Krill oil, vitamin D and Lactobacillus reuteri cooperate to reduce gut inflammation.

Current research into original therapies to treat intestinal inflammation is focusing on no-drug therapies. KLD is a mixture of krill oil (KO), probiotic Lactobacillus reuteri (LR), and vitamin D (VitD3). The aim of this study was to assess in vitro and in vivo the potential cooperative effects of KLD in reducing gut inflammation. Colorectal adenocarcinoma cell lines, CACO2 and HT29, and C57BL/6 mice were used for in vitro and in vivo analyses, respectively. Cells were exposed to cytomix (interferon gamma + tumour necrosis factor alpha (TNF-α)) to induce inflammation or co-exposed to cytomix and KO, LR and VitD3 alone or to cytomix and KLD. Animals were treated for 7 days with dextran sodium sulphate (DSS) to induce colitis or with DSS and KLD. In vitro assays: F-actin expression was analysed by immunofluorescence; scratch test and trans-epithelial electric resistance test were performed to measure wound healing; adhesion/invasion assays of adhesive and invasive Escherichia coli (AIEC) bacteria were made; mRNA expression of TNF-α, interleukin (IL)-8 and vitamin D receptor (VDR) was detected by quantitative PCR. In vivo assays: body weight, clinical score, histological score and large intestine weight and length were estimated; mRNA expression of TNF-α, IL-1ß, IL-6, IL-10 by quantitative PCR; VDR expression was detected by quantitative PCR and immunohistochemistry. In vitro: KLD restores epithelial cell-cell adhesion and mucosal healing during inflammation, while decreases the adhesiveness and invasiveness of AIEC bacteria and TNF-α and IL-8 mRNA expression and increases VDR expression. In vivo: KLD significantly improves body weight, clinical score, histological score and large intestine length of mice with DSS-induced colitis and reduces TNF-α, IL-1ß and IL-6 mRNA levels, while increases IL-10 mRNA and VDR levels. KLD has significant effects on the intestinal mucosa, strongly decreasing inflammation, increasing epithelial restitution and reducing pathogenicity of harmful commensal bacteria.

Expression of p89(c-Mybex9b), an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells.

The c-Myb gene encodes the p75(c-Myb) isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p(c-Mybex9b), which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein-protein interactions and negative regulation. In hematopoietic cells, expression of p(c-Mybex9b) accounts for 10-15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p(c-Mybex9b) and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75(c-Myb), p(c-Mybex9b) is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p(c-Mybex9b) enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p(c-Mybex9b) reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34(+) cells, without affecting the levels of p75(c-Myb). Together, these studies indicate that expression of the low-abundance p(c-Mybex9b) isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells.

Silencing of endogenous IGFBP-5 by micro RNA interference affects proliferation, apoptosis and differentiation of neuroblastoma cells.

Signal transduction through the IGF axis is implicated in proliferation, differentiation and survival during development and adult life. The IGF axis includes the IGF binding proteins (IGFBPs) that bind IGFs with high affinity and modulate their activity. In neuroblastoma (NB), a malignant childhood tumor, we found that IGFBP-5 is frequently expressed. Since NB is an IGF2-sensitive tumor, we investigated the relevance and the function of endogenous IGFBP-5 in LAN-5 and in SY5Y(N) cell lines transfected with micro and small interfering RNAs directed to IGFBP-5 mRNA. Cells in which IGFBP-5 expression was suppressed were growth-inhibited and more prone to apoptosis than the parental cell line and controls. Apoptosis was further enhanced by X-ray irradiation. The ability of these cells to undergo neuronal differentiation was impaired after IGFBP-5 inhibition but the effect was reversed by exposure to recombinant IGFBP-5. Together, these data demonstrate the importance of IGFBP-5 for NB cell functions and suggest that IGFBP-5 might serve as a novel therapeutic target in NB.

Cyclin D1-dependent regulation of B-myb activity in early stages of neuroblastoma differentiation.

Levels of the transcription factor B-myb must be down-regulated to allow terminal differentiation of neuroectodermal cells and yet its constitutive expression induces early markers of neural differentiation. Thus, we investigated potential mechanisms of enhanced B-myb activity in early stages of neural differentiation. We report here that B-myb expression does not decrease, cyclin A and Sp1 levels remain constant while p21 levels increase continuously upon retinoic acid-induced differentiation of the LAN-5 neuroblastoma cell line. In contrast, cyclin D1 expression is down-regulated at the onset of the differentiative process by protein destabilization. Luciferase assays of promoter activity indicate that B-myb-dependent transactivation is enhanced in LAN-5 cells treated with retinoic acid (RA) for 24 h. The enhancement is independent from cyclin A but is suppressed by a degradation-resistant mutant form of cyclin D1. The importance of cyclin D1 in controlling B-myb activity is further suggested by co-immunoprecipitation experiments, showing that the amount of cyclin D1 co-immunoprecipitated with B-myb decreased after RA treatment. Thus, B-myb may play an active role in the early stages of differentiation when its transactivation activity is enhanced as a consequence of cyclin D1 down-modulation.

