Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells

Impaired function of apoptosis-related genes is deeply involved in oncogenesis and the progression of cancers, and caspase-3 plays a critical role as an executioner of apoptosis. We introduced the caspase-3 gene via an adenovirus (Adv) vector into Alexander hepatoma cells, MCF-7 breast cancer cells, and U251 and U-373MG glioma cells which have different endogenous levels of caspase-3 expression. None of the cell lines underwent apoptosis by overexpression of caspase-3, indicating that induction of caspase-3 alone is not applicable for cancer gene therapy. Next, we investigated whether overexpression of caspase-3 could enhance Fas ligand-mediated apoptosis in these four cell lines. In U-373MG cells, which showed the highest level of expression of surface Fas among the four cell lines, coinfection of the Adv for caspase-3 (Adv-caspase-3) and the Adv for Fas ligand (Adv-FL) induced a remarkably increased degree of apoptosis compared with that induced by the single infection of either Adv-caspase-3 or Adv-FL. Similar results were obtained by cotreatment with anti-Fas antibody in U-373MG cells. These data suggest that when strong proapoptotic upstream stimuli are induced, the level of caspase-3 expression determines the degree of apoptosis in cancer cell lines. In conclusion, overexpression of caspase-3 alone did not induce apoptosis in cancer cells. Both a strong proapoptotic signal and a high expression of caspase-3 were required to induce drastic apoptosis in cancers. This strategy would be highly beneficial for selected cancer patients.     

Sensitivity of human glioma U-373MG cells to radiation and the protein kinase C inhibitor, calphostin C

We assessed the radiosensitivity of the grade III human glioma cell line U-373MG by investigating the effects of radiation and the specific protein kinase C inhibitor, calphostin C on the cell cycle and cell proliferation. Irradiated glioma U-373MG cells progressed through G1-S and underwent an arrest in G2-M phase. The radiosensitivity of U-373MG cells to graded doses of either photons or electrons was determine by microculture tetrazolium assay. The data was fitted to the linear-quadratic model. The proliferation curves demonstrated that U-373MG cells appear to be highly radiation resistant since 8 Gy was required to achieve 50% cell mortality. Compared to radiation alone, exposure to calphostin C (250 nM) 1 h prior to radiation decreased the proliferation of U-373MG by 76% and calphostin C provoked a weakly synergistic effect in concert with radiation. Depending on the time of application following radiation, calphostin C produced an additive or less than additive effect on cell proliferation. We postulate that the enhanced radiosensitivity observed when cells are exposed to calphostin C prior to radiation may be due to direct or indirect inhibition of protein kinase C isozymes required for cell cycle progression.     

Alkylphospholipids deregulate cholesterol metabolism and induce cell-cycle arrest and autophagy in U-87 MG glioblastoma cells

Glioblastoma is the most common malignant primary brain tumour in adults and one of the most lethal of all cancers. Growing evidence suggests that human tumours undergo abnormal lipid metabolism, characterised by an alteration in the mechanisms that regulate cholesterol homeostasis. We have investigated the effect that different antitumoural alkylphospholipids (APLs) exert upon cholesterol metabolism in the U-87 MG glioblastoma cell line. APLs altered cholesterol homeostasis by interfering with its transport from the plasma membrane to the endoplasmic reticulum (ER), thus hindering its esterification. At the same time they stimulated the synthesis of cholesterol from radiolabelled acetate and its internalisation from low-density lipoproteins (LDLs), inducing both 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and LDL receptor (LDLR) genes. Fluorescent microscopy revealed that these effects promoted the accumulation of intracellular cholesterol. Filipin staining demonstrated that this accumulation was not confined to the late endosome/lysosome (LE/LY) compartment since it did not colocalise with LAMP2 lysosomal marker. Furthermore, APLs inhibited cell growth, producing arrest at the G2/M phase. We also used transmission electron microscopy (TEM) to investigate ultrastructural alterations induced by APLs and found an abundant presence of autophagic vesicles and autolysosomes in treated cells, indicating the induction of autophagy. Thus our findings clearly demonstrate that antitumoural APLs interfere with the proliferation of the glioblastoma cell line via a complex mechanism involving cholesterol metabolism, cell-cycle arrest or autophagy. Knowledge of the interrelationship between these processes is fundamental to our understanding of tumoural response and may facilitate the development of novel therapeutics to improve treatment of glioblastoma and other types of cancer.     

