Glucocorticoid alternative effects on proliferating and differentiated mammary epithelium are associated to opposite regulation of cell-cycle inhibitor expression

Glucocorticoids influence post-natal mammary gland development by sequentially controlling cell proliferation, differentiation, and apoptosis. In the mammary gland, it has been demonstrated that glucocorticoid treatment inhibits epithelial apoptosis in post-lactating glands. In this study, our first goal was to identify new glucocorticoid target genes that could be involved in generating this effect. Expression profiling, by microarray analysis, revealed that expression of several cell-cycle control genes was altered by dexamethasone (DEX) treatment after lactation. Importantly, it was determined that not only the exogenous synthetic hormone, but also the endogenous glucocorticoids regulated the expression of these genes. Particularly, we found that the expression of cell cycle inhibitors p21CIP1, p18INK4c, and Atm was differentially regulated by glucocorticoids through the successive stages of mammary gland development. In undifferentiated cells, DEX treatment induced their expression and reduced cell proliferation, while in differentiated cells this hormone repressed expression of those cell cycle inhibitors and promoted survival. Therefore, differentiation status determined the effect of glucocorticoids on mammary cell fate. Particularly, we have determined that p21CIP1 inhibition would mediate the activity of these hormones in differentiated mammary cells because over-expression of this protein blocked DEX-induced apoptosis protection. Together, our data suggest that the multiple roles played by glucocorticoids in mammary gland development and function might be at least partially due to the alternative roles that these hormones play on the expression of cell cycle regulators.     

Sensitivity to DNA damage is a common component of hormone-based strategies for protection of the mammary gland

An early full-term pregnancy significantly reduces the risk of getting breast cancer in women. In animals, this protection can be mimicked by a short-term exposure to physiologic doses of estrogen plus progesterone. Sensitization of p53 and up-regulation of transforming growth factor beta are believed to be important aspects of the mechanism by which protection is imparted. Little is known, however, about the use of this pathway in response to other chemopreventive agents. In this article, we investigated the ability of retinoids, such as 9-cis retinoic acid, all-trans retinoic acid, and N-4-hydroxyphenylretinamide (4-HPR), to sensitize the ductal epithelial cells of virgin mammary glands to DNA damage responses. Using a whole-organ culture system, we observed enhanced cell death in response to gamma-irradiation in the virgin tissues treated with retinoids for 72 hours. These retinoids were partially dependent on p53 and transforming growth factor beta to exert their radiosensitizing effects. However, 4-HPR seemed to sensitize other cells or activate these pathways in a different manner as costimulation with ovarian hormones and 4-HPR was additive, whereas coculture of ovarian hormones and the natural retinoids did not increase amount of death. Taken together, these data suggest that sensitization of the mammary epithelium to p53-dependent apoptosis is a common pathway, which is engaged by retinoids as well as ovarian hormones.     

Developmental role of the SNF1-related kinase Hunk in pregnancy-induced changes in the mammary gland

The steroid hormones 17 beta-estradiol and progesterone play a central role in the pathogenesis of breast cancer and regulate key phases of mammary gland development. This suggests that developmental regulatory molecules whose activity is influenced by ovarian hormones may also contribute to mammary carcinogenesis. In a screen designed to identify protein kinases expressed in the mammary gland, we previously identified a novel SNF1-related serine/threonine kinase, Hunk (hormonally upregulated Neu-associated kinase). During postnatal mammary development, Hunk mRNA expression is restricted to a subset of mammary epithelial cells and is temporally regulated with highest levels of expression occurring during early pregnancy. In addition, treatment of mice with 17 beta-estradiol and progesterone results in the rapid and synergistic upregulation of Hunk expression in a subset of mammary epithelial cells, suggesting that the expression of this kinase may be regulated by ovarian hormones. Consistent with the tightly regulated pattern of Hunk expression during pregnancy, mammary glands from transgenic mice engineered to misexpress Hunk in the mammary epithelium manifest temporally distinct defects in epithelial proliferation and differentiation during pregnancy, and fail to undergo normal lobuloalveolar development. Together, these observations suggest that Hunk may contribute to changes in the mammary gland that occur during pregnancy in response to ovarian hormones.     

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells.     

Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland

Milk production during lactation is a consequence of the suckling stimulus and the presence of glucocorticoids, prolactin, and insulin. After weaning the glucocorticoid hormone level drops, secretory mammary epithelial cells die by programmed cell death and the gland is prepared for a new pregnancy. We studied the role of steroid hormones and prolactin on the mammary gland structure, milk protein synthesis, and on programmed cell death. Slow-release plastic pellets containing individual hormones were implanted into a single mammary gland at lactation. At the same time the pups were removed and the consequences of the release of hormones were investigated histologically and biochemically. We found a local inhibition of involution in the vicinity of deoxycorticosterone- and progesterone-release pellets while prolactin-release pellets were ineffective. Dexamethasone, a very stable and potent glucocorticoid hormone analogue, inhibited involution and programmed cell death in all the mammary glands. It led to an accumulation of milk in the glands and was accompanied by an induction of protein kinase A, AP-1 DNA binding activity and elevated c-fos, junB, and junD mRNA levels. Several potential target genes of AP-1 such as stromelysin-1, c-jun, and SGP-2 that are induced during normal involution were strongly inhibited in dexamethasone-treated animals. Our results suggest that the cross-talk between steroid hormone receptors and AP-1 previously described in cells in culture leads to an impairment of AP-1 activity and to an inhibition of involution in the mammary gland implying that programmed cell death in the postlactational mammary gland depends on functional AP-1.     

Pim-1 kinase expression during murine mammary development

Pim-1 kinase phosphorylates substrates whose activities are linked to proliferation, survival, differentiation, and apoptosis. Although pim-1 is induced by hormones and cytokines, the hormonal control and contribution of Pim-1 to mammary gland development have not been evaluated. We examined Pim-1 expression in mammary cell lines, investigated whether Pim-1 levels could be altered in breast epithelia by mammogenic hormones, and evaluated Pim-1 expression during mammary development. We found that Pim-1 was elevated in most mammary carcinoma cell lines and progesterone increased Pim-1 protein to some extent in non-tumorigenic mammary epithelia. Pim-1 expression in situ was consistent with the documented profile of progesterone activity in mouse mammary glands. Pim-1 nuclear localization correlated with cytoplasmic distribution for its substrate, p21(CIP/Waf1), and we found that Pim-1 and p21 associate in vitro. Our results suggest that Pim-1 expression may be regulated by progesterone during mammary development and Pim-1 associates with p21 in mammary epithelial cells.     

Role of steroid hormones and prolactin in canine mammary cancer

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.     

Vitamin D(3) receptor ablation alters mammary gland morphogenesis

Postnatal mammary gland morphogenesis is achieved through coordination of signaling networks in both the epithelial and stromal cells of the developing gland. While the major proliferative hormones driving pubertal mammary gland development are estrogen and progesterone, studies in transgenic and knockout mice have successfully identified other steroid and peptide hormones that impact on mammary gland development. The vitamin D(3) receptor (VDR), whose ligand 1,25-dihydroxyvitamin D(3) is the biologically active form of vitamin D(3), has been implicated in control of differentiation, cell cycle and apoptosis of mammary cells in culture, but little is known about the physiological relevance of the vitamin D(3) endocrine system in the developing gland. In these studies, we report the expression of the VDR in epithelial cells of the terminal end bud and subtending ducts, in stromal cells and in a subset of lymphocytes within the lymph node. In the terminal end bud, a distinct gradient of VDR expression is observed, with weak VDR staining in proliferative populations and strong VDR staining in differentiated populations. The role of the VDR in ductal morphogenesis was examined in Vdr knockout mice fed high dietary Ca(2+) which normalizes fertility, serum estrogen and neonatal growth. Our results indicate that mammary glands from virgin Vdr knockout mice are heavier and exhibit enhanced growth, as evidenced by higher numbers of terminal end buds, greater ductal outgrowth and enhanced secondary branch points, compared with glands from age- and weight-matched wild-type mice. In addition, glands from Vdr knockout mice exhibit enhanced growth in response to exogenous estrogen and progesterone, both in vivo and in organ culture, compared with glands from wild-type mice. Our data provide the first in vivo evidence that 1,25-dihydroxyvitamin D(3) and the VDR impact on ductal elongation and branching morphogenesis during pubertal development of the mammary gland. Collectively, these results suggest that the vitamin D(3) signaling pathway participates in negative growth regulation of the mammary gland.     