Caspase cleavage enhances the apoptosis-inducing effects of BAD.

The function of BAD, a proapoptotic member of the Bcl-2 family, is regulated primarily by rapid changes in phosphorylation that modulate its protein-protein interactions and subcellular localization. We show here that, during interleukin-3 (IL-3) deprivation-induced apoptosis of 32Dcl3 murine myeloid precursor cells, BAD is cleaved by a caspase(s) at its N terminus to generate a 15-kDa truncated protein. The 15-kDa truncated BAD is a more potent inducer of apoptosis than the wild-type protein, whereas a mutant BAD resistant to caspase 3 cleavage is a weak apoptosis inducer. Truncated BAD is detectable only in the mitochondrial fraction, interacts with BCL-X(L) at least as effectively as the wild-type protein, and is more potent than wild-type BAD in inducing cytochrome c release. Human BAD, which is 43 amino acids shorter than its mouse counterpart, is also cleaved by a caspase(s) upon exposure of Jurkat T cells to anti-FAS antibody, tumor necrosis factor alpha (TNF-alpha), or TRAIL. Moreover, a truncated form of human BAD lacking the N-terminal 28 amino acids is more potent than wild-type BAD in inducing apoptosis. The generation of truncated BAD was blocked by Bcl-2 in IL-3-deprived 32Dcl3 cells but not in Jurkat T cells exposed to anti-FAS antibody, TNF-alpha, or TRAIL. Together, these findings point to a novel and important role for BAD in maintaining the apoptotic phenotype in response to various apoptosis inducers.

Neuroblastoma specific effects of DR-nm23 and its mutant forms on differentiation and apoptosis.

DR-nm23 belongs to a gene family which includes nm23-H1, originally identified as a candidate metastasis suppressor gene. Nm23 genes are expressed in different tumor types where their levels have been alternatively associated with reduced or increased metastatic potential. Nm23-H1, -H2, DR-nm23 and nm23-H4 all possess NDP kinase activity. Overexpression of DR-nm23 inhibits differentiation and promotes apoptosis in hematopoietic cells. By contrast, it induces morphological and biochemical changes associated with neural differentiation in neuroblastoma cells. In this study, we show that mutations in the catalytic domain and in the serine 61 phosphorylation site, possibly required for protein-protein interactions, impair the ability of DR-nm23 to induce neural differentiation. Moreover, neuroblastoma cells overexpressing wild-type or mutant DR-nm23 are less sensitive to apoptosis triggered by serum withdrawal. By subcellular fractionation, wild-type and mutant DR-nm23 localize in the cytoplasm and prevalently in the mitochondrial fraction. In co-immunoprecipitation experiments, wild-type DR-nm23 binds other members of nm23 family, but mutations in the catalytic and in the RGD domains and in serine 61 inhibit the formation of hetero-multimers. Thus, the integrity of the NDP kinase activity and the presence of a serine residue in position 61 seem essential for the ability of DR-nm23 to trigger differentiation and to bind other Nm23 proteins, but not for the anti-apoptotic effect in neuroblastoma cells. These studies underline the tissue specificity of the biological effects induced by DR-nm23 expression.

BCR-ABL prevents c-jun-mediated and proteasome-dependent FUS (TLS) proteolysis through a protein kinase CbetaII-dependent pathway.