Transfection of 2,6 and 2,3-sialyltransferase genes and GlcNAc-transferase genes into human glioma cell line U-373 MG affects glycoconjugate expression and enhances cell death

Human glioma cell line U-373 MG expresses CMP-NeuAc : Galbeta1,3GlcNAc alpha2,3-sialyltransferase [EC No. 2.4.99.6] (alpha2,3ST), UDP-GlcNAc : beta-d-mannoside beta1,6-N-acetylglucosaminyltransferase V [EC 2.4.1.155] (GnT-V) and UDP-GlcNAc3: beta-d-mannoside beta1,4-N-acetylglucosaminyltransferase III [EC 2.4.1.144] (GnT-III) but not CMP-NeuAc : Galbeta1,4GlcNAc alpha2,6-sialyltransferase [EC 2.4.99.1] (alpha2,6ST) under normal culture conditions. We have previously shown that transfection of the alpha2,6ST gene into U-373 cells replaced alpha2,3-linked sialic acids with alpha2,6 sialic acids, resulting in a marked inhibition of glioma cell invasivity and a significant reduction in adhesivity. We now show that U-373 cells, which are typically highly resistant to cell death induced by chemotherapeutic agents (< 10% death in 18 h), become more sensitive to apoptosis following overexpression of these four glycoprotein glycosyltransferases. U-373 cell viability showed a three-fold decrease (from 20 to 60% cell death) following treatment with staurosporine, C2-ceramide or etoposide, when either alpha2,6ST and GnT-V genes were stably overexpressed. Even glycosyltransferases typically raised in cancer cells, such as alpha2,3ST and GnT-III, were able to decrease viability two-fold (from 20 to 40% cell death) following stable overexpression. The increased susceptibility of glycosyltransferase-transfected U-373 cells to pro-apoptotic drugs was associated with increased ceramide levels in Rafts, increased caspase-3 activity and increased DNA fragmentation. In contrast, the same glycosyltransferase overexpression protected U-373 cells against a different class of apoptotic drugs, namely the phosphatidylinositol 3-kinase inhibitor LY294002. Thus altered surface protein glycosylation of a human glioblastoma cell line can lead to lowered resistance to chemotherapeutic agents.     

cAMP differentially regulates gamma-globin gene expression in erythroleukemic cells and primary erythroblasts through c-Myb expression

Our previous studies demonstrated roles of cyclic nucleotides in gamma-globin gene expression. We recently found that, upon activation of the cAMP pathway, expression of the gamma-globin gene is inhibited in K562 cells but induced in adult erythroblasts. Here we show that c-Myb, a proto-oncogene product that plays a role in cell growth and differentiation, is involved in the cAMP-mediated differential regulation of gamma-globin gene expression in K562 cells and primary erythroblasts. Our studies found that c-Myb is expressed at a high level in K562 cells compared to primary erythroblasts, and that c-Myb expression is further increased following the treatment with forskolin, an adenylate cyclase activator. The induction of gamma-globin gene expression was also inhibited in K562 cells by raising the levels of c-Myb expression. Importantly, forskolin-induced erythroid differentiation in K562 cells, as determined by the expression of glycophorins and CD71, suggesting that high-level expression of c-Myb may not be sufficient to inhibit the differentiation of erythroid cells. In contrast, c-Myb was not expressed in adult erythroblasts treated with forskolin and primary erythroblasts may lack the c-Myb-mediated inhibitory mechanism for gamma-globin gene expression. Together, these results show that the cAMP pathway blocks gamma-globin gene expression in K562 cells by increasing c-Myb expression and c-Myb plays a role in defining the mode of response of the gamma-globin gene to fetal hemoglobin inducers in erythroid cells.     

