Biomedical subjects

Younghee Lee

Publications and source records attributed to Younghee Lee.

27 records · Page 2Linked to original sources

PubMed2003

Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2.

Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death.

Apoptosis↗

PubMed2003

Transgenic expression of stromal cell-derived factor-1/CXC chemokine ligand 12 enhances myeloid progenitor cell survival/antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo.

Hemopoiesis is regulated in part by survival/apoptosis of hemopoietic stem/progenitor cells. Exogenously added stromal cell-derived factor-1 ((SDF-1)/CXC chemokine ligand (CXCL)12) enhances survival/antiapoptosis of myeloid progenitor cells in vitro. To further evaluate SDF-1/CXCL12 effects on progenitor cell survival, transgenic mice endogenously expressing SDF-1/CXCL12 under a Rous sarcoma virus promoter were produced. Myeloid progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte) from transgenic mice were studied for in vitro survival in the context of delayed addition of growth factors. SDF-1-expressing transgenic myeloid progenitors were enhanced in survival and antiapoptosis compared with their wild-type littermate counterparts. Survival-enhancing effects were due to release of low levels of SDF-1/CXCL12 and mediated through CXCR4 and G(alpha)i proteins as determined by ELISA, an antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin. Transgenic effects of low SDF-1/CXCR4 may be due to synergy of SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes with low concentrations of other cytokines to enhance survival of normal mouse myeloid progenitors. Consistent with in vitro results, progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow and splenic myelopoiesis: greatly increased progenitor cell cycling and significant increases in progenitor cell numbers. These results substantiate survival effects of SDF-1/CXCL12, now extended to progenitors engineered to endogenously produce low levels of this cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition to cell trafficking.

Adjuvants, Immunologic↗

PubMed2003

Distinct requirements for Stat4 and Stat6 in hematopoietic progenitor cell responses to growth factors and chemokines.

Hematopoietic progenitor cell (HPC) homeostasis is critical in maintaining innate immunity and healing processes. Recently, we demonstrated that Th1 cells regulate HPC homeostasis, partly based on altered homeostasis in Stat4- and Stat6-deficient mice. To explore changes in HPC responsiveness in altered T helper cell environments, we directly examined growth factor-stimulated colony formation and chemokine-induced myelosuppression of HPC in Stat4- and Stat6-deficient bone marrow cells. Stat6-deficient cells have increased responses to the synergy between granulocyte-macrophage colony-stimulating factor (GM-CSF) and steel factor (SLF), compared to wild-type and Stat4-deficient cells. Increased responses are eliminated by in vivo depletion of CD4 cells. Whereas Stat6-deficient bone marrow cells respond to chemokine-mediated myelosuppression, Stat4-deficient bone marrow cells are refractory to the suppressive effects of chemokines. Thus, T helper cell development affects HPC homeostasis through several mechanisms, including the sensitivity to growth factor stimulation and chemokine suppression of HPC colony formation. Since Stat4 and Stat6 regulate opposing programs of T helper differentiation, there are distinct requirements for Stat4 and Stat6 in regulation of growth factor and chemokine responses of HPC.

Animals↗

PubMed2003

Stromal cell-derived factor-1/CXCL12 directly enhances survival/antiapoptosis of myeloid progenitor cells through CXCR4 and G(alpha)i proteins and enhances engraftment of competitive, repopulating stem cells.

Stromal cell-derived factor-1 (SDF-1/CXCL12) enhances survival of myeloid progenitor cells. The two main questions addressed by us were whether these effects on the progenitors were direct-acting and if SDF-1/CXCL12 enhanced engrafting capability of competitive, repopulating mouse stem cells subjected to short-term ex vivo culture with other growth factors. SDF-1/CXCL12 had survival-enhancing/antiapoptosis effects on human bone marrow (BM) and cord blood (CB) and mouse BM colony-forming units (CFU)-granulocyte macrophage, burst-forming units-erythroid, and CFU-granulocyte-erythroid-macrophage-megakaryocyte with similar dose responses. The survival effects were direct-acting, as assessed on colony formation by single isolated human BM and CB CD34(+++) cells. Effects were mediated through CXCR4 and G(alpha)i proteins. Moreover, SDF-1/CXCL12 greatly enhanced the engrafting capability of mouse long-term, marrow-competitive, repopulating stem cells cultured ex vivo with interleukin-6 and steel factor for 48 h. These results extend information on the survival effects mediated through the SDF-1/CXCL12-CXCR4 axis and may be of relevance for ex vivo expansion and gene-transduction procedures.

Animals↗

PubMed2002

3-pyrrolines are mechanism-based inactivators of the quinone-dependent amine oxidases but only substrates of the flavin-dependent amine oxidases.

We previously reported that 3-pyrroline and 3-phenyl-3-pyrroline effect a time-dependent inactivation of the copper-containing quinone-dependent amine oxidase from bovine plasma (BPAO) (Lee et al. J. Am. Chem. Soc. 1996, 118, 7241-7242). Quinone cofactor model studies suggested a mechanism involving stoichiometric turnover to a stable pyrrolylated cofactor. Full details of the model studies are now reported along with data on the inhibition of BPAO by a family of 3-aryl-3-pyrrolines (aryl = substituted phenyl, 1-naphthyl, 2-naphthyl), with the 4-methoxy-3-nitrophenyl analogue being the most potent. At the same time, the parent 3-phenyl analogue is a pure substrate for the flavin-dependent mitochondrial monoamine oxidase B from bovine liver. Spectroscopic studies (including resonance Raman) on BPAO inactivated by the 4-methoxy-3-nitrophenyl analogue are consistent with covalent derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The distinction of a class of compounds acting as an inactivator of one amine oxidase family and a pure substrate of another amine oxidase family represents a unique lead to the development of selective inhibitors of the mammalian copper-containing amine oxidases.

