigated using the anti-hormones RU-486 and hydroxyflutamide (FLU). Cell growth was evaluated in epithelial and fibroblastenriched cultures using3H-thymidine and/or autoradiography and immunocytochemistry. The results indicate that cell growth is directly stimulated by MPA and Pg at concentrations ranging from 10−11 to 10−7 M. RU486 prevented MPA-induced stimulation in concentrations 10 to 100 fold lower than those of MPA. When used alone, it inhibited cell proliferation only in concentrations higher than 10−11 M. At nM concentrations, neither DEXA nor DHT stimulated3H-thyrnidine uptake except DEXA at 100 nM. MPA-induced stimulation was not reverted by micromolar concentrations of FLU. As for E2 (10−7–10−9 M) it prevented MPA stimulation only in cultures of estrogen-sensitive tumors. Progesterone receptors (PR) (475 ± 115 fmoles/105 cells, n = 5) and estrogen receptors (ER) (ND-115 fmoles/105 cells, n = 5) were detected only in epithelial-enriched cultures. Serum from 7 day-MPA-treated mice induced a significant increase of3H-thymidine uptake; an increase was also obtained with serum from untreated ovariectomized animals to which 1 nM-100 nM concentrations of MPA had been added. The stimulatory effect of the exogenous MPA was much lower than that of the serum obtained from MPA-treated animals.It is concluded that MPA stimulates cell growth of primary cultures of MPA-induced PD tumors via PR. The results provide support for a direct effect of MPA which may be mediated or potentiated by serum factors.

α2-Adrenoceptor action on cell proliferation and mammary tumour growth in mice

Breast cancer, the most common cancer in women in most countries, is a highly stressful disease. Catecholamines released during stress bind to adrenoceptors and we have recently described α2‐adrenoceptors in human breast cell lines, linked to enhanced cell proliferation. The purpose was to assess the in vivo effects of compounds acting on α2‐adrenoceptors in a reliable model of breast cancer.

Involvement of α2- and β2-adrenoceptors on breast cancer cell proliferation and tumour growth regulation.

BACKGROUND AND PURPOSE β‐Adrenoceptors are expressed in human and experimental animal breast cancer cells. However, the effect of the agonists and antagonists reported on cell proliferation and tumour growth was paradoxical, precluding their utilization as possible adjuvant therapy, mainly in the cases of refractory tumours.

Progesterone receptor involvement in independent tumor growth in MPA-induced murine mammary adenocarcinomas

We have developed a model of hormonal carcinogenesis in BALB/c female mice, in which MPA induced ductal mammary adenocarcinomas, expressing high levels of estrogen and progesterone receptors (ER and PR). A series of tumor lines, retaining both PR and ER expression, were obtained from selected tumors, which are maintained by syngeneic passages. In this model progesterone behaves as the growth-stimulating hormone (progesterone-dependent or PD tumors), whereas estrogens induce tumor regression. Through selective treatments we were able to derive a series of progesterone-independent (PI) variants. These lines do not require progesterone treatment to grow in ovariectomized female BALB/c mice, but retain, however, the expression of ER and PR. The aim of this paper is to investigate a possible regulatory role of the progesterone receptor (PR) on PI tumor growth. ER and PR were detected by immunocytochemistry in all lines studied. They were also characterized using biochemical assays and Scatchard plots. No differences in Kd of PR or ER were detected in PI variants. AR or GR were not detected in tumor samples using biochemical assays. Estradiol (5 mg silastic pellet) induced complete tumor regression in all tumors tested. We also evaluated the effects of different antiprogestins on tumor growth. Onapristone (10 mg/kg/day) and mifepristone (4.5 mg/kg/day) were able to induce complete tumor regression. The antiandrogen flutamide (5 mg silastic pellet) had no effect on tumor growth in agreement with the lack of androgen receptors. We used an in vitro approach to corroborate that the antiprogestin-induced inhibition was not attributable to an intrinsic effect. Cultures of a selected PI line were treated with PR antisense oligodeoxynucleotides (ASPR) to inhibit in vitro cell proliferation. A significant decrease of 3H-thymidine uptake was observed in cells of a PI line growing in the presence of 2.5% charcoalized fetal calf serum and 0.8-20 microg/ml ASPR. It can be concluded that the PR pathway is an essential path in the growth stimulation of PI tumors.