Novel role for RbAp48 in tissue-specific, estrogen deficiency-dependent apoptosis in the exocrine glands

Although tissue-specific apoptosis in the exocrine glands in estrogen-deficient mice may contribute to the development of autoimmune exocrinopathy, the molecular mechanism responsible for tissue-specific apoptosis remains obscure. Here we show that RbAp48 overexpression induces p53-mediated apoptosis in the exocrine glands caused by estrogen deficiency. RbAp48-inducible transfectant results in rapid apoptosis with p53 phosphorylation (Ser9) and alpha-fodrin cleavage. Reducing the expression of RbAp48 through small interfering RNA inhibits the apoptosis. Prominent RbAp48 expression with apoptosis was observed in the exocrine glands of C57BL/6 ovariectomized (OVX) mice but not in OVX estrogen receptor alpha(-/-), p53(-/-), and E2F-1(-/-) mice. Indeed, transgenic expression of the RbAp48 gene induced apoptosis in the exocrine glands but not in other organs. These findings indicate that estrogen deficiency initiates p53-mediated apoptosis in the exocrine gland cells through RbAp48 overexpression and exerts a possible gender-based risk of autoimmune exocrinopathy in postmenopausal women.     

Mitogenic activity of pituitary hormones on cell cultures of normal and carcinogen-induced tumor epithelium from rat mammary glands

Cell suspension containing normal or tumor epithelium were readily obtained by enzymatically digesting rat mammary glands from perphenazine-treated (prolactin-hypersecreting) cycling, female virgin animals or hormone- responsive mammary tumors from animal treated with dimethylbenzanthracene. Cell suspensions were fractioned into predominantly epithelial and predominantly stromal cells by their differential rates of attachment to culture dishes. Both normal mammary and tumor epithelial cells were characterized by the presence of specific cell-junctional complexes, desmosome-like structures, surface microvilli, and their ability to synthesize casein. Serum-dependent protease activity was greater in cultures derived from tumors, and cells from such cultures grew in agarose whereas those from the non-neoplastic gland did not. The addition of prolactin to the culture medium stimulated DNA synthesis in primary or secondary epithelial cultures from tumors, whereas additional insulin and hydrocortisone with prolactin were required for similar levels of DNA synthesis in cultures from non-neoplastic glands. The fraction of cells synthesizing DNA was, however, smaller than that with 10 percent serum measured in the same time period. Both growth hormone and epidermal growth factor stimulated DNA synthesis but to a lesser extent than did prolactin. Prolactin with hydrocortisone and insulin were relatively inactive in promoting DNA synthesis of the nonepithelial cells whereas pituitary fibroblast growth factor was more active. These mitogenic effects were obtained when the hormones were added to the medium at near physiological concentrations, and paralleled the known activities of the hormones in control of mammary gland growth and development in the rat.     

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.     

MDM2 is required for suppression of apoptosis by activated Akt1 in salivary acinar cells

Chronic damage to the salivary glands is a common side effect following head and neck irradiation. It is hypothesized that irreversible damage to the salivary glands occurs immediately after radiation; however, previous studies with rat models have not shown a causal role for apoptosis in radiation-induced injury. We report that etoposide and gamma irradiation induce apoptosis of salivary acinar cells from FVB control mice in vitro and in vivo; however, apoptosis is reduced in transgenic mice expressing a constitutively activated mutant of Akt1 (myr-Akt1). Expression of myr-Akt1 in the salivary glands results in a significant reduction in phosphorylation of p53 at serine(18), total p53 protein accumulation, and p21(WAF1) or Bax mRNA following etoposide or gamma irradiation of primary salivary acinar cells. The reduced level of p53 protein in myr-Akt1 salivary glands corresponds with an increase in MDM2 phosphorylation in vivo, suggesting that the Akt/MDM2/p53 pathway is responsible for suppression of apoptosis. Dominant-negative Akt blocked phosphorylation of MDM2 in salivary acinar cells from myr-Akt1 transgenic mice. Reduction of MDM2 levels in myr-Akt1 primary salivary acinar cells with small interfering RNA increases the levels of p53 protein and renders these cells susceptible to etoposide-induced apoptosis in spite of the presence of activated Akt1. These results indicate that MDM2 is a critical substrate of activated Akt1 in the suppression of p53-dependent apoptosis in vivo.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.     

Mammary gland Ca2+-binding (-dependent) proteins: Identification as calelectrins and calpactin I/p36

