Clusterin overexpression is responsible for the anti-apoptosis effect in a mouse neuroblastoma cell line,B103

The functional role of clusterin in apoptosis was examined using flow cytometry. Clusterin cDNA was transfected into the mouse neuroblastoma cell line, B103, in order to determine if clusterin overexpression inhibits apoptosis. The increased clusterin expression level in the B103 cells tended to suppress the apoptotic index. This suggests an association of clusterin gene expression with apoptosis inhibition. These results support the conclusion that clusterin expression in B103 cells has an anti-apoptotic influence.     

Extracellular proteinase inhibitor-accelerated apoptosis is associated with B cell activating factor in mammary epithelial cells

The expression of extracellular proteinase inhibitor (Expi) gene was induced during the involution of mammary gland, when apoptosis occurs in this tissue. Transient transfection of Expi gene partially induced apoptosis of mammary epithelial HC11 cells. We developed the stable cell lines overexpressing Expi gene and found that overexpression of Expi accelerated apoptosis of mammary epithelial cells under serum starvation. To understand apoptosis pathway involved in the Expi overexpression, we examined the gene expression profile by using apoptosis gene array containing 243 genes. The subsequent confirmation of the altered gene expression by northern analysis demonstrated that overexpression of the Expi gene induced expression of several genes, which included B cell activating factor (BAFF), Bax, cytochrome c, caspase-9, caspase-3, caspase-6, and CIDE-A. From this study, we first demonstrate that BAFF is involved in mammary apoptosis. Furthermore, we have found that the Expi-accelerated apoptosis is mediated via BAFF receptor among three known BAFF receptors: BAFF receptor, tumor necrosis factor (TNF) receptor homologue TACI (transmembrane activator and CAML-interactor), and BCMA (another TNFR homologue, B cell maturation antigen). Our studies also demonstrate that the use of apoptosis array provides an efficient tool to identify apoptosis pathway involved in gene transfection.     

Role of DDC-4/sFRP-4, a secreted frizzled-related protein,at the onset of apoptosis in mammary involution

Using differential display, we isolated DDC-4, a secreted frizzled-related protein (sFRP), which is induced in the physiological apoptosis of hormonally regulated, reproductive tissues such as mammary gland, prostate, corpus luteum and uterus. The role of this gene in apoptosis was studied in animals overexpressing ectopic DDC-4/sFRP-4. Transgenic mice bearing the DDC-4/sFRP-4 cDNA under the control of the MMTV-LTR promoter showed lactational insufficiency and many apoptotic cells in the alveoli between day 19 of pregnancy and day 4 of lactation as demonstrated by TUNEL reaction and the presence of activated caspase-3. We performed a PKB/Akt kinase assay and studied several of its substrates using phosphorylation-specific antibodies to show reduced phosphorylation in PKB/Akt itself, as well as in glycogen synthetase kinase-3beta (GSK-3beta), BAD, and Forkhead. Taken together, our results show a role for DDC-4/sFRP-4 in abrogating an epithelial cell survival pathway at the onset of mammary gland involution.     

The 24p3 gene is induced during involution of the mammary gland and induces apoptosis of mammary epithelial cells

To understand the molecular mechanism of mammary gland involution we identified involution-induced clones by differential screening of a mouse mammary gland cDNA library. Characterization of clones by sequencing and Northern analysis showed that expression of 24p3 was induced during involution of the mammary gland. RNA in situ hybridization showed that it was mainly expressed in the secretory epithelial cells surrounding the lumen of the mammary gland alveoli. Induction of 24p3 was also observed in apoptotic HC11 mammary epithelial cells under serum starvation. In these cells, dexamethasone increased 24p3 gene expression four-fold. Transient expression of 24p3 increased the percentage of apoptotic cells 3- to 4-fold over a period of 3 days after transfection. This study provides evidence that overexpression of 24p3 gene can induce apoptosis of mammary epithelial cells.     

