Knockdown of apolipoprotein B, an atherogenic apolipoprotein, in HepG2 cells by lentivirus-mediated siRNA

ApoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target for lowering atherogenic lipoproteins. In the present study, we used a lentiviral vector to express short hairpin RNAs for inhibition of apoB production in HepG2 cells. We first demonstrated that lentivirus could efficiently deliver transgene into HepG2 cells by using GFP lentivirus. We then made three lentiviral siApoB constructs, two of which were highly efficient for silencing apoB expression in HepG2 cells. We showed that siApoB lentivirus specifically knocked down apoB but had no effects on other proteins such as apoAI and albumin. Consequently, the secretion of apoB was reduced markedly. The silencing effect of siApoB lentivirus appeared to be permanent. Knocking down apoB did not alter the expression of cytoplasmic stress proteins (HSP70 and HSP90) and their ER homologues (GRP78 and GRP94). Furthermore, neither IKKalpha and JNK nor phosphorylated IKK and JNK were increased in long-term apoB-deficient hepatocytes as compared to the control cells. Consistent with these findings, apoB-deficient hepatocytes responded to insulin to a similar extent as the control cells as determined by measuring insulin-induced phosphorylation of IRS and ERK. Our studies indicate that lentiviral siRNAs provide an excellent approach for delivering siRNA into HepG2 cells and may be used for gene therapy for hyperlipidemia.     

Invariant chain alters the malignant phenotype of MHC class II+ tumor cells.

T lymphocytes usually recognize endogenously encoded Ag in the context of MHC class I molecules, whereas exogenous Ag is usually presented by MHC class II molecules. In vitro studies in model systems suggest that presentation of endogenous Ag by class II molecules is inhibited by the association of class II with its invariant chain (Ii). In the present study we test this hypothesis in an in vivo system in which endogenously encoded tumor peptides are presented by tumor cell MHC class II molecules. In this system, transfection of syngeneic MHC class II genes (Aak and Abk) into a highly malignant, Ii negative, mouse tumor (SaI sarcoma) produces an immunogenic tumor (SaI/Ak) that is rejected by the autologous host. The class II+ transfectants also effectively immunize autologous A/J mice against a subsequent challenge of wild-type class II- tumor cells. We have hypothesized that the SaI/Ak transfectants induce protective immunity because they function as APC for endogenously synthesized tumor peptides, and thereby stimulate tumor-specific Th cells, by-passing the need for professional APC. To test the role of Ii as an inhibitor of presentation of endogenous peptides, SaI/Ak tumor cells were supertransfected with Ii gene (SaI/Ak/Ii cells), and the tumorigenicity of the resulting cells determined. Nine SaI/Ak/Ii clones were tested, and their malignancy compared with that of SaI/Ak and SaI cells. Seven of the nine class II+/Ii+ tumor cells are more malignant than class II+/Ii- tumor cells in autologous A/J mice. Expression of Ii therefore restores the malignant phenotype, presumably by preventing presentation of endogenously synthesized tumor peptides. Ii therefore regulates Ag presentation and can be a critical parameter for in vivo tumor immunity.     

A high-throughput assay for siRNA-based circadian screens in human U2OS cells

The advent of siRNA-based screens has revolutionized the efficiency by which functional components of biological processes are identified. A notable exception has been the field of mammalian circadian rhythms. Here, we outline a medium- to high-throughput siRNA-based approach that, in combination with real-time bioluminescence measurement of a circadian reporter gene, can be utilized to elucidate the effects of gene knockdown across several days in human cells.     

Knockdown of zebrafish crim1 results in a bent tail phenotype with defects in somite and vascular development

The Crim1 gene encodes a transmembrane protein containing six cysteine-rich repeats similar to those found in the BMP antagonist, chordin (chd). To investigate its physiological role, zebrafish crim1 was cloned and shown to be both maternally and zygotically expressed during zebrafish development in sites including the vasculature, intermediate cell mass, notochord, and otic vesicle. Bent or hooked tails with U-shaped somites were observed in 85% of morphants from 12 hpf. This was accompanied by a loss of muscle pioneer cells. While morpholino knockdown of crim1 showed some evidence of ventralisation, including expansion of the intermediate cell mass (ICM), reduction in head size bent tails and disruption to the somites and notochord, this did not mimic the classically ventralised phenotype, as assessed by the pattern of expression of the dorsal markers chordin, otx2 and the ventral markers eve1, pax2.1, tal1 and gata1 between 75% epiboly and six-somites. From 24 hpf, morphants displayed an expansion of the ventral mesoderm-derived ICM, as evidenced by expansion of tal1, lmo2 and crim1 itself. Analysis of the crim1 morphant phenotype in Tg(fli:EGFP) fish showed a clear reduction in the endothelial cells forming the intersegmental vessels and a loss of the dorsal longitudinal anastomotic vessel (DLAV). Hence, the primary role of zebrafish crim1 is likely to be the regulation of somitic and vascular development.     

