Presence Of G418 Research Articles

USP11 Stabilizes HPV-16E7 and Further Modulates the E7 Biological Activity

HPV-16E7 is a major transforming protein, which has been implicated in the development of cervical cancer. The stability of E7 is thus important to ensure its fully functional status. Using the yeast two-hybrid system, we found that USP11 (ubiquitin-specific protease 11), a member of a protein family that cleaves polyubiquitin chains and/or ubiquitin precursors, interacts and forms a specific complex with HPV-16E7. Our results indicate that the USP11 can greatly increase the steady state level of HPV-16E7 by reducing ubiquitination and attenuating E7 degradation. In contrast, a catalytically inactive mutant of USP11 abolished the deubiquitinating ability and returned E7 to a normal rate of degradation. Moreover, USP11 not only protected E7 from ubiquitination but also influenced E7 function as a modulator of cell growth status. These results suggest that USP11 plays an important role in regulating the levels of E7 protein and subsequently affects the biological function of E7 as well as its contribution to cell transformation by HPV-16E7.

Production of functional antibody Fab fragment by recombinant insect cells

The production of an Fab fragment of the catalytic antibody 6D9 in stably transformed lepidopteran insect cells was investigated. On the basis of an expression vector that utilizes the Bombyx mori cytoplasmic actin promoter, from which foreign gene expression is stimulated with the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer, two plasmid vectors were constructed which contain either a neomycin or a blasticidin resistance gene for use as a selectable marker. The genes encoding the heavy chain (Hc; Fd fragment) and light chain (Lc) of the 6D9 Fab fragment were inserted separately into the expression vectors. After cotransfection with the resulting plasmids to introduce the Hc and Lc genes and the two different antibiotic resistance genes, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418 and blasticidin. Colonies of cells resistant to the antibiotics were obtained around 2 weeks after cotransfection. Western blotting and enzyme-linked immunosorbent assay (ELISA) of the cell culture supernatant suggested that the resistant cells stably secrete an Fab fragment which retains an antigen-binding activity. High yields of over 300 μg/ml of Fab fragment were achieved in simple batch shake-flask culture of transfected insect cells. These results indicate that recombinant insect cells may offer a novel approach for efficient production of antibody molecules.

Inactivation of NuRD Component Mta2 Causes Abnormal T Cell Activation and Lupus-like Autoimmune Disease in Mice

Dynamic changes in chromatin structure through ATP-dependent remodeling and covalent modifications on histones play important roles in transcription regulation. Among the many chromatin modifiers identified, the NuRD (nucleosome remodeling histone deacetylase) complex is unique because it possesses both nucleosome remodeling and histone deacetylase activities. To understand the biological function of the NuRD complex, we generated a knock-out mouse model of the Mta2 (metastasis-associated protein 2) gene, which encodes a NuRD-specific component. Mta2 null mice exhibited partial embryonic lethality. The surviving mice developed lupus-like autoimmune symptoms including skin lesions, bodyweight loss, glomerulonephritis, liver inflammation, and production of autoantibodies. Transplantation of bone marrow cells from Mta2 null mice recapitulated some of the symptoms including skin lesion and bodyweight loss in the recipient mice. Mta2 null T lymphocytes showed normal development but hyperproliferation upon stimulation, which correlates with hyperinduction of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma. T cell hyperproliferation, but not other autoimmune symptoms, was observed in T cell-specific Mta2 knock-out mice. Mta2 null T cells produced more IL-4 and IFN-gamma under Th2 activation conditions, but normal levels of IL-4 and IFN-gamma under Th1 activation conditions. Furthermore, we found that IL-4 is a direct target gene of Mta2. Our study suggests that Mta2/NuRD is involved in modulating IL-4 and IFN-gamma expression in T cell immune responses, and gene expression in non-T cells plays an important role in controlling autoimmunity.

Characterization of Some Molecular Mechanisms Governing Autoactivation of the Catalytic Domain of the Anaplastic Lymphoma Kinase

NPM/ALK is an oncogenic fusion protein expressed in approximately 50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr --> Phe) three potential autophosphorylation sites contained in the "YXXXYY" motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK.

