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Supplementary Methods and Figures 1-3 from Hedgehog Signaling and Bmi-1 Regulate Self-renewal of Normal and Malignant Human Mammary Stem Cells

The epithelial components of the mammary gland are thought to arise from stem cells with a capacity for self-renewal and multilineage differentiation. Furthermore, these cells and/or their immediate progeny may be targets for transformation. We have used both in vitro cultivation and a xenograft mouse model to examine the role of hedgehog signaling and Bmi-1 in regulating self-renewal of normal and malignant human mammary stem cells. We show that hedgehog signaling components PTCH1, Gli1, and Gli2 are highly expressed in normal human mammary stem/progenitor cells cultured as mammospheres and that these genes are down-regulated when cells are induced to differentiate. Activation of hedgehog signaling increases mammosphere-initiating cell number and mammosphere size, whereas inhibition of the pathway results in a reduction of these effects. These effects are mediated by the polycomb gene Bmi-1. Overexpression of Gli2 in mammosphere-initiating cells results in the production of ductal hyperplasia, and modulation of Bmi-1 expression in mammosphere-initiating cells alters mammary development in a humanized nonobese diabetic-severe combined immunodeficient mouse model. Furthermore, we show that the hedgehog signaling pathway is activated in human breast “cancer stem cells” characterized as CD44+CD24−/lowLin−. These studies support a cancer stem cell model in which the hedgehog pathway and Bmi-1 play important roles in regulating self-renewal of normal and tumorigenic human mammary stem cells. (Cancer Res 2006; 66(12): 6063-71)

This paper deals with the application of Glass Fiber Reinforce Polymer (GFRP) to shear reinforcement for deep beam. Instead of steel stirrup, GFRP shear reinforcement was fabricated in the form of plate with openings and embedded in concrete. An experimental study was performed to evaluate the shear behavior of eight shear reinforced concrete deep beam. Shear test was conducted in which the shear span-to-depth ratio were 1.1, 1.3 and 1.6. Also, shear reinforcement area, and effective depth were considered as variables. Crack patterns, failure modes, and load-displacement were compared in order to evaluate shear strength of the specimens. The effects of these variables on the shear strength of the deep beam were examined. The test results in terms of the shear span-to-depth ratio showed that shear strength increased when the ratio decreased. Also, it showed that shear strength increased as the reinforcement area and the effective depth increased.

Sulforaphane is an antioxidant and a potent stimulator of natural detoxifying enzyme and associated with lowered risk of cancer that is associated with the consumption of cruciferous vegetables. The chemopreventive effects of SFN was investigated using the MCF-7 human breast cancer cells and the M13SV1-immortalized human breast luminal epithelial cells. Sulforaphane reduced proliferation in MCF-7 cells and inhibited cyclooxygenase-2 expression in M13SV1 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). The chemopreventive effects of sulforaphane were associated with p38 mitogen-activated protein kinase suggest its important role in cell survival/apoptosis regulation and stabilization of cyclooxygenase-2. Sulforaphane upregulates p38 in MCF-7 cells and prevented TPA-reduced phosphorylation of p38 in M13SV1 cells, but activated caspase-7 associated with apoptosis in MCF-7 cells. These results suggest that sulforaphane may be an alternative candidate for targeted prevention of ER-positive and cyclooxygenase-2-induced phenotypes and breast cancer.

Oligonol is a novel catechin-rich biotechnology product. The role of oligonol in modulating intracellular signaling mechanisms was investigated with the view of demonstrating its potential chemopreventive effect and the ability to inhibit cell proliferation using the estrogen-responsive MCF-7 and the estrogen-unresponsive MDA-MB-231 human breast cancer cell lines. Cell survival assay indicated that Oligonol was cytotoxic to both cells. Oligonol triggered apoptosis as revealed by the morphological features typical of nucleus staining and the accumulation of sub-G1 peak. Treatment with 25 microg/ml Oligonol resulted in an activation of caspase-7 and up-regulation of Bad on MCF-7 cells, while the Oligonol (20 microg/ml) induced up-regulation of Bcl-2 protein in a time-response manner on MDA-MB-231 cells. ERK1/2 in both cells were inactivated after Oligonol treatment in a time-dependent manner, and also inactivated upstream MEK1/2. Oligonol triggers apoptosis in MCF-7 and MDA-MB-231 cells through the modulation of pro-apoptotic Bcl-2 family proteins and MEK/ERK signaling pathway.

Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a variety of carcinogenic and noncarcinogenic effects in experimental animals, its role in human carcinogenicity remain controversial. A simian virus 40-immortalized cell line from normal human breast epithelial cells with stem cells and luminal characteristics (M13SV1) was used to study whether TCDD can induce AIG positive colony formation and cause increased cell numbers in a inverted U-shaped dose-response manner. TCDD activated Akt, ERK2, and increased the expression of CYP1A1, PAI-2, IL-lb mRNA, and ERK2 protein levels. TCDD was able to increased phosphorylation and expression of ERK2 in same dose-response manner as AIG positive colony formation. Thus, TCDD induced tumorigenicity in M13SV1, possibly through the phosphorylation of ERK2 and/or Akt. Further, cDNA microarray with 7448 sequence-verified clones was used to profile various gene expression patterns after treatment of TCDD. Three clear patterns could be delineated: genes that were dose-dependently up-regulated, genes expressed in either U-shape and/or inverted U-shape. The fact that these genes are intrinsically related to breast epithelial cell proliferation and survival clearly suggests that they may be involved in the TCDD-induced breast tumorigenesis.

Gap junctional intercellular communications (GJIC) contributes to neural function in development and differentiation of CNS. In this study, we have investigated the expression of GJIC during the differentiation of neuronal stem cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neuronal stem cell-derived cells from rat brain. During neuronal stem cell differentiation, expressions of Cx43 and 32 were increased for the duration of 72 hr, however the effect were decreased on the 7d. In the neuronal stem cell-derived cells, pretreatments with p38 MAP kinase inhibitor, SB203580, and MEK inhibitor, PD98059, could protect GJIC against TPA-induced inhibition of GJIC. Our data suggest that GJIC plays an important role during neuronal stem cell differentiation, and ERK1/2 and p38 MAP kinase signaling pathway may be closely related functionally to regulate gap junction in rat neuronal stem cell-derived cells.

The primary factors that affecting concrete sulfate resistance are the chemistry of the Portland cement and the chemistryandreplacementlevelofmineraladmixtures. In order to investigate the effect of those on the sulfate attack the testing program involved the testing of several different mortar mixes using the standardized test, ASTM C 1012. four different cements were evaluated including one Type I cement, two Type I-II cements, and one Type V cement. Mortar mixes were also made with mineral admixtures as each cement was combined with three different types of mineral admixtures. One Class F fly ash and one Class C fly ash was added in various percent volumetric replacement levels. The expansion measurements of mortar bars were taken and compared with expansion criteria recommended from past experience to investigate the effect of each factor.

The epithelial components of the mammary gland are thought to arise from stem cells with a capacity for self-renewal and multilineage differentiation. Furthermore, these cells and/or their immediate progeny may be targets for transformation. We have used both in vitro cultivation and a xenograft mouse model to examine the role of hedgehog signaling and Bmi-1 in regulating self-renewal of normal and malignant human mammary stem cells. We show that hedgehog signaling components PTCH1, Gli1, and Gli2 are highly expressed in normal human mammary stem/progenitor cells cultured as mammospheres and that these genes are down-regulated when cells are induced to differentiate. Activation of hedgehog signaling increases mammosphere-initiating cell number and mammosphere size, whereas inhibition of the pathway results in a reduction of these effects. These effects are mediated by the polycomb gene Bmi-1. Overexpression of Gli2 in mammosphere-initiating cells results in the production of ductal hyperplasia, and modulation of Bmi-1 expression in mammosphere-initiating cells alters mammary development in a humanized nonobese diabetic-severe combined immunodeficient mouse model. Furthermore, we show that the hedgehog signaling pathway is activated in human breast “cancer stem cells” characterized as CD44+CD24−/lowLin−. These studies support a cancer stem cell model in which the hedgehog pathway and Bmi-1 play important roles in regulating self-renewal of normal and tumorigenic human mammary stem cells. (Cancer Res 2006; 66(12): 6063-71)