Proteome profile changes that are differentially regulated by lipid and protein phosphatase activities of tumor suppressor PTEN in PTEN-expressing U-87 MG human glioblastoma cells

The phosphatase and tensin homolog tumor suppressor (PTEN) belongs to a class of "gatekeeper" tumor suppressors together with p53, retinoblastoma and adenomatous polyposis. It is considered one of the most important tumor suppressors in the post p53 era. Previously to identify the molecules involved in the signaling network regulated by PTEN using proteomic tools, we reported global proteome profiles at different time points using the PTEN inducible NIH3T3 cells (Kim, S.-y., Kim, Y. S., Bahk, Y. Y., Mol. Cells 2003, 15, 396-405). However, the system had a critical limitation that NIH3T3 cell has endogenous wild-type PTEN and, thus to be exact, the induced PTEN could not give the answer about the real physiological roles of this tumor suppressor. Here, to find out PTEN-related protein network we have established various PTEN (wild-type, an activity inert C124G, and a lipid phosphatase deficient G129E)-expressing cell clones in U-87 MG human glioblastoma cells lacking detectable PTEN as a result of genetic lesions. In this biological context, we compared their morphological and expression patterns, and proteome images of each PTEN-expressing cell clone by 2-DE followed by identification with MALDI-TOF MS. We obtained some pieces of evidence that morphological change by PTEN expression is mediated by its protein phosphatase activity and their growth rate by the lipid phosphatase activity. The proteomic approaches showed that 30 proteins possibly correlated with PTEN's protein phosphatase activity (13 down-regulated and 17 up-regulated) and 20 with the lipid phosphatase activity (14 down-regulated and 6 up-regulated) were identified. Taken together, we conclude that the comparative analysis of proteome from various PTEN-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly caused by individual phosphatase activities of PTEN in vivo.     

Clozapine, a neuroleptic agent, inhibits Akt by counteracting Ca2+/calmodulin in PTEN-negative U-87MG human glioblastoma cells

Clozapine (CZP), a dibenzodiazepine derivative with a piperazinyl side chain, is in clinical use as an antipsychotic drug. This study investigated the effect of CZP on the modulation of the PI3K/Akt/GSK-3beta pathway in PTEN-negative U-87MG glioblastoma cells. Treatment with CZP rapidly inhibited the basal and EGF-induced phosphorylation of Akt. The inhibition of Akt resulted in the dephosphorylation of GSK-3beta and increased GSK-3beta kinase activity. A voltage-sensitive Ca(2+) channel blocker and calmodulin (CaM) antagonists inhibited Akt phosphorylation, whereas elevation of the intracellular Ca(2+) concentration prevented CZP-induced dephosphorylation of Akt and GSK-3beta, suggesting that Ca(2+)/CaM participates in the inhibition of Akt by CZP in U-87MG cells. In addition, similar to LY294002, CZP arrested cell cycle progression at G0/G1 phase, which was accompanied by decreased expression of cyclin D1. The reduction in the cyclin D1 level induced by CZP was abrogated by the inhibition of GSK-3beta, the inhibition of proteasome-dependent proteolysis, or an increase in the intracellular Ca(2+) concentration. These results suggest that the antipsychotic drug CZP modulates the PI3K/Akt/GSK-3beta pathway by counteracting Ca(2+)/CaM in PTEN-negative U-87MG glioblastoma cells.     

Extracellular matrix regulates whey acidic protein gene expression by suppression of TGF-alpha in mouse mammary epithelial cells: studies in culture and in transgenic mice

Whey acidic protein (WAP) is an abundant rodent milk protein. Its expression in mouse mammary epithelial cell cultures was previously found to require the formation of an extracellular matrix (ECM)-induced three-dimensional alveolar structure. In the absence of such structures, cells were shown to secrete diffusible factors leading to suppression of WAP expression. We demonstrate here that (a) TGF-alpha production and secretion by mammary cells is downregulated by the basement membrane-dependent alveolar structure, and (b) compared with beta-casein, WAP expression is preferentially inhibited both in culture and in transgenic mice when TGF-alpha is added or overexpressed. Thus, (c) the enhanced TGF-alpha production when cells are not in three- dimensional structures largely accounts for the WAP-inhibitory activity found in the conditioned medium. Since this activity can be abolished by incubating the conditioned medium with a function blocking antibody to TGF-alpha. The data suggest that ECM upregulates WAP by downregulating TGF-alpha production. We also propose that changes in TGF-alpha activity during mouse gestation and lactation could contribute to the pattern of temporal expression of WAP in the gland. These results provide a clear example of cooperation among lactogenic hormones, ECM, and locally acting growth factors in regulation of tissue-specific gene expression.     