Exacerbating Role of γδ T Cells in Chronic Colitis of T-Cell Receptor α Mutant Mice

T-cell receptor (TCR) gammadelta T cells are an important component of the mucosal immune system and regulate intestinal epithelial homeostasis. Interestingly, there is a significant increase in gammadelta T cells in the inflamed mucosa of patients with ulcerative colitis (UC). However, the role of gammadelta T cells in chronic colitis has not been fully identified. TCRalpha-deficient mice, which spontaneously develop chronic colitis with many features of human UC including an increase in gammadelta T-cell population, represent an excellent model to investigate the role of gammadelta T cells in UC-like colitis. To identify the role of gammadelta T cells in this colitis, we herein have generated TCRgamma-deficient mice through deletion of all TCR Cgamma genes (Cgamma1, Cgamma2, Cgamma3, and Cgamma4) using the Cre/loxP site-specific recombination system and subsequently crossing these mice with TCRalpha-deficient mice. An increase in colonic gammadelta T cells was associated with the development of human UC as well as UC-like disease seen in TCRalpha-deficient mice. Interestingly, the newly established TCRalpha(-/-) x TCRgamma(-/-) double mutant mice developed significantly less severe colitis as compared with TCRalpha-deficient mice. The suppression of colitis in TCRalpha(-/-) x TCRgamma(-/-) double mutant mice was associated with a significant reduction of proinflammatory cytokine and chemokine productions and a decrease in neutrophil infiltration. gammadelta T cells are involved in the exacerbation of UC-like chronic disease. Therefore, gammadelta T cells may represent a promising therapeutic target for the treatment of human UC.

High-Throughput Analysis of Promoter Occupancy Reveals Direct Neural Targets of FOXP2, a Gene Mutated in Speech and Language Disorders

We previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders.

Disruption of CXC Motif Chemokine Ligand-14 in Mice Ameliorates Obesity-induced Insulin Resistance

In obese individuals, white adipose tissue (WAT) is infiltrated by large numbers of macrophages, resulting in enhanced inflammatory responses that contribute to insulin resistance. Here we show that expression of the CXC motif chemokine ligand-14 (CXCL14), which targets tissue macrophages, is elevated in WAT of obese mice fed a high fat diet (HFD) compared with lean mice fed a regular diet. We found that HFD-fed CXCL14-deficient mice have impaired WAT macrophage mobilization and improved insulin responsiveness. Insulin-stimulated phosphorylation of Akt kinase in skeletal muscle was severely attenuated in HFD-fed CXCL14+/- mice but not in HFD-fed CXCL14-/- mice. The insulin-sensitive phenotype of CXCL14-/- mice after HFD feeding was prominent in female mice but not in male mice. HFD-fed CXCL14-/- mice were protected from hyperglycemia, hyperinsulinemia, and hypoadiponectinemia and did not exhibit increased levels of circulating retinol-binding protein-4 and increased expression of interleukin-6 in WAT. Transgenic overexpression of CXCL14 in skeletal muscle restored obesity-induced insulin resistance in CXCL14-/- mice. CXCL14 attenuated insulin-stimulated glucose uptake in cultured myocytes and to a lesser extent in cultured adipocytes. These results demonstrate that CXCL14 is a critical chemoattractant of WAT macrophages and a novel regulator of glucose metabolism that functions mainly in skeletal muscle.

SLP-65 Signal Transduction Requires Src Homology 2 domain-mediated Membrane Anchoring and a Kinase-independent Adaptor Function of Syk

The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.

Receptor-mediated Endocytosis Is Not Required for Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.

Control of Splicing Efficiency by the Mouse Histone H2a Element in a Murine Leukemia Virus–based Retroviral Vector

While using various human complementary DNA (cDNA) sequences in the context of the murine leukemia virus (MLV)-based retroviral vector, it was found that a retroviral vector containing some human cDNA sequences produces unusually low viral titer. One of those sequences is that for the human IL-1 receptor antagonist protein (IL1RN). The RNA analysis showed that a cryptic splice acceptor sequence is present in the middle of its coding region, resulting in the deletion of the packaging signal sequence and the removal of some coding sequences that lead to low viral titer and a low level of the transgene product. We tested whether the mouse Hist2h2aa1 element (mH2aE), previously shown to suppress the splicing function, could inhibit the cryptic splicing in the context of MLV-based retroviral vectors. It was found that the mH2aE could efficiently suppress such unwanted splicing event, thus increasing the amount of unspliced transcript, which eventually led to the increase in the level of IL1RN expression and viral titer. The mH2aE could also be used to control unusually high splicing activity. Our data suggested that the mH2aE could be used for the fine-tuning of the splicing process, thus improving the level of gene expression and viral titer in the context of retroviral vectors.