Sodium butyrate (NaBu) is known to exhibit anti-cancer effects via the differentiation and apoptosis of various carcinoma cells. However, the mechanism by which NaBu induces apoptosis and the involvement of protein kinases during apoptosis is not completely understood. To investigate the underlying pathways, we performed cell culture experiments in androgen-independent human prostate cancer (DU145 cells) focusing on various protein kinases. NaBu causes concentration-dependent cell detachment and growth inhibition. Exposure of DU145 cells to NaBu for 24 h caused a strong apoptotic effect with 26% nuclear fragmentation and condensation. In addition, NaBu induced caspase-3 and poly-ADP ribose polymerase cleavage and up-regulation of bax, suggesting that mitochondrial damage is involved in NaBu-induced caspase-dependent apoptosis. Interestingly, NaBu stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) activation, but not extracellular signal-regulated kinase 1/2 activation during apoptosis. Furthermore, NaBu up-regulated total protein levels and phospho forms of MAPK kinase 3 (MKK3) and MAPK kinase 4 (MKK4) as the upstream kinases of p38 MAPK and JNK independently of oxidative stress. Taken together, it is suggested that NaBu can be a promising chemopreventive agent for prostate cancer and the p38 MAPK and JNK pathways have critical roles in NaBu-induced apoptosis in DU145 cells.

Sodium butyrate (NaBu) has an inhibitory effect on histone deacetylases (HDACs). The mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAP, kinase are known to be modulated during NaBu-induced apoptosis. In the present study, we showed that low concentrations of NaBu could induce apoptosis synergistically with the inhibition of p38 MAP kinase as proven by using specific p38 MAP kinase inhibitor and dominant negative p38 transfection in a ras-transformed rat liver epithelial cell line (WB-ras). There were no changes in HDAC1, suggesting that NaBu might be able to kill transformed cells bypassing the HDAC inhibitory effect. We further demonstrated that inhibition of p38 MAP kinase potentiated apoptotic cascades, including cleavage of poly(ADP-ribose) polymerase, caspase-3, and decrease in Bcl-2/Bax ratio even at a lower concentration of NaBu. Thus, p38 MAP kinase played inhibitory roles in NaBu-induced apoptosis, and simultaneous modulation of MAP kinases in NaBu treatment could increase the efficiency of the chemotherapeutic effect of NaBu.

The histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and sodium butyrate (NaBu) are considered as potent therapeutic agents for cancer treatment presenting therapeutic benefits with less risk of side effects. The microbial metabolite, TSA is a potent reversible and highly specific inhibitor of mammalian histone deacetylases. NaBu causes hyperacetylation of core histones with effects similar to TSA but it is not a specific inhibitor of HDACs. The gap junction is a channel in the plasma membrane of most cell types which allows direct communication (gap junctional intercellular communication; GJIC) of small molecules and ions. Modulation of GJIC is a known cellular event associated with tumor promotion. The effects of NaBu and TSA on the H(2)O(2)- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced GJIC inhibition of WB cells and the mechanisms involved in the process were assessed. TSA and NaBu exerted differential preventive effects on the H(2)O(2) and TPA-induced inhibition of GJIC as well as hyperphosphorylation of connexin43 (Cx43) in WB-F344 rat liver epithelial cells (WB cells). NaBu prevented the TPA-induced GJIC inhibition via ERK1/2 inactivation whilst TSA restored the H(2)O(2)-induced GJIC inhibition and Cx43 hyperphosphorylation by preventing p38 MAP kinase. The inhibition of tyrosine phosphorylation and down-regulation of src protein observed may also contribute to Connexin 43 dephosphorylation and GJIC restoration by TSA and NaBu partly through depletion of src protein pool. Thus, TSA and NaBu exert differential effects on chemically induced GJIC inhibition via modulation of MAP kinases and partly, tyrosine kinases.

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK. These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis.

Histone deacetylase inhibitors such as TSA, SAHA, and NaBu etc. are prospective cancer therapeutics of growing interest. Here, we demonstrated that oncogenic ras-transformed rat liver epithelial (WB-ras) cells were specifically undergone apoptosis by 48 h treatment of NaBu. During this, inhibition of ras proteins, especially farnesylated form of ras, and down-regulation of ERK1/2 were observed, which suggest ras/raf/MEK/ERK down-regulation, while p38 MAP kinase was maintained up-regulated. In addition, up-regulation of pro-apoptotic proteins such as p53 and p21CIP1/WAF1, and down-regulation of cell cycle regulator/anti-apoptotic proteins such as cdk2, -4 and phosphorylated Akt were observed concurrently with an increase in apoptotic cell portion. A phosphatase inhibitor, sodium orthovanadate (SOV), efficiently blocked apoptosis and restored responsible proteins for each phenomenon including ERK1/2 while SB203580, a specific p38 MAP kinase inhibitor, showed minor effect on them. Thus, ras/ERK signaling pathway can be considered in chemotherapeutic strategies of NaBu regardless of its inhibitory action on histone deacetylase.