α-TEA cooperates with MEK or mTOR inhibitors to induce apoptosis via targeting IRS/PI3K pathways.

BACKGROUND: α-Tocopherol ether-linked acetic acid (α-TEA) is a promising agent for cancer prevention/therapy based on its antitumour actions in a variety of cancers. METHODS: Human breast cancer cells, MCF-7 and HCC-1954, were used to study the effect of α-TEA using Annexin V/PI staining, western blot analyses, and siRNA knockdown techniques. RESULTS: α-Tocopherol ether-linked acetic acid suppressed constitutively active basal levels of pAKT, pERK, pmTOR, and their downstream targets, as well as induced both cell types to undergo apoptosis. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin suppressed pAKT, pERK, pmTOR, and their downstream targets, indicating PI3K to be a common upstream mediator. In addition, α-TEA induced increased levels of pIRS-1 (Ser-307), a phosphorylation site correlated with insulin receptor substrate-1 (IRS-1) inactivation, and decreased levels of total IRS-1. Small interfering RNA (siRNA) knockdown of JNK blocked the impact of α-TEA on pIRS-1 and total IRS-1 and impeded its ability to downregulate the phosphorylated status of AKT, ERK, and mTOR. Combinations of α-TEA+MEK or mTOR inhibitor acted cooperatively to induce apoptosis and reduce basal levels of pERK and pmTOR. Importantly, inhibition of MEK and mTOR resulted in increased levels of pAKT and IRS-1, and α-TEA blocked them. CONCLUSIONS: Downregulation of IRS-1/PI3K pathways via JNK are critical for α-TEA and α-TEA+MEK or mTOR inhibitor-induced apoptosis in human MCF-7 and HCC-1954 breast cancer cells.

Monoclonal antibody to chicken fetal antigens on normal erythroid cells and hematopoietic-lymphoid tumor cell lines.

Hybridoma cell lines secreting antibodies to chicken fetal antigens (CFAs) were generated by the fusion of mouse P3X63Ag8 myeloma cells with spleen cells from a mouse immunized with intact SC chicken strain one-day-hatched red blood cells. Immunodepletion studies show monoclonal anti-CFA to be detecting a subset of the Mr 50,000 CFA molecules recognized by polyclonal anti-CFA. Monoclonal anti-CFA is erythroid specific against in vivo-derived hematopoietic-lymphoid cells. Exceptions to the erythroid specificity of monoclonal anti-CFA include failure to react with avian erythroblastosis virus-transformed erythroid cells both before and after butyric acid-induced differentiation and reactions with reticuloendotheliosis virus-transformed immature lymphoid cells and chicken embryo cells. Immunofluorescence and 125I binding analyses utilizing monoclonal anti-CFA show reticuloendotheliosis virus cells to possess high levels of CFA even though the CFA determinant does not appear to be a 125I-labeled immunoprecipitable Mr 50,000 molecule. The unique property of monoclonal anti-CFA that permits it to distinguish among surface membrane antigens of normal and neoplastic cells of the same lineage makes it an important tool for future investigations of normal and abnormal cell differentiation.

Immune abnormalities in avian erythroblastosis virus-infected chickens.

Infection of animals with retroviruses frequently leads to immunosuppressed states. The immune status of chickens injected with the replication-defective avian erythroblastosis virus (AEV), with its naturally occurring subgroup B helper virus (avian erythroblastosis-associated virus; AEAV), was evaluated daily and compared to the immune status of age-matched uninfected control chickens. Spleen cells from AEV-infected chickens gave depressed responses to concanavalin A, phytohemagglutinin, and pokeweed mitogen beginning 3 days after injection of the virus and continuing until death. Spleen cells from AEV-infected chickens suppressed the T-cell mitogen-induced blastogenic responses of spleen cells from uninfected chickens. The ability of spleen cells from infected chickens to suppress mitogen-induced blastogenic responses of spleen cells from normal chickens in coculture was transient beginning 4 days following viral inoculation, reaching peak levels of suppression on day 7 and disappearing by day 12. Cytolysis of splenic cells from AEV-infected chickens with polyclonal anti-T-cell-serum removed the suppressor activity. Addition of conditioned medium rich in T-cell growth factor resulted in a partial restoration of the blastogenic responsiveness of splenic cells from 6-day post-AEV-infected chickens. Addition of exogenous T-cell growth factor had no effect on the suppressed blastogenic responsiveness of spleen cells from 12-day post-AEV-infected chickens, and it had no effect on coculture suppression. In addition to suppressed T-cell responses to polyclonal mitogen-induced proliferation in vitro and transiently expressed T-suppressor cells, thymic atrophy and structural disruption was observed in AEV-infected chickens.