Expression of Wnt-3a in Myeloma Cells Inhibits the Osteolytic Phenotype In-Vivo.

Wnt signaling is a highly conserved signal transduction pathway involved in embryonic development. Inappropriate canonical Wnt signaling resulting in beta-catenin stabilization, is associated with several types of human cancers. Multiple myeloma plasma cells express Wnt receptors, Wnt ligands and soluble Wnt inhibitors. Wnt signaling is central to osteoblast and osteoclasts development and secretion of Wnt signaling inhibitors by myeloma cells is thought to contribute to the osteolytic phenotype seen in this disease and prostate cancer. While it is now clear that MM cells can signal through both canonical and non-canonical mechanisms, there are conflicting data as to the direct role of Wnt signaling in myeloma cell biology. Others have shown that Wnts cause proliferation of myeloma cells; while we have shown that canonical Wnts cause morphological changes and migration, but not cell proliferation. To further elucidate the role of canonical Wnt signaling in myeloma and myeloma bone disease we used limiting dilutions in the presence of G418 to create two independent stable clones of the myeloma cell line NCI-H929 expressing Wnt-3A (H929/W3A), which is not expressed in myeloma, and an empty vector (H929/EV). Because Wnt antibodies are not available we cloned Wnt-3A as a fusion protein with hemagglutinin (HA). Western blots against HA revealed a positive band of the expected size only in the H929/W3A clones. GST-E-cadherin binding assay and Western blot analysis revealed elevated levels of total and free beta-catenin in H929/W3A relative to H929/EV, however, there this was not associated with increased growth or proliferation by MTT assay. To determine the in-vivo growth characteristics and effects on bone resorption of Wnt-3A producing cells, we transplanted the lines into a human bone implanted the flank of SCID mice. Tumor growth rate as determined by increased production of human immunoglobulin in mice serum was significantly slower in the Wnt-3A transfected cells relative to controls (P < .05). Loss of bone mineral density (BMD) of the implanted bones engrafted with H929/W3A cells was lower than in bones engrafted with H929/EV cells (P < .05). Reduced tumor burden and BMD loss was also visualized on x-ray radiographs. Taken together these data indicate that all factors promoting bone resorption produced by or elicited by the myeloma cell line H929 are subordinate to canonical Wnt signaling and that prevention of bone destruction may help control myeloma progression.

The Effect of Human IL-17F on the Growth of Human Hepatocarcinoma Xenograft Tumors in Nude Mice

The human interleukin-17F (hIL-17F) gene was amplified by RT-PCR from PHA-activated human peripheral blood mononuclear cells (PBMCs). It was then subcloned into the retrovirus vector pSIV-1. The pSIV-1/hIL-17F together with its two-helper virus vectors pHIT456 and pHIT60 cotransfected into the package cell 293T by lipofectin to produce mature recombinant retrovirus, which was then used to infect SMMC-7721 hepatocarcinoma cells (HCCs), and the cells were selected in the presence of G418. The integration, transcription, and expression of hIL-17F gene in SMMC-7721 cells were identified by PCR, RT-PCR, and Western blot, respectively. MTT and FCM showed that hIL-17F could not alter the proliferation and cell cycle of SMMC-7721 cells, but ELISA showed that it could down-regulate IL-6, IL-8, and VEGF expressions. The effect of rhIL-17F supernatant on the growth suppression of ECV304 cells was observed by MTT. The experiment on human hepatocarcinoma xenografts tumor in nude mice showed that the formation and growth rates of hIL-17F-transgenic SMMC-7721 HCCs showed an obvious decline, and VEGF and CD34 expressions and angiogenesis of the transgenic neoplasms were also evidently reduced. hIL-17F can inhibit markedly the growth of human hepatocarcinoma xenograft tumors in nude mice by antiangiogenesis. This study provided an experimental evidence for further conducting the tumor gene therapy by targeting vascularity and exploiting antiangiogenic novel medicine related to hIL-17F.

Mouse Homologue of Skin-specific Retroviral-like Aspartic Protease Involved in Wrinkle Formation

Retroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization.