Adrenoceptors: Non Conventional Target for Breast Cancer?

Epinephrine and Norepinephrine, typically released during stress bind to nine different adrenoceptors (AR) which classically control the cardiovascular and respiratory systems. New targets were described for the many agonists and antagonists developed for these AR, as the central nervous system. During the last three decades, AR expression and action on the mammary gland/breast were extensively investigated. In the cow mammary gland, good milkability was associated with low density of beta(2)-AR and high density of alpha(2)-AR. In the rat normal mammary gland, beta-AR are expressed in the epithelial cells, alveoli, ducts, and adipocytes showing an exquisite regulation by steroid hormones and prolactin. In rat dimethylbenz(a)anthracene (DMBA) tumors, a close correlation was observed between tumor growth and beta-AR concentration. beta(2)-AR were described in numerous human cell lines and breast tumors. The action of beta-adrenergic compounds on cell proliferation is contradictory. While some authors found that beta-agonists significantly inhibit cancer cell proliferation and tumor growth in mice, others described a significant reduction in DNA synthesis by beta-blockers. Also, positive effects of beta-AR on human carcinoma cell migration have been described. alpha(2)-AR are expressed in human breast cancer and non-cancer cell lines, their stimulation being associated with increased cell proliferation. In vivo clonidine increased tumor growth and alpha (2)-adrenergic antagonists completely reversed this effect. When administered alone, rauwolscine inhibited tumor growth behaving as an inverse agonist. Therefore, the numerous adrenergic beta- and alpha-AR agonists or antagonists could prove to be unexpected therapeutic options for mammary gland/ breast and mainly breast cancer.

PI3K/AKT pathway regulates phosphorylation of steroid receptors, hormone independence and tumor differentiation in breast cancer

Using a model of medroxyprogesterone acetate (MPA)-induced mouse mammary tumors that transit through different stages of hormone dependence, we previously reported that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT (protein kinase B) pathway is critical for the growth of hormone-independent (HI) mammary carcinomas but not for the growth of hormone-dependent (HD) mammary carcinomas. The objective of this work was to explore whether the activation of the PI3K/AKT pathway is responsible for the changes in tumor phenotype and for the transition to autonomous growth. We found that the inhibition of the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway suppresses HI tumor growth. In addition, we were able to induce mammary tumors in mice in the absence of MPA by inoculating HD tumor cells expressing a constitutively active form of AKT1, myristoylated AKT1 (myrAKT1). These tumors were highly differentiated and displayed a ductal phenotype with laminin-1 and cytokeratin 8 expression patterns typical of HI tumors. Furthermore, myrAKT1 increased the tumor growth of IBH-6 and IBH-7 human breast cancer cell lines. In the estrogen-dependent IBH-7 cell line, myrAKT1 induced estrogen-independent growth accompanied by the expression of the adhesion markers focal adhesion kinase and E-cadherin. Finally, we found that cells expressing myrAKT1 exhibited increased phosphorylation of the progesterone receptor at Ser190 and Ser294 and of the estrogen receptor α at Ser118 and Ser167, independently of exogenous MPA or estrogen supply. Our results indicate that the activation of the PI3K/AKT/mTOR pathway promotes tissue architecture remodeling and the activation of steroid receptors.

Three Novel Hormone-Responsive Cell Lines Derived From Primary Human Breast Carcinomas: Functional Characterization

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 (T3), 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 α‐ and β‐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.

Prolactin binding sites in the brain and kidneys of the toad, Bufo arenarum Hensel

Summary(125I)-ovine prolactin (oPRL) binding was found in several brain areas of the toad,Bufo arenarum Hensel. The olfactory bulb, cerebral hemispheres, and both dorsal and ventral mesencephalic regions showed saturable, high affinity, (125I)-oPRL binding, ranging between 5.6 to 29.9 fmol/mg protein, while the association constant (Ka) by Scatchard analysis was between 4.0 to 8.7×109 M−1. This binding was compared with the Scatchard plot of the kidney, which has been already described by other groups, and gave 41.7 fmol/mg protein andKa 2.5×109 M−1. Liver showed no binding and in the cerebral hemispheres (125I)-oPRL was not displaced by non-lactogenic hormones, indicating that binding was hormone and tissue specific.

