AMR-Me inhibits PI3K/Akt signaling in hormone-dependent MCF-7 breast cancer cells and inactivates NF-κB in hormone-independent MDA-MB-231 cells.

AMR-Me, a C-28 methylester derivative of triterpenoid compound Amooranin isolated from Amoora rohituka stem bark and the plant has been reported to possess multitude of medicinal properties. Our previous studies have shown that AMR-Me can induce apoptosis through mitochondrial apoptotic and MAPK signaling pathways by regulating the expression of apoptosis related genes in human breast cancer MCF-7 cells. However, the molecular mechanism of AMR-Me induced apoptotic cell death remains unclear. Our results showed that AMR-Me dose-dependently inhibited the proliferation of MCF-7 and MDA-MB-231 cells under serum-free conditions supplemented with 1 nM estrogen (E2) with an IC50 value of 0.15 µM, 0.45 µM, respectively. AMR-Me had minimal effects on human normal breast epithelial MCF-10A + ras and MCF-10A cells with IC50 value of 6 and 6.5 µM, respectively. AMR-Me downregulated PI3K p85, Akt1, and p-Akt in an ERα-independent manner in MCF-7 cells and no change in expression levels of PI3K p85 and Akt were observed in MDA-MB-231 cells treated under similar conditions. The PI3K inhibitor LY294002 suppressed Akt activation similar to AMR-Me and potentiated AMR-Me induced apoptosis in MCF-7 cells. EMSA revealed that AMR-Me inhibited nuclear factor-kappaB (NF-κB) DNA binding activity in MDA-MB-231 cells in a time-dependent manner and abrogated EGF induced NF-κB activation. From these studies we conclude that AMR-Me decreased ERα expression and effectively inhibited Akt phosphorylation in MCF-7 cells and inactivate constitutive nuclear NF-κB and its regulated proteins in MDA-MB-231 cells. Due to this multifactorial effect in hormone-dependent and independent breast cancer cells AMR-Me deserves attention for use in breast cancer prevention and therapy.

The hormonal herbicide, 2,4-dichlorophenoxyacetic acid, inhibits Xenopus oocyte maturation by targeting translational and post-translational mechanisms.

The widely used hormonal herbicide, 2,4-dichlorophenoxyacetic acid, blocks meiotic maturation in vitro and is thus a potential environmental endocrine disruptor with early reproductive effects. To test whether maturation inhibition was dependent on protein kinase A, an endogenous maturation inhibitor, oocytes were microinjected with PKI, a specific PKA inhibitor, and exposed to 2,4-D. Oocytes failed to mature, suggesting that 2,4-D is not dependent on PKA activity and likely acts on a downstream target, such as Mos. De novo synthesis of Mos, which is triggered by mRNA poly(A) elongation, was examined. Oocytes were microinjected with radiolabelled in vitro transcripts of Mos RNA and exposed to progesterone and 2,4-D. RNA analysis showed progesterone-induced polyadenylation as expected but none with 2,4-D. 2,4-D-activated MAPK was determined to be cytoplasmic in localization studies but poorly induced Rsk2 phosphorylation and activation. In addition to inhibition of the G2/M transition, 2,4-D caused abrupt reduction of H1 kinase activity in MII phase oocytes. Attempts to rescue maturation in oocytes transiently exposed to 2,4-D failed, suggesting that 2,4-D induces irreversible dysfunction of the meiotic signaling mechanism.

Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle.

High levels of mos protooncogene product are expressed during oocyte meiotic maturation and Mos has been implicated in formation of the spindle and spindle pole. Here, we show that in Swiss 3T3 cells with 4N DNA content, high levels of Mos lead to the production of binucleated cells. The Swiss 3T3 cells in mitosis, before binucleation occurs, are anastral and the spindle poles are juxtaposed to the cell membrane. These phenotypes may be related to the meiotic process of attachment of the spindle pole to the oocyte membrane during polar body formation. The production of binucleated somatic cells could result from attachment of the altered mitotic spindle pole to the cell membrane that interferes with cytokinesis but not karyokinesis. This can explain at least one form of genetic instability that leads to altered DNA content in tumor cells.

Expression of the v-Mos oncogene in male meiotic germ cells of transgenic mice results in metaphase arrest.

