Biomedical subjects

Weiping Shen

Publications and source records attributed to Weiping Shen.

8 recordsLinked to original sources

PubMed2005

Modulation of CD4 Th cell differentiation by ganglioside GD1a in vitro.

Cell surface gangliosides are shed by tumors into their microenvironment. In this study they inhibit cellular immune responses, including APC development and function, which is critical for Th1 and Th2 cell development. Using human dendritic cells (DCs) and naive CD4(+) T cells, we separately evaluated Th1 and Th2 development under the selective differentiating pressures of DC1-inducing pertussis toxin (PT) and DC2-inducing cholera toxin (CT). High DC IL-12 production after PT exposure and high DC IL-10 production after CT exposure were observed, as expected. However, when DCs were first preincubated with highly purified G(D1a) ganglioside, up-regulation of costimulatory molecules was blunted, and PT-induced IL-12 production was reduced, whereas CT-induced IL-10 production was increased. The combination of these effects could contribute to a block in the Th1 response. In fact, when untreated naive T cells were coincubated with ganglioside-preincubated, Ag-exposed DCs, naive Th cell differentiation into Th effector cells was reduced. Both the subsequent DC1-induced T cell production of IFN-gamma (Th1 marker) and DC2-induced T cell IL-4 production (Th2) were inhibited. Thus, ganglioside exposure of DC impairs, by at least two distinct mechanisms, the ability to induce Th differentiation, which could adversely affect the development of an effective cellular antitumor immune response.

Cell Differentiation↗

PubMed2005

Target selection of soluble protein complexes for structural proteomics studies.

BACKGROUND: Protein expression in E. coli is the most commonly used system to produce protein for structural studies, because it is fast and inexpensive and can produce large quantity of proteins. However, when proteins from other species such as mammalian are produced in this system, problems of protein expression and solubility arise 1. Structural genomics project are currently investigating proteomics pipelines that would produce sufficient quantities of recombinant proteins for structural studies of protein complexes. To investigate how the E. coli protein expression system could be used for this purpose, we purified apoptotic binary protein complexes formed between members of the Caspase Associated Recruitment Domain (CARD) family. RESULTS: A combinatorial approach to the generation of protein complexes was performed between members of the CARD domain protein family that have the ability to form hetero-dimers between each other. In our method, each gene coding for a specific protein partner is cloned in pET-28b (Novagen) and PGEX2T (Amersham) expression vectors. All combinations of protein complexes are then obtained by reconstituting complexes from purified components in native conditions, after denaturation-renaturation or co-expression. Our study applied to 14 soluble CARD domain proteins revealed that co-expression studies perform better than native and denaturation-renaturation methods. In this study, we confirm existing interactions obtained in vivoin mammalian cells and also predict new interactions. CONCLUSION: The simplicity of this screening method could be easily scaled up to identify soluble protein complexes for structural genomic projects. This study reports informative statistics on the solubility of human protein complexes expressed in E.coli belonging to the human CARD protein family.

Journal Article↗

PubMed2003

IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion.

Our previous studies have shown that insulin-like growth factor binding protein 2 (IGFBP-2) is frequently overexpressed in the highly invasive glioblastoma multiforme (GBM). By using a yeast two-hybrid system, we identified a gene, invasion inhibitory protein 45 (IIp45), whose protein product bound to IGFBP-2 through the thyroglobulin-RGD region of the C terminus of IGFBP-2. The IIp45 gene is located on chromosome 1p36 and has nine exons. The IIp45 protein has three SEG (segment of low compositional complexity) domains and an integrin-binding RGD motif. The IIp45 protein was not expressed in some GBMs. Functional studies showed that IIp45 inhibited GBM cell invasion both in vitro and in xenograft model. Gene expression profiling studies showed that IIp45 consistently inhibited the expression of cell invasion-associated genes, such as the transcriptional NFkappaB, and its downstream target gene, intercellular adhesion molecule 1. Thus, we report here the isolation and characterization of a gene, IIp45, whose protein product binds to IGFBP-2 and inhibits glioma cell invasion.

Adult↗

PubMed2003

Mechanisms of ganglioside inhibition of APC function.

Gangliosides shed by tumor cells exert potent inhibitory effects on cellular immune responses. Here we have studied ganglioside inhibition of APC function. When human monocytes were preincubated in 50 micro M highly purified ganglioside G(D1a), pulsed with tetanus toxoid (TT), and washed, the expected Ag-induced proliferative response of autologous normal T cells added to these monocytes was inhibited by 81%. Strikingly, there was also almost complete (92%) and selective inhibition of the up-regulation of the monocyte costimulatory molecule CD80, while I-CAM-1, LFA-3, HLA-DR, and CD86 expression were unaffected. Purified LPS-stimulated monocytes that had been preincubated in G(D1a) likewise showed inhibition of CD80 up-regulation (59%) as well as down-regulation of CD40 (54%) and impaired release of IL-12 and TNF-alpha (reduced by 59 and 51%). G(D1a)-preincubated human dendritic cells (DC) were also affected. They had reduced constitutive expression of CD40 (33%) and CD80 (61%), but not CD86, and marked inhibition of release of IL-6 (72%), IL-12 (70%), and TNF-alpha (46%). Even when pulsed with TT, these ganglioside-preincubated DC remained deficient in costimulatory molecule expression and cytokine secretion and were unable to induce a normal T cell proliferative response to TT. Finally, significant inhibition of nuclear localization of NF-kappaB proteins in activated DC suggests that disruption of NF-kappaB activation may be one mechanism contributing to ganglioside interference with APC expression of costimulatory molecules and cytokine secretion, which, in turn, may diminish antitumor immune responses.