The DNA binding activity of FUS (also known as TLS), a nuclear pro-oncogene involved in multiple translocations, is regulated by BCR-ABL in a protein kinase CbetaII (PKCbetaII)-dependent manner. We show here that in normal myeloid progenitor cells FUS, although not visibly ubiquitinated, undergoes proteasome-dependent degradation, whereas in BCR-ABL-expressing cells, degradation is suppressed by PKCbetaII phosphorylation. Replacement of serine 256 with the phosphomimetic aspartic acid prevents proteasome-dependent proteolysis of FUS, while the serine-256-to-alanine FUS mutant is unstable and susceptible to degradation. Ectopic expression of the phosphomimetic S256D FUS mutant in granulocyte colony-stimulating factor-treated 32Dcl3 cells induces massive apoptosis and inhibits the differentiation of the cells escaping cell death, while the degradation-prone S256A mutant has no effect on either survival or differentiation. FUS proteolysis is induced by c-Jun, is suppressed by BCR-ABL or Jun kinase 1, and does not depend on c-Jun transactivation potential, ubiquitination, or its interaction with Jun kinase 1. In addition, c-Jun-induced FUS proteasome-dependent degradation is enhanced by heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and depends on the formation of a FUS-Jun-hnRNP A1-containing complex and on lack of PKCbetaII phosphorylation at serine 256 but not on FUS ubiquitination. Thus, novel mechanisms appear to be involved in the degradation of FUS in normal myeloid cells; moreover, the ability of the BCR-ABL oncoprotein to suppress FUS degradation by the induction of posttranslational modifications might contribute to the phenotype of BCR-ABL-expressing hematopoietic cells.

The nucleoside diphosphate kinase activity of DRnm23 is not required for inhibition of differentiation and induction of apoptosis in 32Dcl3 myeloid precursor cells.

DRnm23 belongs to a multigene family which includes nm23-H1, the first bona fide metastasis suppressor gene, nm23-H2, nm23-H4, and nm23-H5. Like nm23-H1, nm23-H2, and nm23-H4, DRnm23 possesses nucleoside diphosphate kinase (NDPK) activity. Upon overexpression in myeloid precursor 32Dcl3 cells, DRnm23 inhibits granulocytic differentiation and promotes apoptosis. Two specific mutants of DRnm23 (H134Q and S136P), at residues required for the NDPK activity, inhibit differentiation and promote apoptosis of 32Dcl3 cells. By contrast, substitution of serine 61 with proline (S61P) or deletion of the RGD domain (DeltaRGD) abrogates the effects of wild-type DRnm23. Like wild-type DRnm23, all four mutants show a predominantly mitochondrial subcellular localization. These studies indicate that the enzymatic activity of DRnm23 is not required for the effects observed in 32Dcl3 cells. Moreover, the inability of the S61P and DeltaRGD DRnm23 mutants to inhibit differentiation and promote apoptosis may be due to defective protein-protein interactions at the mitochondria, the predominant site of DRnm23 subcellular localization.

Expression of B-myb in neuroblastoma tumors is a poor prognostic factor independent from MYCN amplification.

The transcription factors of the Myb family are expressed in several tissues and play an important role in cell proliferation, differentiation, and survival In this study, the expression of A-myb, B-myb, and c-myb was investigated in a group of 64 neuroblastomas at different dinical stages by a sensitive reverse transcription-PCR tchnique and correlated with patients' survival. All of the myb genes were frequently expressed in neuroblastoma tumors. Interestingly, the expression of B-myb, which was detected in 33 cases, was associated with an increased risk of death (P = 0.027 in a univariate analysis), whereas there was no correlation with A-myb and c-myb expression. In addition, in a multivariate Cox regression analysis that included myb gene expression, MYCN status, age at diagnosis, and tumor staging, MYCN amplification and B-myb expression were independently associated to an increased risk (P < 0.01 and P = 0.015, respectively). In overall survival curves obtained by stratifying the neuroblastoma cases on the basis of MYCN status and B-myb expression, the group of patients without MYCN amplification and positive for B-myb expression had worse survival probability than that without MYCN amplification and nonexpressing B-myb (P < 0.01). In summary, these findings provide the first demonstration that B-myb expression can be a useful prognostic marker in human neuroblastoma. Moreover, B-myb expression has a prognostic value complementary to MYCN amplification and can identify a group of high-risk patients that would not be predicted on the basis of the MYCN status only.

Stabilization of glycoenzymes by cross-linking of their carbohydrate chains.

The results presented here demonstrate the potential applicability of the described cross-linking method for preparation of soluble glycoenzyme derivatives. The high level of the retained enzyme activity supports the assumption that this approach is superior to the cross-linking through the protein part. In this study, high mannose-type glycoproteins were used. However, the complex-type glycoproteins that are spread among glycoproteins of higher eukaryotes also can be cross-linked by this procedure because, at the least, the terminal monosaccharide will be oxidized by periodate. Moreover, this approach may be applicable when dealing with partially purified glycoenzymes because the protein impurities are not expected to interfere with the cross-linking reaction. Besides cross-linking, other kinds of chemical modifications of glycoenzymes through the carbohydrate part are possible. This, in turn, could lead to changes in the physicochemical and catalytic properties of the enzymes, thereby opening a new field of glycoenzyme engineering.