The headpiece domain of dematin regulates cell shape, motility, and wound healing by modulating RhoA activation

RhoA is known to participate in cytoskeletal remodeling events through several signaling pathways, yet the precise mechanism of its activation remains unknown. Here, we provide the first evidence that dematin functions upstream of RhoA and regulates its activation. Primary mouse embryonic fibroblasts were generated from a dematin headpiece domain null (HPKO) mouse, and the visualization of the actin morphology revealed a time-dependent defect in stress fiber formation, membrane protrusions, cell motility, and cell adhesion. Rescue experiments using RNA interference and transfection assays revealed that the observed phenotypes are due to a null effect and not a gain of function in the mutant fibroblasts. In vivo wounding of adult HPKO mouse skin showed a decrease in wound healing (reepithelialization and granulation) compared to the wild-type control. Biochemical analysis of the HPKO fibroblasts revealed a sustained hyperphosphorylation of focal adhesion kinase (FAK) at tyrosine 397 as well as a twofold increase in RhoA activation. Inhibition of both RhoA and FAK signaling using C3 toxin and FRNK (focal adhesion kinase nonrelated kinase), respectively, revealed that dematin acts upstream of RhoA. Together, these results unveil a new function of dematin as a negative regulator of the RhoA activation pathway with physiological implications for normal and pathogenic signaling pathways.     

GABA differentially regulates the gene expression of proopiomelanocortin in rat intermediate and anterior pituitary

The GABAergic regulation of proopiomelanocortin messenger RNA (POMC mRNA) levels in rat pituitary was investigated using molecular hybridization of DNA complementary to POMC mRNA. Endogenous GABA levels increased, in vivo, by inhibiting the GABA catabolic enzyme GABA-transaminase (GAT) with ethalonamine-O-sulfate (EOS) or with vinyl-GABA (VG). Rats were treated with VG (100 mg/kg or 800 mg/kg) or EOS (100 mg/kg), administered each second day. GABA levels in the neurointermediate lobe (NIL) and anterior lobe (AL) of the hypophysis and in the hypothalamus were significantly increased following 4 days of VG treatment (800 mg/kg). All treatments resulted in a 40-60% decrease in POMC mRNA levels after 4 days in the NIL but not in the AL. A similar decrease of about 60% in POMC mRNA levels in the NIL was seen when EOS was given in the drinking water (5 mg/ml). In this set of experiments the time course of alteration of POMC mRNA in the NIL and the concentration of alpha-MSH, a POMC-derived peptide, were analysed. After one day of EOS treatment, when POMC levels had already decreased by 40%, alpha-MSH levels were significantly elevated (34% above controls), possibly reflecting an inhibition of alpha-MSH secretion. However, after 4 and 8 days, POMC mRNA levels and tissue alpha-MSH levels had significantly decreased. When tested in vitro, on primary cultures of IL cells, GABA (10 microM) reduced POMC mRNA levels by 40% after 3 days of treatment. These results show that GABA exerts a direct inhibitory effect on POMC gene expression in the intermediate lobe.     

Growth hormone differentially regulates growth and growth-related gene expression in closely related fish species

Zebrafish (Danio rerio) have become an important model organism for developmental biology and human health studies. We recently demonstrated differential growth patterns between the zebrafish and a close relative the giant danio (Danio aequipinnatus), where the giant danio appears to exhibit indeterminate growth similar to most fish species important for commercial production, while zebrafish exhibit determinate growth more similar to mammalian growth. This study focused on evaluating muscle growth regulation differences in adult zebrafish and giant danio utilizing growth hormone treatment as a mode of growth manipulation. Growth hormone treatment resulted in increased overall growth in giant danio, but failed to increase growth in the zebrafish. Growth hormone treatment increased muscle IGF-I and GHrI gene expression in both species, but to a larger degree in the giant danio. In contrast, zebrafish exhibited a larger increase in IrA and IGF-IrB gene expression in muscle in response to GH treatment. In addition muscle myostatin levels were differentially regulated between the two species, with a down-regulation observed in rapidly growing, GH-treated giant danio and an up-regulation in zebrafish not actively growing in response to GH. This is the first report of differential expression of growth-regulating genes in closely related fish species exhibiting opposing growth paradigms. These results further support the role that the zebrafish and giant danio can play important model organisms for determinate and indeterminate growth.     