Amine Oxidase (Copper-Containing)↗

PubMed2002

Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines.

Stromal cell-derived factor 1 (SDF-1/CXCL12) is a multifunctional cytokine. We previously reported that myelopoiesis was enhanced in SDF-1 alpha transgenic mice, probably due in part to SDF-1 alpha enhancement of myeloid progenitor cell (MPC) survival. To understand signaling pathways involved in this activity, we studied the effects on factor-dependent cell line MO7e cells incubated with SDF-1 alpha alone or in combination with other cytokines. SDF-1 alpha induced transient activation of extracellular stress-regulated kinase (ERK1/2), ribosomal S6 kinase (p90RSK) and Akt, molecules implicated in cell survival. Moreover, ERK1/2, p90RSK, and Akt were synergistically activated by SDF-1 alpha in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor (SLF), or thrombopoietin (TPO). Similar effects were seen after pretreatment of MO7e cells with SDF-1 alpha followed by stimulation with the other cytokines, suggesting a priming effect of SDF-1 alpha. Nuclear factor-kappa B (NF-kappa B) did not appear to be involved in SDF-1 alpha actions, alone or in combination with other cytokines. These intracellular effects were consistent with enhanced myeloid progenitor cell survival by SDF-1 alpha after delayed addition of growth factors. SDF-1 alpha alone supported survival of highly purified human cord blood CD34(+++) cells, less purified human cord blood, and MO7e cells; this effect was synergistically enhanced when SDF-1 alpha was combined with low amounts of other survival-promoting cytokines (GM-CSF, SLF, TPO, and FL). SDF-1 may contribute to maintenance of MPCs in bone marrow by enhancing cell survival alone and in combination with other cytokines.

Bone Marrow Cells↗

PubMed2002

C-kit associated with the transmembrane 4 superfamily proteins constitutes a functionally distinct subunit in human hematopoietic progenitors.

The transmembrane 4 superfamily (TM4SF) has come into prominence for its association with a wide range of cell surface molecules, especially integrins. We report that TM4SF molecules CD9, CD63, and CD81 are physically associated with c-kit receptor tyrosine kinase in the human factor-dependent myeloid cell line, MO7e. We characterized this complex using coimmunoprecipitation and colocalization methods. The c-kit coimmunoprecipitated with anti-TM4SF antibodies showed several distinct phenotypes compared to the total c-kit immunoprecipitated with anti-c-kit antibody. These included: (1) higher basal level of tyrosine phosphorylation without elevated kinase activity in the absence of Steel factor (SLF), (2) deficient enhancement of tyrosine phosphorylation and kinase activity in response to SLF, (3) elevated binding rate of SLF shown in chemical cross-linking studies, and (4) little internalization and degradation after SLF treatment. Cocapping studies in living cells showed that c-kit colocalized with TM4SF molecules after SLF stimulation, suggesting confirmation of the biochemical data obtained by the coimmunoprecipitation studies. Colocalization of c-kit with CD81 by SLF was also observed in cord blood CD34(+) cells, suggesting the existence of functional units of c-kit in TM4SF complexes in primary hematopoietic cells. This suggests that some TM4SF members may negatively modulate function of c-kit receptor tyrosine kinase and thus regulate receptor sensitivity to SLF in hematopoietic progenitors.

Antigens, CD↗

PubMed2002

S100A6 protein as a marker for differential diagnosis of cholangiocarcinoma from hepatocellular carcinoma.

A new marker useful for differential diagnosis of cholangiocarcinoma (CC) from hepatocellular carcinoma (HCC) has been identified and investigated. cDNA clone for S100A6 (calcyclin) was identified as a upregulated gene in intrahepatic tumors by differential dot hybridization and Northern blot analysis using normal and tumor intrahepatic tissues. Significant increase in the expression level of S100A6 mRNA was recognized in intrahepatic tissues in three of three CC cases but none of six primary HCC. Immunohistochemical examination of the tumor tissues using monoclonal antibody specific to S100A6 showed that 14 of 18 (77.8%) CC tissue samples were positively stained but only two of 20 (10%) specimens from the patients with primary HCC were weakly stained. These results suggest that the levels of S100A6 mRNA expression and protein in the tissue specimens examined by Northern blot and immunohistochemical staining may be additional useful marker for the differentiation of CC from HCC.

Journal Article↗

PubMedDate not supplied

P21waf-1-Chk1 pathway monitors G1 phase microtubule integrity and is crucial for restriction point transition.

Microtubule-disruption (MTD) is often thought to arrest the mammalian cell cycle only during mitosis. However, MTD has also been demonstrated to arrest cells during interphase at a G(1)-phase point we call G(1)MTA. Microtubule integrity is now shown to be required for progression past G(1)MTA and the mammalian restriction-point. Neither p21(waf1) nor p27(kip1) are required for MTD-induced G(1)-arrest. Only p21(waf1) is crucial for normal G(1)MTA passage. The p21(waf1)-Chk1-cdc25C-cdc2-checkpoint-pathway is implicated in monitoring this passage. P21(waf1) deletion deregulates G(1)MTA transition and decreases MTD-G(1) arrest, possibly via Chk1 disregulation. Oncogene-induced overexpression of p21(waf1) produced opposite effects on the Chk1-cdc25C-cdc2 pathway and enhanced MTD-G(1) arrest. G(1)MTA thus represents a novel facet of mammalian G(1)/S checkpoint.

Animals↗