α2-Adrenoceptors Enhance Cell Proliferation and Mammary Tumor Growth Acting Through both the Stroma and the Tumor Cells

We have previously described enhanced human breast cancer cell proliferation and mouse mammary tumor growth induced by α(2)-adrenoceptor (α(2)-AR) expression in epithelial cells. The aim of the present work was to assess if stromal fibroblasts can contribute to this effect. α(2)-AR expression was assessed by immunocytochemistry and immunohistochemistry, cell proliferation by [(3)H]-Thymidine incorporation and tumor growth by measuring with caliper. All tested mouse and human fibroblasts expressed at least two α(2)-AR subtypes and α(2)-adrenergic agonists enhanced fibroblast proliferation. In vivo, the α(2)-adrenergic agonist clonidine significantly enhanced tumor growth. The α(2)-adrenergic antagonist rauwolscine reversed this effect, but when administered alone, significantly inhibited tumor growth. Clonidine significantly stimulated cell proliferation in the epithelial-enriched fraction, the cancer associated fibroblast-enriched fraction and the co-culture of both fractions in primary cultures from both tumors (IBH-4 and IBH-6). Rauwolscine reversed clonidine stimulation in every fraction. However, when incubated alone, the inhibitory effect was observed in fractions from IBH-4 tumors but not from IBH-6 tumors. These experiments show that fibroblasts from tumor stroma are also influenced by α(2)-adrenergic compounds through the α(2)-ARs expressed in these cells. Moreover, the α(2)-adrenergic antagonist rauwolscine could eventually block in both epithelial and stromal cells, the mitogenic effect of catecholamines released during stress, providing a potential additional treatment for breast cancer patients. Chemists synthesizing adrenergic compounds should consider their action in breast cancer patients.

Dosage-dependent regulation of cell proliferation and adhesion through dual β2-adrenergic receptor/cAMP signals

The role of β‐adrenergic receptors (β‐ARs) remains controversial in normal and tumor breast. Herein we explore the cAMP signaling involved in β ‐AR‐dependent control of proliferation and adhesion of nontumor human breast cell line MCF‐10A. Low concentrations of a β‐agonist, isoproterenol (ISO), promote cell adhesion (87.5% cells remaining adherent to the plastic dishes following specific detachment vs. 35.0% in control, P<0.001), while increasing concentrations further engages an additional 36% inhibition of Erk1/2 phosphorylation (p‐Erk1/2)‐dependent cell proliferation (P<0.01). Isoproterenol dose response on cell adhesion was fitted to a 2‐site curve (EC50(1): 16.5±11.5 fM, EC50(2): 4.08 ±3.09 nM), while ISO significantly inhibited p‐Erk1/2 according to a 1‐site model (EC50: 0.25 ±0.13 nM). Using β‐AR‐selective agonist/antagonists and cAMP analogs/inhibitors, we identified a dosage‐dependent signaling in which low ISO concentrations target a β2‐AR population localized in raft microdomains and stimulate a Gs/cAMP/Epac/ adhesion‐signaling module, while higher concentrations engage a concomitant activation of another β2‐AR population outside rafts and inhibit the proliferation by a Gs/cAMP/PKA‐dependent signaling module. Our data provide a new molecular basis for the dose‐dependent switch of β‐AR signaling. This study also sheds light on a new cAMP pathway core mechanism with a single receptor triggering dual cAMP signaling controlled by PKA or Epac but with different cellular outputs.—Bruzzone, A., Saulière, A., Finana, F., Sénard, J.‐M., Lüthy, I., Galés, C. Dosage‐dependent regulation of cell proliferation and adhesion through dual β2‐adrenergic receptor/cAMP signals. FASEB J. 28, 1342–1354 (2014). www.fasebj.org