In vitro analysis of mouse neural stem cells genetically modified to stably express human NGF by a novel multigenic viral expression system

Objectives: The purpose of this study was to characterize mouse neural stem cells (NSC) transduced by a multigenic lentiviral vector (LV) and stably express recombinant human nerve growth factor (rhNGF). We obtained NSC-derived cell lines which express human NGF in relevant amount to exploit their ability for therapeutic applications.Methods: We constructed advanced multigenic LV vectors which contain a tricistronic cassette to express simultaneously up to three independent genes: (1) rhNGF (β subunit); (2) EGFP (enhanced green fluorescent protein) and (3) NeoR (neomycin antibiotic resistance gene). Lentiviruses were obtained by transfecting LV constructs plus helper plasmids in human embryonic kidney (HEK)-293T packaging cells. Lentiviral virions were released in culture media and subsequent used to infect mouse NSC. Genetycin 418-resistant NSC were obtained after 1 month of selection in the presence of antibiotic (G418). Levels of human NGF secreted by rhNGF-NSC were determined by ELISA (enzyme-linked immunosorbent assay). Features of multipotentiality of engineered NSC-derived cell lines versus naive cells (control-NSC) were assessed by immunocytochemical analysis in differentiation conditions. Self-renewal of NSC was tested by neurospheres assay (NSA).Results: Levels of secreted human NGF, from conditioned media obtained by rhNGF-NSC cultures, were found to be elevated in either proliferation or differentiation conditions as compared with control cells. Moreover, released hNGF demonstrated biologic activity on PC12 cells by a functional test of neurite outgrowth. Immunocytochemical analysis revealed that engineered NSC showed to be all positives for EGFP. After thirty passages in vitro in the presence of G418, engineered cells versus naive NSC cultures maintained their multipotentiality to differentiate into neurons, astrocytes and oligodendrocytes. Furthermore, it was found that rhNGF-NSC-derived neurons expressed choline acetyltransferase (ChAT) and displayed an enhanced axonal growth. NSA showed an altered sphere forming frequency either in rhNGF-NSC or both group of control NSC.Discussion: Lentivirus-mediated rhNGF gene transfer into NSC was achieved using a new version of LV vectors. We obtained rhNGF-NSC-derived cell lines which released hNGF to high levels in the culture medium. The expression of neural differentiation markers, like microtubule associated protein 2 (MAP2) (a/b), glial fibrillary acidic protein (GFAP) and chondroitin sulphate proteoglycan (NG2), was not enhanced in rhNGF-NSC compared with control cells. Secreted hNGF increased axonal sprouting by rhNGF-NSC-derived neurons which was associated with ChAT expression. rhNGF-NSC may prospectively be good candidates for the treatment of either neurodegenerative diseases such as Alzheimer disease or central nervous system injuries.

HCV NS2 protein inhibits cell proliferation and induces cell cycle arrest in the S-phase in mammalian cells through down-regulation of cyclin A expression

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2 protein is a HCV hydrophobic transmembrane protein that associates with several cellular proteins in mammalian cells. In this report, we investigated the functions of NS2 protein by examining its effects on cell growth and cell cycle progression. Stable NS2-expressing HeLa and Vero cell lines were established by transfection of the cells with pcDNA3.1(−)-NS2 followed by selection of the transfected cells in the presence of G418. We found that the proliferation rates of both NS2-expressing cell lines were inhibited by 40–50% compared with the control cells that were transfected with pcDNA3.1(−) control vector. Cell cycle analysis of these NS2-expressing cell lines shows that the proportion of cells in the S-phase increased significantly compared to that of control cells that do not express NS2 protein, suggesting NS2 protein induces cell cycle arrest in the S-phase. Further studies showed that the induction of cell cycle arrest in the S-phase by NS2 protein is associated with the decrease of cyclin A level. In contrast, the expression of NS2 protein does not affect the levels of cyclin-dependent kinase CDK2, CDK4, cyclin D1, or cyclin E. Our results suggest that HCV NS2 protein inhibits cell growth and induces the cell cycle arrest in the S-phase through down-regulation of cyclin A expression, which may be beneficial to HCV viral replication. Our findings not only provide information in the understanding mechanism of HCV infection, but also provide guidance for the future development of potential therapeutics for the prevention and treatment of the viral infection.