Immunochemical characterization of differentiation and age-related cell surface antigens expressed by chicken erythrocytes.

Hematopoietic-lymphoid membrane antigens that are related to cell differentiation and development, referred to as chicken fetal antigen (CFA) and chicken adult antigen (CAA) were immunochemically characterized; Mr 220,000; Mr 170,000; Mr 130,000; Mr 99,000; Mr 88,000; Mr 50,000; and Mr 24,000 CFA molecules are detected on embryonic RBC, and Mr 210,000; Mr 130,000; Mr 102,000; Mr 56,000; Mr 48,000; and Mr 43,000 CAA molecules are detected on adult RBC. Limited peptide mapping analyses showed all of the CFA and CAA molecules to be distinct entities. Both the Mr 50,000 CFA and the Mr 43,000 CAA molecules exhibited multiple isomorphic variants when analyzed by 2-dimensional electrophoresis. Analyses involving neuraminidase treatments and limited peptide mapping showed the Mr 50,000 CFA isomorphic variants to be chemically identical with the isoelectric point variations being due to sialic acid differences. In addition to multiple isomorphic variants, the molecular weight and charge differences of which were diminished by neuraminidase treatments, the Mr 43,000 CAA molecules exhibited a doublet pattern suggesting that the polyclonal antisera may be detecting chicken major histocompatibility complex products. Analyses of the Mr 50,000 CFA molecules immunoprecipitated with monoclonal antibody 190-4 confirmed that the monoclonal antibody recognizes a serological subset of the Mr 50,000 CFA molecules but showed that it did not recognize a unique molecularly detectable subset among the 18 isomorphic variants discernable by 2-dimensional electrophoretic analyses. Cocapping analyses with splenic lymphocytes showed CFA and CAA to occur as distinct membrane entities on lymphocytes.

Chicken fetal and adult antigen expression on erythroleukemia cells before and after induced differentiation.

Avian erythroblastosis virus strain R (AEV)-transformed, cloned erythroleukemia cells from three different ages of SC strain chickens were analyzed before and after differentiation induced by 1.0 mM butyric acid for expression of chicken fetal antigens (CFAs) and chicken adult antigens (CAAs) and for hemoglobin expression. Immunofluorescent analyses show the loss of individual CFA determinants from erythroleukemia cells with induced differentiation, although there appeared to be no correlation between CFA loss and onset of hemoglobin production. Erythroleukemia cells were examined by cell surface labeling followed by immunoprecipitation with antisera specific to CFAs and CAAs. Erythroleukemia cells expressed CFAs and CAAs on their membranes that are not reported to be expressed by the target cell of AEV. The expression of CAAs and the enhanced expression of CFAs by erythroleukemia cells may be due to limited cellular differentiation, alterations in regulatory controls of genes coding for CFAs and CAAs, or increased levels of production of previously undetected CFAs and CAAs following AEV transformation. Control and induced erythroleukemia cells expressed CFAs and CAAs that differed both quantitatively and qualitatively from normal erythroid cells. Molecular weight variations of CFAs and CAAs observed in the erythroleukemia cells may represent glycolyzation differences between AEV-transformed cells and normal erythroid cells.

Vitamin E succinate (VES) induces Fas sensitivity in human breast cancer cells: role for Mr 43,000 Fas in VES-triggered apoptosis.

Fas (CD95/APO-1) is an important mediator of apoptosis. We show that Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 human breast cancer cells become responsive to anti-Fas (CD95) agonistic antibody-triggered apoptosis after pretreatment or cotreatment with vitamin E succinate (VES; RRR-alpha-tocopheryl succinate). In contrast, no enhancement of anti-Fas agonistic antibody-triggered apoptosis was observed following VES pretreatment or cotreatment with Fas-sensitive primary cultures of human mammary epithelial cells, immortalized MCF-10A cells, or T47D human breast cancer cells. Although VES is itself a potent apoptotic triggering agent, the 6-h pretreatment procedure for Fas sensitization did not initiate VES-mediated apoptosis. The combination of VES plus anti-Fas in pretreatment protocols was synergistic, inducing 2.8-, 3.0-, and 6.3-fold enhanced apoptosis in Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells, respectively. Likewise, cotreatment of Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells with VES plus anti-Fas enhanced apoptosis 1.9-, 2.0-, and 2.6-fold, respectively. Functional knockout of Fas-mediated signaling with either Fas-neutralizing antibody (MCF-7-, MDA-MB-231-, and MDA-MB-435-treated cells) or Fas antisense oligomers (MDA-MB-435-treated cells only), reduced VES-triggered apoptosis by approximately 50%. Analyses of whole cell extracts from Fas-sensitive cells revealed high constitutive expression of Mr 43,000 Fas, whereas Fas-resistant cells expressed low levels that were confined to the cytosolic fraction. VES treatment of the Fas-resistant cells caused a depletion of cytosolic Mr 43,000 Fas with a concomitant increase in Mr 43,000 membrane Fas. These data show that VES can convert Fas-resistant human breast cancer cells to a Fas-sensitive phenotype, perhaps by translocation of cytosolic Mr 43,000 Fas to the membrane and show that VES-mediated apoptosis involves Mr 43,000 Fas signaling.

