Cripto-1 induces apoptosis in HC-11 mouse mammary epithelial cells.

Cripto-1 (CR-1) is an epidermal growth factor (EGF)-related protein. CR-1 can inhibit beta-casein and whey acidic protein expression in mouse mammary epithelial cells. The present study demonstrates that CR-1 can induce apoptosis in HC-11 mouse mammary epithelial cells, as measured by bis-benzimide stained nuclei, TUNEL assay and cell death ELISA. Apoptosis could be observed after 2 days of exposure of confluent HC-11 cells to CR-1 in the absence of the survival factors EGF and insulin, with maximum apoptosis occurring at 3 days. A reduction in poly(ADP-ribose) polymerase (PARP) expression and an increase in beta-catenin cleavage was found after 18 h of exposure to CR-1 suggesting that apoptosis was preceded by the induction of a caspase activity since the caspase inhibitor ZFAD.FMK could block the CR-1-induced reduction in PARP expression and CR-1-induced apoptosis. CR-1 was found to increase the expression of caspase-3-like protease. Although, the levels of p27kip1 and p21Bax did not change after exposure to CR-1 for 18 h, the levels of Bcl-xL became undetectable. These studies suggest that CR-1 promotes apoptosis by mediating the induction of caspase-3-like protease and downregulating the expression of Bcl-xL.

Myelin basic protein domains involved in the interaction with actin.

A fluorescence assay was used to measure the interaction of myelin basic protein (MBP) with monomeric actin labeled with a fluorescent compound (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal dependence on the concentration of MBP and the fluorescence maximum is reached at a MBP:actin molar ratio of 1:20. The fluorescence maximum in absence of Ca2+ and ATP is 4 times lower than that in their presence although it is reached at the same MBP:actin molar ratio. Similar behavior is observed when synapsin replaces MBP, while acetylated MBP and bovine serum albumin fail to induce any fluorescence change. To define possible interacting domains on MBP involved in the actin-MBP interaction, experiments were performed using MBP-derived peptides obtained under controlled proteolysis of the whole molecule. The fluorescence changes induced by the different peptides depend on their location in the native protein and can not be explained simply by a difference in the net charge of the peptides. The results suggest that two sites are involved in the interaction. A Ca2+/ATP-dependent site located in the amino-terminal region (peptide 1-44) and a Ca2+/ATP-independent one near the carboxyl terminus of the MBP molecule. The actin-MBP interaction was also observed using immunoblot and ELISA techniques.

Experimental allergic encephalomyelitis: characterization of T lymphocytes that bind myelin basic protein and synapsin.

The immuno phenotypic profile of the mononuclear cells that bind myelin basic protein (MBP) and synapsin was investigated in lymph node cells from rats with experimental allergic encephalomyelitis induced by injection with MBP. Using a double immunofluorescent labeling technique, purified cells that bind one or both antigens were analyzed in different stages of the disease. The total MBP-bound lymphocytes increased at 14 days post-inoculation (dpi), had a CD4+/CD8+ ratio of two and were present until 29 dpi. Conversely, the apportionment of cells specific for MBP that also recognize synapsin reached a maximum value at 14 dpi coincidentally with the expression of the paralysis symptoms and then, they disappeared when the animal began to recover. This population amounted to about 40% of the total lymph node MBP-bound cells and had a CD4+/CD8+ ratio of one, indicating that the lymphocytes with MBP-synapsin crossreactivity could be principally implicated in a cytotoxic or suppressor activity.

Cellular immune crossreactivity between myelin basic protein and synapsin in rats with experimental allergic encephalomyelitis.

We previously demonstrated that antibodies against myelin basic protein (MBP) obtained from animals with experimental allergic encephalomyelitis (EAE), induced with MBP and purified by affinity chromatography, have the property to recognize a neuronal protein, synapsin Ia and Ib. To investigate whether this crossreactivity also occurs at the cellular level, we purified spleen and lymph node mononuclear cells from rats sensitized with MBP or synapsin using polystyrene plates coated with the respective antigen. We observed that animals injected with MBP have T lymphocytes that bind both antigens. Using the same system, each purified cell population was confronted again to the studied antigens. The anti-MBP cells recognized once more epitopes of MBP and about 40% of them also recognized synapsin. On the other hand, cells that first were attached to synapsin, in the second exposure to antigens bound to MBP and synapsin in similar amounts. Double immunofluorescent labeling of the mononuclear cells isolated from animals injected with bovine myelin or MBP showed that the same lymphocyte was able to recognize MBP as well as synapsin. In both experimental systems the quantitative results were similar indicating that in bovine myelin- or MBP-sensitized animals practically all the cells that recognize synapsin are anti-MBP cells, and of the total cells raised against MBP, around 40% of them show this crossreactivity. On the contrary, animals injected with synapsin have cells that bind to this protein but not to MBP indicating that the described crossreactivity, as observed at humoral level, is only in one way.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness.

Cripto-1 inhibits beta-casein expression in mammary epithelial cells through a p21ras-and phosphatidylinositol 3'-kinase-dependent pathway.

Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that does not directly activate any of the known erbB type 1 tyrosine kinase receptors. Also, CR-1 stimulates the growth of HC-11 mouse mammary epithelial cells. We found that prior treatment of HC-11 cells with exogenous CR-1 induced a competency response to the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) with respect to the induction of the milk protein beta-casein. In contrast, simultaneous treatment of mouse HC-11 cells with CR-1 in the presence of DIP inhibited beta-casein expression. The inhibitory effects of CR-1 on beta-casein expression in response to DIP were not unique to this mouse mammary epithelial cell line, because beta-casein and whey acidic protein expression in primary mouse mammary explant cultures established from midpregnant mice were also differentially inhibited by several epidermal growth factor-related peptides including CR-1. The mitogenic and differentiation effects of CR-1 are mediated by the binding of CR-1 to a cell surface receptor that is known to activate the ras/raf/mitogen-activated protein kinase (MAPK)/MAPK kinase pathway. The inhibitory response of CR-1 in HC-11 cells on beta-casein expression after treatment with DIP can be attenuated by B581, a peptidomimetic farnesyltransferase inhibitor that blocks p21ras farnesylation and activation, and by the phosphatidylinositol 3'-kinase (PI3k) inhibitor LY 294002 but not by PD 98059, a MAPK kinase inhibitor that blocks MAPK activation. These data suggest that the ability of CR-1 to block lactogenic hormone-induced expression of beta-casein is mediated through a p21ras-dependent, PI3k-mediated pathway. This is further substantiated by the observation that CR-1 is able to stimulate the tyrosine phosphorylation of the p85 PI3k regulatory subunit and to increase the activity of PI3k in HC-11 cells.