Expression and distribution of HSP27 in response to G418 in different human breast cancer cell lines

Heat shock proteins (HSPs) play an important role in folding, intracellular localization and degradation of cellular proteins. However, the cellular role of HSP27 is not completely understood. The conflicting results have been reported regarding stress-induced nuclear translocation of HSP27. In this study, human breast cancer cells transiently and stably expressing HSP27-EGFP chimera were utilized to observe the intracellular localization of HSP27. The data show that the transient and stable expression of HSP27-EGFP displayed distinguishingly cellular localization. The nuclear translocalization of HSP27-EGFP was correlated with the presence of G418. Experiments carried out with different human breast cancer cell lines revealed clearly different distribution patterns of endogenous HSP27. The subcellular distribution of endogenous HSP27 appeared diffuse throughout the cytoplasm in MDA435 cells. In MCF-7 and SKBR3 cells, the accumulation of the protein was distinctly seen along the cell membrane and around nucleus. Moreover, the nuclear translocation of endogenous HSP27 was stimulated by G418 only in MDA435 cells, but not in MCF-7 and SKBR3 cells. Overexpression of HSP27 has been associated with resistance to cisplatin and doxorubicin. The correlation of the expression pattern of HSP27 with the drug resistance may need to be investigated. Further studies on the intracellular function of HSP27 may take into account its interaction proteins in the cells. It may provide useful information for the identification of sensitivity of carcinoma cells to the chemotherapeutic drugs and development of more specific agents to circumvent HSP27.

Interrelated Roles for Mcl-1 and BIM in Regulation of TRAIL-mediated Mitochondrial Apoptosis

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1. Direct degradation of Mcl-1 by TRAIL-activated caspase-8 or caspase-3 produces Mcl-1-free Bim that mediates a Bax-dependent apoptotic cascade. Using Mcl-1 or Bim RNAi, we demonstrate that a loss in Mcl-1 expression significantly enhances the mitochondrial apoptotic response to TRAIL that is now mediated by freed Bim. Whereas overexpression of Mcl-1 contributes to the preservation of the mitochondrial membrane potential, Mcl-1 knockdown facilitates the Bim-mediated dissipation of this potential. Loss of Mcl-1 contributes to an increased level of caspase activity downstream of the mitochondrial response to TRAIL. Furthermore, the Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome c, Smac, and HtrA2 in response to Bim. These are the first findings to demonstrate the involvement of Bim in the TRAIL-mediated mitochondrial cascade. They also suggest that Mcl-1 may serve as a direct substrate for TRAIL-activated caspases implying the existence of a novel TRAIL/caspase-8/Mcl-1/Bim communication mechanism between the extrinsic and the intrinsic apoptotic pathways.

Identification and Characterization of an 8-kDa Light Chain Associated with Dictyostelium discoideum MyoB, a Class I Myosin

Dictyostelium discoideum MyoB is a single-headed class I myosin. Analysis of purified MyoB by SDS-PAGE indicated the presence of an approximately 9-kDa light chain. A tryptic digest of MyoB yielded a partial sequence for the light chain that exactly matched a sequence in a 73-amino acid, 8,296-Da protein (dictyBase number DDB0188713). This protein, termed MlcB, contains two EF-hand motifs and shares approximately 30% sequence identity with the N- and C-terminal lobes of calmodulin. FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD. Recombinant MlcB bound Ca2+ with a Kd value of 0.2 microm and underwent a Ca2+-induced change in conformation that increased alpha-helical content and surface hydrophobicity. Mutational analysis showed that the first EF-hand was responsible for Ca2+ binding. In the presence and absence of Ca2+ MlcB was a monomer in solution and bound to a MyoB IQ motif peptide with a Kd value of approximately 0.5 microm. A MyoB head-neck construct with a Ser to Glu mutation at the TEDS site bound MlcB and displayed an actin-activated Mg2+ ATPase activity that was insensitive to Ca2+. We conclude that MlcB represents a novel type of small myosin light chain that binds to IQ motifs in a manner comparable with a single lobe of a typical four-EF-hand protein.

Inoculation of the cells transfected with IP10 inhibited experimental tumor metastases in vivo