Activation of extracellular signal-regulated kinase and c-Jun-NH(2)-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells.

RRR-alpha-tocopherol succinate (vitamin E succinate, VES) is a potent, selective apoptotic agent for cancer cells but not normal cells. VES has been shown to inhibit the growth of a wide variety of tumor cells in cell culture and animal models. Studies addressing mechanisms of action of VES-induced apoptosis have identified transforming growth factor-beta, Fas/CD95-APO-1, and mitogen-activated protein kinase (MAPK) signaling pathway involvement. Here we show that MAPKs, the extracellular signal-regulated kinases (ERK), and the stress-activated protein kinases, c-Jun NH2-terminal kinases (JNK), but not p38, are critical mediators in VES-induced apoptosis of human breast cancer MDA-MB-435 cells. VES activates ERK1/2 and JNK both in level and duration of kinase activity. Expression of dominant negative mutants of ERK1, MAPK/ERK activator-1, or JNK1 but not p38 blocked phosphorylation of the substrate glutathione S-transferase-c-Jun and inhibited VES-induced apoptosis. Increased phosphorylation and transactivation activity of nuclear transcription factors c-Jun, ATF-2, and Elk-1 are observed after VES treatments; however, only c-Jun and ATF-2 appear to be involved in VES-induced apoptosis based on antisense blockage experiments. Collectively, these results imply a critical role for ERK1 and JNK1 but not p38 in VES-induced apoptosis of human MDA-MB-435 breast cancer cells.

c-Jun involvement in vitamin E succinate induced apoptosis of reticuloendotheliosis virus transformed avian lymphoid cells.

Previous studies have shown that treatment of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells with 10 microg/ml (18.8 microM) of RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) for 3 days induced approximately 50% of the cells to undergo apoptosis. Elevated and prolonged expression of c-jun mRNA and protein was temporally correlated with VES-induced cell death. Data presented in this paper show that the elevated and prolonged expression of c-jun message and protein are not accounted for by enhanced stability, and show the involvement of c-Jun in VES-induced apoptosis in this lymphoblastoid cell type. C4-1 cells infected with a virus carrying a dominant, negatively acting mutant form of c-Jun, supjun-1, exhibited: (i) 71% reduction in VES-induced apoptosis, (ii) a 2.0-2.5-fold decrease in wildtype, endogenous c-Jun expression, and (iii) a 2.4-2.6-fold reduction in AP-1 binding activity. Additionally, cells co-treated with VES plus RRR-alpha-tocopherol, exhibited a 70% reduction in apoptosis, a marked reduction in c-Jun expression and a 1.6-fold reduction in AP-1 binding activity. These studies suggest that c-Jun plays a crucial role in VES-induced apoptosis in C4-1 cells, and add to our understanding of mechanisms of action involved in VES-mediated tumor cell growth inhibition.

Chicken fetal antigen (CFA) expression on the primitive erythroid maturation series.

Indirect immunofluorescence was utilized to study the expression of chicken fetal antigens (CFA), developmentally phasic membrane antigens, on the primitive erythroid series during chick embryo development. Cells of this series expressed CFA determinants at all four stages of cellular maturation. However, the relative intensity of fluorescent staining (measurement of CFA determinants) varied with development. CFA determinant loss was correlated with the age of the embryo, and the stage of cellular maturation. Indirect immunofluorescence utilizing antisera capable of detecting eight individual CFA determinants revealed that all eight CFA determinants were present on early polychromatic erythrocytes of the primitive series, but that a selective and sequential loss of CFA determinants occurred in later stages. The data suggest that certain CFA determinants are differentially expressed during primitive erythroid cellular maturation in the developing chick embryo.

EA-rosette-forming cells in chickens: presence in spleens of bursectomized chickens and relationships to B-lymphocytes.

Spleen cells from 3-month-old chickens which had been surgically and chemically bursectomized were examined for rosette-forming lymphocytes (RFL). Finding no correlation between the degree of agammaglobulinemia in bursectomized chickens and the percentage of RFL in their spleens demonstrated a dissociation between two B-cell dependent functions, namely, immunoglobulin production and rosette-forming ability. In fact, the percentage of RFL in the spleens of agammaglobulinemic chickens was found to be slightly higher than those of normal chickens. The data indicate that RFL in spleens of normal chickens are B cells, and suggest the existence of a population of rosette-forming B lymphocytes in agammaglobulinemic bursectomized chickens.