Calcium-binding (-dependent) proteins (CBPs) associated with the spreading of mammary epithelial cell cultures have been identified as various calelectrins and calpactins (p36). In immunoblot analysis, the CBPs of 30–36 kD and 68–70 KD variously react with different calelectrin and calpactin I monomer/p36 antisera. The same immunoreactive proteins were shown to be present in virgin mammary glands and collagen gel mouse mammary epithelial cell cultures. The mammary CBPs show extensive immunochemical relatedness; however, they fail to show cross-reaction with antiserum to calpactin II (lipocortin) antiserum. These immunoreactive CBPs comigrate in electrophoresis with ³⁵S-methionine-labeled CBPs isolated from mammary epithelial cell cultures. Unlike calmodulin, the mammary CBPs that correspond to calelectrins and calpactin I monomer/p36 are not stable to thermal denaturation. The mammary CBPs bind to epithelial cell membranes in a Ca²⁺-dependent manner and are differentially released from ruptured cells, compared with calmodulin, suggesting subcellular localization. Phenothiazineagarose and phenylagarose are equivalent in their ability to bind the mammary CBPs. Thus, mammary gland CBPs of 30–36 kD and 68–70 kD have been shown to be related or equivalent to the calelectrins and to calpactin I monomer/p36. Since these proteins are known to bind Ca²⁺, we conclude that the mammary gland CBPs are also Ca²⁺-binding proteins. The mammary gland CBPs are immunologically related and probably represent members of a larger family of related proteins.     

The lack of G1/S arrest of teratocarcinoma F9 cells is determinated by degradation of cyclin-dependent kinases inhibitor p21waf1/cip1

We have studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints following gamma-irradiation. Wild-type p53 protein is rapidly accumulated in F9 cells after gamma-irradiation, however this is not followed by G1/S arrest; there is just a reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells we investigated the levels of regulatory cell cycle proteins: G1-cyclins, cyclin dependent kinases and kinase inhibitor p21WAF1/CIP1. We have shown that in spite of p53-dependent activation of p21WAF1/CIP1 promoter, p21WAF1/CIP1 protein is not revealed by different polyclonal and monoclonal antibodies, either by immunoblotting or by immunofluorescent staining. However, when cells are treated with specific proteasome inhibitor lactacystin, p21WAF1/CIP1 protein is revealed. We therefore suggest that p21WAF1/CIP1 protein is subjected to proteasome degradation in F9 cells and probably the lack of G1/S arrest after gamma-irradiation is due to this degradation. Thus, it is the combination of functionally active p53 with low level expression of p21WAF1/CIP1 that causes a short delay of the cell cycle progression in G2/M, rather than the G1-arrest after gamma-irradiation of F9 cells.     

Metabolism of the chemopreventive retinoid N-(4-hydroxyphenyl)retinamide by mammary gland in organ culture.

N-(4-Hydroxyphenyl)retinamide (4-HPR) is considered to be the most effective chemopreventive retinoid for chemically induced mammary carcinogenesis in rats. However, the mechanism of 4-HPR action in mammary cells is poorly understood. In the present study we examined the metabolism of 4-HPR in the mouse mammary gland in organ culture. Mammary glands excised from BALB/c mice were incubated with 4-HPR in the presence of insulin, prolactin and steroid hormones for 6 days. The glands were extracted with chloroform/methanol (2:1, v/v), and the metabolites were separated on a reversed-phase h.p.l.c. column. Three metabolites were separated in addition to 4-HPR; one of the metabolites, M2, was co-eluted with 13-cis-4-HPR, M3 was co-eluted with N-(4-methoxyphenyl)retinamide (4-MPR) and M1 remains unidentified. There appeared to be some hormonal regulation in the distribution of metabolites in the glands. Increased levels of 4-MPR and M1 were observed in insulin-plus-prolactin-treated glands as compared with the glands incubated with steroid hormones. Furthermore, it was observed that M1 isolated from the livers of 4-HPR-treated rats competed for the cellular retinoic acid-binding protein (CRABP) sites; however, 4-HPR did not bind to CRABP. These results indicate that mouse mammary gland can metabolize 4-HPR and that the metabolites which compete for CRABP sites may have physiological significance in the retinoid inhibition of mammary carcinogenesis.     

Cell cycle progression and apoptosis after irradiation in an acidic environment

We investigated the effect of an acidic environment on the radiation-induced G2/M arrest and apoptosis using RKO.C human colorectal cancer cells expressing wild-type p53 and RC10.1 cells, a subline of RKO.C cells deficient in p53 as well as p53+/+ MEFs and p53-/- MEFs (mouse embryonic fibroblasts). The cells were irradiated with 4 Gy or 12 Gy of gamma-rays in pH 7.5 medium or pH 6.6 medium. p53 accentuated the progression of cells from radiation-induced G2/M arrest to apoptosis and the pH 6.6 environment suppressed the progression of cells through G2/M-phase to apoptosis after irradiation. Further analysis indicated that the radiation-induced G2/M arrest was due mainly to G2 arrest in both pH 7.5 and pH 6.6. Therefore, it was concluded that p53 enhances, and an acidic environment suppresses, the exit of cells from radiation-induced G2 arrest by altering cyclin B1-Cdc2 kinase activity.