To explore the inhibitory effects of IP10 on the experimental tumor metastases of a mammary carcinoma cell line 4T1 in vivo. 4T1 cells were transfected with pcDNA3-IP10 plasmid and the positive clones (IP10-4T1) were screened in the presence of G418. The parental 4T1 cells and 4T1 cells transfected with pcDNA3 (pcDNA3-4T1) were used as controls. The expression of IP10 mRNA was examined with RT-PCR. The chemotactic activity of the cell-cultured supernatant for the activated lymphocytes was assessed with chemotaxis assay. All BALB/c mice were divided into 3 equal groups: IP10-4T1, parental 4T1 and pcDNA3-4T1 (6 mice in each group). Seven days after mice were inoculated with 2 x 10(6) 4T1, pcDNA3-4T1 or IP10-4T1 cells, 1 x 10(5) 4T1 cells were injected into mice in tail vein. On day 14 tumors were sectioned. On day 28 mice were sacrificed and the lung weight, metastatic forci on the lung surface, disseminated metastases in the lungs and the number of clonogenic metastases of 4T1 cells were observed. The splenocytes were isolated from tumor-bearing mice, and the cytotoxicity of the splenocytes was evaluated by CFSE/7-AAD method. The transcription of IP10 mRNA increased in IP10-4T1 cells compared to parental 4T1 and pcDNA3-4T1 cells (P = 0.002). Moreover, accumulated lymphocytes migrated to the supernatants of IP10-4T1 cells, which can be abrogated by anti-CXCR3 (P < 0.05). Compared to controls, inoculation of IP10-4T1 cells resulted in the decreased disseminated metastases as indicated by dramatically reduced lung metastatic forci on the surface of the lungs; the lung weight was lighter (IP10-4T1, 0.27 +/- 0.02 g v.s. 4T1, 0.48 +/- 0.08 g and pcDNA3-4T1, 0.43 +/- 0.16 g, P = 0.021); the number of clonogenic metastatic 4T1 cells enumerated in ten fields decreased greatly (IP10-4T1, 2.6 +/- 1.7 v.s. 4T1, 34 +/- 6.3 and pcDNA3-4T1, 33 +/- 2.3, P < 0.05); histological assay showed that metastasis was not found in the lungs of the 4/6 of mice in IP10-4T1 group, and was seen in all the mice of parental 4T1 and pcDNA3-4T1 groups. The splenocytes from the mice inoculated with IP10-4T1 cells exhibited stronger cytotoxic activity against 4T1 target cells at different ratios of effector versus target cells (3:1, 9:1, 27:1) than controls (P < 0.05). IP10 expressed locally could inhibit disseminated metastasis of circulating 4T1 tumor cells by enhancing anti-tumor cellular immune responses.

Enhanced growth in NS0 cells expressing aminoglycoside phosphotransferase is associated with changes in metabolism, productivity, and apoptosis

Prior work has reported that cotransfecting a gene of interest with the selectable marker neo can seriously perturb a number of cellular processes. In this study the influence of the neo gene on the growth, death, and metabolism of a murine myeloma NS0 cell line, expressing a chimeric antibody, was investigated. A pool of neo transfectants, 6A1-NEO, was selected with 500 microg/mL G418. Quantitative PCR analysis revealed that 6A1-NEO contained, on average, three copies of the neo gene per cell. Batch cultivation of 6A1-NEO showed that there was a 36% increase in maximum viable cell concentration, a 20% increase in the maximum apparent growth rate, and a 134% increase in cumulative cell hours as compared with the parent, 6A1-(100)3. Batch cultivation of five randomly selected clones illustrated that 6A1-NEO's advantage over the parent was not due to clonal variation. Neither the use of G418 during the selection process nor the cultivation of cells in the presence of G418 were responsible. This implied that the neo gene product, APH(3')-II, was causing the changes in proliferative capacity. Analysis of the cell cycle revealed that there were no differences in the distribution of cells in the G(1), S, and G(2) phases. When cell growth was synchronized, there were no observed differences in cell-cycle duration. 6A1-NEO resisted the onset of apoptosis during the growth phase. Consequently, there was a larger viable population of 6A1-NEO cells available for proliferation as compared with the parent. However, 6A1-NEO died at the same rate as the parent when resuspended in spent media or after treatment with staurosporin. Expression of the anti-apoptotic protein Bcl-2 was upregulated in 6A1-NEO, indicating that APH(3')-II could be acting by modulating endogenous gene expression. Analysis of key metabolites showed that 6A1-NEO's specific glucose consumption rate was 133% higher, whilst its specific glutamate consumption rate was 45% lower than the parent. 6A1-NEO's efficient utilization of glutamate and shift towards glucose metabolism may have contributed to the rise in proliferative capacity. However, this was accompanied by a 70% drop in the specific antibody production rate. These results show that the increase in growth rate and proliferative capacity caused by the expression of recombinant APH(3')-II was associated with changes in metabolism, apoptosis, and endogenous gene expression.