Characterization and developmental expression of the chicken B-G heterodimer.

Monoclonal antibody R7-3 recognized an erythroid specific cell surface molecule with a m.w. of approximately 98 kilodaltons (Kd) under nonreducing conditions and molecules of 40 and 44 Kd under reducing conditions on both embryonic- and adult-derived peripheral RBC. Immunochemical characterization, including limited peptide map analyses of these molecules, provided evidence that mAb R7-3 was recognizing the MHC coded B-G heterodimer. This is the first report of a monoclonal antibody that recognizes the B-G heterodimer. Affinity binding studies suggested that mAb R7-3 preferentially recognized the 44 Kd molecule. Immune depletion analyses demonstrated the presence of a single population of B-G heterodimers. Endoglycosidase-F and neuraminidase digestions suggested that the 44 and 40 Kd molecules contained very little, if any, carbohydrate. B-G heterodimer expression was examined on primitive and definitive RBC during embryonic development. B-G heterodimer expression was not detected on RBC of other avians.

Chicken transferrin receptor expression during erythroid differentiation and by retrovirus transformed cells.

Antiserum prepared against sucrose gradient purified reticuloendotheliosis virus (REV) recognized the chicken transferrin receptor. Molecules immunoprecipitated from red blood cells (RBC) obtained from embryonic chickens with either the anti-REV reagent or a chicken transferrin immunomatrix were demonstrated to be identical by co-migration in both reducing and nonreducing SDS-polyacrylamide gels and in two-dimensional isoelectric focusing analyses, reciprocal immunodepletion analyses and by peptide mapping. The chicken transferrin receptor was shown to be a 190,000 dalton cell surface membrane molecule consisting of two similar disulfide-bonded subunits of approximately 95,000 daltons. The chicken transferrin receptor was expressed on erythroid cell surface membranes as 95,000 dalton monomers as well as 190,000 dalton dimers. The chicken transferrin receptor was expressed on all differentiation/maturation stages, including mature RBC, of both the primitive and definitive type I erythroid cell series. In adult chickens, the transferrin receptor was expressed by immature erythroid cells in the bone marrow, but not by mature circulating RBC. REV-transformed immature lymphoid cells and avian erythroblastosis virus (AEV)-transformed erythroid cells expressed dimers composed of 95,000 and 110,000 dalton subunits. Comparisons among V8 protease derived peptides from 95,000 dalton transferrin receptors obtained from RBC and REV-transformed lymphoid cells revealed a high degree of homology; however, the 95,000 dalton molecules isolated from REV-transformed lymphoid cells exhibited a 56,000 dalton peptide that was unique. Cloned AEV-transformed erythroleukemia cells induced to differentiate by supplementation of the media with 1 mM butyric acid expressed elevated transferrin receptor levels. Both serological and peptide mapping studies demonstrated the human transferrin receptor on K562 cells and the chicken transferrin receptor to be distinct. However, chicken transferrin was shown to be capable of reacting with the human transferrin receptors on K562 cells.

Modulation of immune suppression and enhanced tumorigenesis in retrovirus tumor challenged chickens treated with vitamin E.

Vitamin E(dl-alpha-tocopherol) dissolved in ethanol with polyethylene glycol as the vehicle and administered by intraperitoneal injections of 0.1 mg/gm body weight at two day intervals, was demonstrated to be a potent immunomodulating reagent in young chickens challenged with avian reticuloendotheliosis virus-transformed tumor cells. Vitamin E treatment enhanced the mitogen-induced proliferative responses of spleen cells from age matched, unchallenged chickens; reduced the tumor cell-induced suppression of host splenic lymphocyte mitogen responses; and eliminated tumor cell-induced suppressor cell activity. However, inspite of an improved immune status the vitamin E treated-tumor cell challenged chickens exhibited enhanced tumorigenesis.

Nutrition, immunology and cancer: an overview.

There is epidemiological, laboratory and some clinical evidence that certain dietary factors play a role in either promoting or inhibiting cancer development. An understanding of the mechanisms whereby specific nutrients are having effects in the promotion or prevention of cancer is beginning to take shape. Research into nutrient effects on the expression of specific genes, especially cytokine and cytokine receptor expression, will help increase our basic knowledge of cancer biology. Since cytokines can either enhance or suppress both immune defense and cancer growth, increased understanding of nutrient effects on the cytokine network will be beneficial. The regulation of specific gene expression by specific nutrients, indeed, identifies a major frontier for present and future nutritional biologists.