Active Transport, Cell Nucleus↗

PubMed2003

Insulin-like growth factor binding protein 2 enhances glioblastoma invasion by activating invasion-enhancing genes.

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastoma (GBM), the most advanced stage of glioma. To determine whether IGFBP2 is involved in the proliferative and invasive nature of GBM, we established stable SNB19 GBM cell lines that overexpress IGFBP2. Although there was no marked difference in the cell growth between IGFBP2 overexpressing SNB19(BP2) lines when compared with the control cells, these clones showed significantly increased invasive rates when compared with the parental or vector transfected SNB19 cells. Total RNAs from controls and SNB19(BP2) clones were used for microarray analysis to detect IGFBP2-mediated alterations in gene expression. When compared with parental or vector-transfected control cells, SNB19(BP2) cells consistently showed 3-5-fold increase in the expression of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes. Increased MMP-2 expression in SNB19(BP2) cells was subsequently confirmed by real time reverse-transcription PCR, Western blotting, and gelatin zymography. Furthermore, consistent with increased MMP-2 expression in SNB19(BP2) cells, transient transfection of a MMP-2 promoter/luciferase reporter also resulted in 3-6-fold higher luciferase activity in SNB19(BP2) cells than in parental or vector-transfected control cells. Finally, tissue microarray analysis of 68 GBM tissue specimens showed a significant correlation between the overexpression of IGFBP2 and elevated MMP-2 expression. Taken together, our data provide evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion.

Gene Expression Regulation, Neoplastic↗

PubMed2003

Dimerization of MLL fusion proteins immortalizes hematopoietic cells.

MLL fusion proteins are leukemogenic, but their mechanism is unclear. Induced dimerization of a truncated MLL immortalizes bone marrow and imposes a reversible block on myeloid differentiation associated with upregulation of Hox a7, a9, and Meis1. Both dimerized MLL and exon-duplicated MLL are potent transcriptional activators, suggesting a link between dimerization and partial tandem duplication of DNA binding domains of MLL. Dimerized MLL binds with higher affinity than undimerized MLL to a CpG island within the Hox a9 locus. However, MLL-AF9 is not dimerized in vivo. The data support a model in which either MLL dimerization/exon duplication or fusion to a transcriptional activator results in Hox gene upregulation and ultimately transformation.

Animals↗

PubMed2002

Ganglioside GD1a impedes lipopolysaccharide-induced maturation of human dendritic cells.

Immunosuppressive membrane gangliosides are released by tumor cells and inhibit normal antigen presenting cell (APC) function. To better understand this process, we have studied the effect of gangliosides on lipopolysaccharide (LPS)-induced maturation of human dendritic cells (DCs). Immature DCs were generated in vitro from human peripheral blood monocytes and were exposed for 72 h to a highly purified ganglioside, G(D1a). During the last 24 h, LPS was added to effect maturation. As assessed by fluorescence activated cell sorting (FACS) analysis, incubation in 50 microM G(D1a) significantly blunted the LPS-induced maturation of the dendritic cells. The expected up-regulation of expression of the co-stimulatory molecules CD80 and CD86 was ablated and that of CD40 was reduced, as were surface CD83 expression and intracellular CD208 production. In addition, ganglioside pretreatment of DC markedly inhibited the allostimulatory capacity and partially prevented the down-regulation of FITC-dextran uptake characteristic of LPS-activated DC. Furthermore, ganglioside-exposed DC also evidenced a broad down-regulation of the cytokine release that is normally initiated by LPS exposure, i.e., there was no increase in IL-1 beta, IL-6, IL-10, IL-12, or tumor necrosis factor (TNF)-alpha release. That a common mechanism may underlie these defects was suggested by the finding that G(D1a) exposure of DC also inhibited the nuclear binding of NF-kappa B that is normally induced by LPS. These results suggest that tumor gangliosides may blunt the anti-tumor immune response in vivo by binding and interfering with dendritic cell maturation.

Antigens, CD↗

PubMedDate not supplied

Apoptotic response to 5-fluorouracil treatment is mediated by reduced polyamines, non-autocrine Fas ligand and induced tumor necrosis factor receptor 2.

5-fluorouracil (5-FU) is the major chemotherapeutic agent for treatment of colorectal carcinoma, but the molecular mechanisms of response and resistance are not understood completely. We therefore studied the 5-FU dose response and time course of gene expression transcriptome changes in colon carcinoma cell lines that are relatively sensitive to or resistant to 5-FU (RKO and HT29, respectively. We identified cellular pathways and corroborated functions of selected pathways. Expression of genes for polyamine biosynthesis, i.e., ornithine decarboxylase (ODC) and spermine and spermidine synthases, was repressed in the sensitive line, while the biosynthesis-inhibiting gene ODC antizyme was induced in the resistant line. The rate-limiting gene in catabolism, spermine/spermidine acetyltransferase, was induced in both lines. Polyamine levels showed corresponding drastic decreases after 5-FU treatment, and polyamine replenishment interfered with 5-FU-induced apoptosis. In the sensitive cells which have wild-type p53, the p53 gene and its downstream genes including p21/WAF1, mdm2, Fas, mic-1, EphA2, and ferredoxin reductase as well as genes in the tumor necrosis factor (TNF) pathway including TNF receptor 2 (TNFR2) were induced, but not Fas ligand (FasL). Exposure to exogenous FasL increased 5-FU-induced apoptosis, and anti-TNFR2 antibody, but not anti-TNFR1, partially protected the sensitive cells. Our combination of gene expression profiling and corroborative functional studies revealed that reduced polyamine levels, non-autocrine FasL originating exogenous to tumor cells, and induced TNFR2 are all functional mediators of apoptosis caused by 5-FU in colon carcinoma cells.

Acetyltransferases↗