Tumor suppression by a proapoptotic calcium-activated chloride channel in mammary epithelium

Little is known of the roles played by ion channels in cancer. Here we describe a pair of closely related calcium-activated chloride channels whose differential regulation in normal, apoptotic, and transformed mouse cells suggests that channel function is proapoptotic and antineoplastic. While mCLCA1 predominates over mCLCA2 under normal physiological conditions, this relationship is reversed by apoptotic stress both in developing mammary gland and in cultured HC11 mammary epithelial cells. Consistent with an apoptosis-promoting role, splicing of mCLCA2 is disrupted in apoptosis-resistant tumor cell lines and in HC11 cells selected for resistance to detachment-induced apoptosis (anoikis). Unexpectedly, mCLCA1 message is also down-regulated in these cells by at least 30-fold. These results suggest that both genes antagonize survival of mammary tumor cells by sensitizing them to anoikis. When MCF7 or HEK293 tumor cells were transfected with plasmids encoding either mCLCA1 or mCLCA2, colony formation was greatly reduced relative to a vector-transfected control, demonstrating that calcium-sensitive chloride channel (CLCA) expression is deleterious to tumor cell survival. Furthermore, mammary epithelial cells overexpressing mCLCA2 had twice the rate of apoptosis of normal cells when subjected to serum starvation and formed multinuclear giants at a high frequency in normal culture, suggesting that mCLCA2 can promote either apoptosis or senescence.     

Differential expression of connexin 43 in mouse mammary cells

In this study we have employed suppressive subtractive hybridization (SSH) analysis to investigate differential gene expression in primary mouse mammary epithelial cells (PMMEC) cultured under mildly apoptotic/quiescent and differentiating conditions. Among a small group of genes whose expression was differentially regulated was connexin 43. In vitro, connexin 43 mRNA and protein were detectable in PMMEC cultured under proliferative or mildly apoptotic conditions. The level of connexin 43 mRNA expression in vivo was also investigated. High levels of expression were found to be associated with the periods of greatest glandular plasticity (pubertal expansion of the mammary tree, early pregnancy and during early involution). Thus, terminally differentiated cells in vivo and in vitro did not express connexin 43 mRNA suggesting that connexin 43 expression, and perhaps facilitated gap junction communication, is associated with undifferentiated progenitor cell populations.     

Regulation of clusterin expression in mammary epithelial cells

Mammary epithelial cells undergo changes in growth, invasion, differentiation, and dedifferentiation throughout much of adult hood, and most strikingly during pregnancy, lactation, and involution. Clusterin is a multifunctional glycoprotein that is involved in the differentiation and morphogenesis of epithelia, and that is important in the regulation of postnatal mammary gland development. However, the mechanisms that regulate clusterin expression are still poorly understood. Here, we show that clusterin is up-regulated twice during mouse mammary gland development, a first time at the end of pregnancy and a second time at the beginning of the involution. These points of clusterin up-regulation coincide with the dramatic phenotypic and functional changes occurring in the mammary gland. Using cell culture conditions that resemble the regulatory microenvironment in vivo, we determined that the factors responsible for the first up-regulation of clusterin levels can include the extracellular matrix component, laminin, and the lactogenic hormones, prolactin and hydrocortisone. On the other hand, the second and most dramatic up-regulation of clusterin can be due to the potent induction by TGF-beta1, and this up-regulation by TGF-beta1 is dependent on beta1 integrin ligand-binding activity. Moreover, the level of expression of beta-casein, a marker of mammary epithelial cell differentiation, was decreased upon treatment of cells with clusterin siRNA. Overall, these findings reveal several novel pathways for the regulation of clusterin expression during mammary gland development, and suggest that clusterin is a morphogenic factor that plays a key role during differentiation.     

Insulin-like growth factor binding protein (IGFBP)-5 is upregulated during both differentiation and apoptosis in primary cultures of mouse mammary epithelial cells

We have previously demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) is upregulated following treatment of the mouse mammary epithelial cell line HC11 with lactogenic hormones (dexamethasone, insulin, and prolactin-DIP). In addition, we have also shown that IGFBP-5 is upregulated in mammary epithelial cells in vivo during involution of the rodent mammary gland. We have, therefore, postulated that there may be a dual regulation of IGFBP-5 expression during the temporally separated processes of differentiation and apoptosis of mammary epithelial cells. To test this hypothesis further, we have used a phenotypically differentiated model, which comprises primary cultures of mouse mammary epithelial cells grown on a layer of EHS (Engelbreth-Holm-Swarm) extracellular matrix. We show that lactogenic hormone treatment (hydrocortisone, insulin, and prolactin-HIP) of these cultures induces the upregulation of IGFBP-5 thus replicating the results obtained with the HC11 cell line. In addition, following the induction of apoptosis in primary cultures of mammary epithelial cells by treatment with TGFbeta-3, IGFBP-5 expression is also upregulated. In parallel with this upregulation of IGFBP-5, there is also an increase in the levels of cleaved caspase-3, a well-characterized marker of cellular apoptosis. These findings confirm previous in vivo work demonstrating an increase in IGFBP-5 expression during involution of the mouse mammary gland. When HC11 cells are cultured under serum-free conditions (a well-characterized apoptotic insult in cell culture), there is also an increase in cleaved caspase-3 levels. Unexpectedly, in the presence of TGFbeta-3, caspase-3 levels are attenuated. In the presence of DIP, caspase-3 levels are also decreased in HC11 cells. As described previously, TGFbeta-3 inhibits beta-casein synthesis in HC11 cells. In the HC11 cell line (in contrast to primary cultures of mammary epithelial cells), there is no evidence for TGFbeta-3 induction of IGFBP-5 under either serum-free or DIP-supplemented conditions. We believe our data with primary cultures of mammary epithelial cells support the hypothesis of dual regulation of IGFBP-5 expression during both differentiation and apoptosis in the mammary gland and emphasizes the importance of using appropriate cell culture models to investigate such phenomena in this tissue. We discuss the possible implications of our observations in relation to the physiological processes of pregnancy, lactation, and involution in the mammary gland and the associated changes in mammary epithelial cell function.     

Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.     

Induction of osteopontin gene expression during mammary gland involution and effects of glucocorticoid on its expression in mammary epithelial cells

To understand the molecular mechanisms of mammary gland involution, an involution-induced clone was identified from a cDNA library of mouse mammary gland by differential screening. Characterization of a clone by sequencing and northern analysis showed that expression of the osteopontin gene was induced during involution of mouse mammary gland. But induction of the osteopontin gene was not observed in apoptotic HC11 mammary epithelial cells under serum starvation. In HC11 cells, dexamethasone treatment from the seeding stage showed five-fold induction of osteopontin gene expression, but the expression was not changed when dexamethasone was added to confluent cells.     

Expression profiles of 109 apoptosis pathway-related genes in 82 mouse tissues and experimental conditions

Apoptosis is an important process in development and tissue homeostasis. To understand the similarities and differences in the apoptosis machinery in different normal, developmental, and diseased tissues, the expression profiles of 109 apoptosis-pathway-related genes in 82 mouse tissues and experimental conditions were examined using Incyte Mouse GEMI cDNA arrays. It has been found that the compositions of the apoptotic machinery vary among different tissues, developmental stages, and disease states, with subsets of apoptotic genes co-ordinately expressed in the 82 tissues and experimental conditions. Additional genes whose expression profiles resemble selected genes from the 109 apoptotic gene list were also identified. This study provides valuable information on possible molecular mechanisms of differential apoptotic responses to developmental signals, environmental stimuli, and therapeutic treatments in tissue-specific manner.     

Plasticity of the response of fetal mouse fibroblast to lactation hormones

In the work described in this article, mouse fetal fibroblasts were treated with three lactation hormones (insulin, progesterone and oxytocin) and the cellular changes were analyzed by RT-PCR-Southern hybridization. A gene-expression pattern characteristic of mammary epithelioid cells was induced by the hormones, as indicated by expression of the marker genes alpha-casein and beta-casein. Two mammary epithelial cell-specific gene expression vectors were constructed with bovine alpha-s1-casein or ovine beta-casein gene promoters directing an EGFP reporter gene. Transient expression of the EGFP gene was observed in cells treated by the hormones but not in control cells. Cell morphology also changed after insulin and oxytocin treatments; the cells resembled epithelial cells rather than fibroblasts. Our results suggest that mouse fetus fibroblasts can be partially induced by lactation hormones to resemble mammary epithelial cells. This procedure might help to increase the efficiency of gene targeting in studies of mammary gland bioreactors.     

Isolation of cell death-associated cDNAs from involuting mouse mammary epithelium

Although apoptosis is important in determining cell fate and maintaining tissue homeostasis, the initiation and control of apoptotic cell death in epithelium is not well understood. Post-lactationai involution of the mammary gland provides both an important developmental process and a normal physiological setting for studying apoptosis of epithelium. We used a differential screening strategy, based on previous studies correlating morphology with gene expression and nucleic acid integrity during mammary gland involution, to isolate genes involved in the regulation and execution of apoptotic cell death in regressing mammary epithelium. This screening strategy yielded a large number of genes the expression of which is significantly altered during mammary gland involution. These include genes associated with cell death processes, tissue remodelling and mesenchymal differentiation. In addition, a number of novel genes have been isolated. We have used Northern analysis and in situ hybridisation to study the expression of a selection of these putative death-associated genes during post-lactational mouse mammary gland involution.     

Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. alpha-Casein and beta-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.     

MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesion-dependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.