Effects of human placenta-derived mesenchymal stem cells with NK4 gene expression on glioblastoma multiforme cell lines

Poor prognosis and low survival are commonly seen in patients with glioblastoma multiforme (GBM). Due to the specific nature of solid tumors such as GBM, delivery of therapeutic agents to the tumor sites is difficult. So, one of the major challenges in the treatment of these tumors is a selection of appropriate method for drug delivery. Mesenchymal stem cells (MSCs) have a unique characteristic in migration toward the tumor tissue. In this regard, the present study examined the antitumor effects of manipulating human placenta-derived mesenchymal stem cells (PDMSCs) with NK4 expression (PDMSC-NK4) on GBM cells. After separation and characterization of PDMSCs, these cells were transduced with NK4 which was known as the antagonist of hepatocyte growth factor (HGF). The results indicated that engineered PDMSCs preferably migrate into GBM cells by transwell coculture system. In addition, the proliferation of the GBM cells significantly reduced after coculture with these cells. In fact, manipulated PDMSCs inhibited growth of tumor cells by induction of apoptosis. Our findings suggested that besides having antitumor effects, PDMSCs can also be applied as an ideal cellular vehicle to target the glioblastoma multiforme.     

PDGF-PDGFR network differentially regulates the fate,migration, proliferation,and cell cycle progression of myogenic cells

Platelet-derived growth factors (PDGFs) regulate embryonic development, tissue regeneration, and wound healing through their binding to PDGF receptors, PDGFRα and PDGFRβ. However, the role of PDGF signaling in regulating muscle development and regeneration remains elusive, and the cellular and molecular responses of myogenic cells are understudied. Here, we explore the PDGF-PDGFR gene expression changes and their involvement in skeletal muscle myogenesis and myogenic fate. By surveying bulk RNA sequencing and single-cell profiling data of skeletal muscle stem cells, we show that myogenic progenitors and muscle stem cells differentially express PDGF ligands and PDGF receptors during myogenesis. Quiescent adult muscle stem cells and myoblasts preferentially express PDGFRβ over PDGFRα. Remarkably, cell culture- and injury-induced muscle stem cell activation altered PDGF family gene expression. In myoblasts, PDGF-AB and PDGF-BB treatments activate two pro-chemotactic and pro-mitogenic downstream transducers, RAS-ERK1/2 and PI3K-AKT. PDGFRs inhibitor AG1296 inhibited ERK1/2 and AKT activation, myoblast migration, proliferation, and cell cycle progression induced by PDGF-AB and PDGF-BB. We also found that AG1296 causes myoblast G0/G1 cell cycle arrest. Remarkably, PDGF-AA did not promote a noticeable ERK1/2 or AKT activation, myoblast migration, or expansion. Also, myogenic differentiation reduced the expression of both PDGFRα and PDGFRβ, whereas forced PDGFRα expression impaired myogenesis. Thus, our data highlight PDGF signaling pathway to stimulate satellite cell proliferation aiming to enhance skeletal muscle regeneration and provide a deeper understanding of the role of PDGF signaling in non-fibroblastic cells.     

1H NMR metabolomics analysis of glioblastoma subtypes: correlation between metabolomics and gene expression characteristics

Glioblastoma multiforme (GBM) is the most common form of malignant glioma, characterized by unpredictable clinical behaviors that suggest distinct molecular subtypes. With the tumor metabolic phenotype being one of the hallmarks of cancer, we have set upon to investigate whether GBMs show differences in their metabolic profiles. (1)H NMR analysis was performed on metabolite extracts from a selection of nine glioblastoma cell lines. Analysis was performed directly on spectral data and on relative concentrations of metabolites obtained from spectra using a multivariate regression method developed in this work. Both qualitative and quantitative sample clustering have shown that cell lines can be divided into four groups for which the most significantly different metabolites have been determined. Analysis shows that some of the major cancer metabolic markers (such as choline, lactate, and glutamine) have significantly dissimilar concentrations in different GBM groups. The obtained lists of metabolic markers for subgroups were correlated with gene expression data for the same cell lines. Metabolic analysis generally agrees with gene expression measurements, and in several cases, we have shown in detail how the metabolic results can be correlated with the analysis of gene expression. Combined gene expression and metabolomics analysis have shown differential expression of transporters of metabolic markers in these cells as well as some of the major metabolic pathways leading to accumulation of metabolites. Obtained lists of marker metabolites can be leveraged for subtype determination in glioblastomas.