To explore the role of pp39mos in male germ cell meiosis, we have constructed transgenic mice carrying either the c-Mos or v-Mos genes linked to the human male germ cell-specific phosphoglycerate kinase-2 promoter. All male transgenic mice bearing the v-Mos but not the c-Mos construct were sterile due to arrest of germ cells at metaphase I. Immunocytochemistry performed on sections from control and c-Mos transgenic testes with eight different monoclonal and polyclonal antisera against either alpha-, beta- or gamma-tubulins demonstrated that all could recognize MI spermatocyte spindles from control and c-Mos transgenics, but only one monoclonal anti-microtubule sera decorated the spindles of v-Mos-arrested meiotic figures. Western blot analyses with this one serum revealed a change in proteins in the v-Mos samples. Immunocytochemistry with the MPM-2 monoclonal antibody, which is specific for epitopes phosphorylated during mitosis, demonstrated an increase in cytoplasmic and spindle-associated phosphoproteins in arrested v-Mos spermatocytes. Western analysis with MPM-2 showed an increase in a M(r) 50,000-55,000 and a M(r) 25,000-29,000 protein in Mos transgenic testes when compared to controls. An anti-MAP kinase antibody demonstrated an increase in all four MAP kinases in testes of transgenic mice. Thus, overexpression of pp39v-mos during male germ cell meiosis resulted in an alteration of various cell cycle related kinases and cytostatic factor-like arrest at MI.

Influence of exogenous ras and p53 on P-glycoprotein function in immortalized rodent fibroblasts.

The ability of ras oncogenes and mutant p53 to activate reporter gene expression from human and rodent mdr1 gene promoters was described, although functional significance of this finding was unclear. We analyzed the influence of various forms of recombinant human ras and p53 on the mdr1 gene expression and P-glycoprotein (Pgp) function in rodent immortalized fibroblasts. The ras genes, in addition to activation of exogenous human mdr1 gene promoter, caused an increase in (i) expression of endogenous mdr1 mRNA, (ii) Pgp activity as determined by flow cytometry analysis of Rhodamine 123 exclusion, and (iii) resistance of cells to the cytotoxic action of colchicine and some other drugs. To elucidate whether the same signalling pathway is responsible for multidrug resistance induced by various oncogenes and protein kinase C (PKC), we tested the effects of v-mos and the PKC agonist 12-O-tetradecanoylphorbol-13-acetate. Similarly to cells transformed by ras, a Rat1 subline transformed by the v-mos oncogene was characterized by decreased drug sensitivity. On the contrary, Rat1 cells treated with the protein kinase C agonist 12-O-tetradecanoylphorbol-13-acetate showed neither increased mdr1 mRNA expression nor stimulation of Pgp function. Introduction by retrovirus-mediated gene transfer of wild-type p53 into Rat1 cells or into murine p53-deficient 10(1) and 10(3) cells did not change the Pgp function significantly, whereas in Rat1 cells transformed by activated N-ras or v-mos, expression of wild-type p53 caused partial reversion of oncogene-induced drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

v-mos suppresses platelet-derived growth factor (PDGF) type-beta receptor autophosphorylation and inhibits PDGF-BB-mediated signal transduction.

The function of mos protein in somatic cells, the mechanisms underlying its ability to transform cells, and its relationship to growth factor autonomy and growth factor-mediated signal transduction are not well defined. This report demonstrates that the expression of transforming mos (v-mos) can block the stimulation of growth-related gene expression mediated by the platelet-derived growth factor (PDGF-BB). This blockade by v-mos of PDGF-BB signal transduction occurs very early in the signalling pathway, at the level of PDGF type-beta receptor autophosphorylation. Although the expression of PDGF type-beta receptor, as detected by Western blot with anti-PDGF type-beta receptor antibody,was not diminished in v-mos transformed BALB/c-3T3 murine fibroblasts, the autophosphorylation of PDGF-beta receptor in response to ligand (recombinant PDGF-BB homodimer) stimulation was profoundly suppressed. This same phenomenon of v-mos-mediated PDGF type-beta receptor autophosphorylation inhibition was also demonstrated in NIH-3T3 fibroblasts. A v-mos mutant gene, which was incapable of binding ATP and was kinase-defective, did not block ligand-mediated receptor autophosphorylation. Factor(s) present in v-mos expressing fibroblasts, and found in the membrane fractions of these cells, dominantly inhibit the autophosphorylation of the PDGF type-beta receptor obtained from normal fibroblasts. This trans-acting factor does not appear to be a protein-tyrosine phosphatase. These findings suggest a role for mos, or a similar serine/threonine kinase, as a control mechanism in one of the earliest steps of the PDGF signal transduction pathway, and may provide a model for the functional interaction of mos with growth factor receptors.