Knockdown of clusterin alters mitochondrial dynamics,facilitates necrosis in camptothecin-induced cancer stem cells

The existence of a well-established drug resistance mechanism in cancer stem cells (CSC) complicates the cancer treatment. Clusterin (CLU) plays a key role in maintaining the integrity of endoplasmic reticulum (ER) during drug-induced stress. Hence, silencing the CLU could significantly reduce the inherent drug resistance mechanism of CSC. The combination of drug-induced cytotoxicity, as well as the suppression of drug resistance in CSC, could circumvent the recurrence capability of the tumor. In the present study, camptothecin (CPT)-induced apoptosis and necrosis in CSC with and without siCLU treatment were simultaneously measured using Qdot-based total internal reflection fluorescence microscope (TIRF). In addition, to elucidate the mechanism of CPT-induced cytotoxicity in CLU-suppressed CSC, expression of Bcl-2, Bax, Bak, and PARP and mitochondrial permeability transition pore (MPTP) were studied. EC₅₀ values of CPT-induced apoptosis and necrosis were significantly reduced (p < 0.01) in CLU-suppressed MCF-7 and CSC. Significantly increased MPTP (p < 0.001) and cytosolic Ca²⁺ (p < 0.001) were observed in CPT-treated CLU-suppressed CSC as compared to the normal CSC. Elevated expression of Bax, Bak, and cleaved PARP and reduced expression of Bcl-2 and cytosolic ATP were observed in CPT-treated CLU-suppressed CSC. Observed results indicate that silencing the expression of CLU could improve the anticancer efficacy of CPT at 128.4-nM concentration by equally inducing necrotic signals along with apoptosis. Furthermore, the developed high content TIRF assay based on the CLU-suppressed CSC could be an ideal and beneficial tool for rapidly analyzing the cytotoxicity of anti-cancer agents.     

Depletion of Neuroguidin/CANu1 sensitizes human osteosarcoma U2OS cells to doxorubicin

Osteosarcoma is a primary bone cancer which occurs mainly in children. Neuroguidin/CANu1 is a nucleolar protein involved in the maintenance of ribosomal structure. In this study, we investigated the effect of Neuroguidin/CANu1 depletion on the response of osteosarcoma cells to doxorubicin. In normal circumstances, Neuroguidin/CANu1 is localized at nucleoli, which translocates to nuclear foci in the presence of doxorubicin. shRNA knockdown of Neuroguidin/CANu1 did not affect cell viability in the absence of doxorubicin, but led to enhanced cytotoxicity in doxorubicin-treated cells. Doxorubicin increased the population of apoptotic cells by 3-fold in Neuroguidin/CANu1-depleted cells compared to that in control cells. Depletion of Neuroguidin/CANu1 mRNA induced the expression of p21 and the cleavage of PARP, leading to increased caspase-3/7 activity. Together, these results suggest that Neuroguidin/CANu1 is required for maintaining cellular homeostasis and may contribute to the improved efficiency of chemotherapy.     

Mastoparan alters subcellular distribution of profilin and remodels F-actin cytoskeleton in cells of maize root apices

Indirect immunofluorescence localization of profilin in cells of maize root apices revealed that this abundant protein was present both in the cytoplasm and within nuclei. Nucleo-cytoplasmic partitioning of profilin exhibits tissue-specific and developmental features. Mastoparan-mediated activation of heterotrimeric G-proteins, presumably through triggering a phosphoinositide-signaling pathway based on phosphatidylinositol-4,5-bisphosphate (PIP(2)), induced relocalization of profilin from nuclei into the cytoplasm of root apex cells. In contrast, PIP(2) accumulated within nuclei of mastoparan-treated root cells. Intriguingly, cytoplasmic accumulation of profilin was associated with remodeling of F-actin arrays in root apex cells. Specifically, dense F-actin networks were dismantled and distinct actin patches became associated with the periphery of small vacuoles. On the other hand, disruption of F-actin with the G-actin sequestering agent latrunculin B does not affect the subcellular distribution of profilin or PIP(2). These data suggest that nuclear profilin can mediate a stimulus-response action on the actin cytoskeleton which is somehow linked to a phosphoinositide-signaling cascade.     

Hesperetin-etoposide combinations induce cytotoxicity in U2OS cells: Implications on therapeutic developments for osteosarcoma

Osteosarcoma chemotherapy has improved survival rates, however, chemoresistance and drug toxicity still limit therapy. Drug combinations may overcome these limitations by allowing fewer chemoresistant cells to survive. The aim of this study was to evaluate the cytotoxic potential of hesperetin to osteosarcoma and to analyze the cell cycle effects of combinations of hesperetin with chemotherapeutic agents. For this, the U2OS human osteosarcoma cell line was exposed to hesperetin or hesperetin combined with etoposide or doxorubicin in defined proportions. Hesperetin was less cytotoxic compared to chemotherapeutic agents, as shown by cell growth, viability and clonogenic assays. Notwithstanding, hesperetin combined with etoposide showed additive effects on the inhibition of cell growth. Furthermore, hesperetin induced G2-phase arrest, associated with decreased gene expression of cyclins B1 and E1 and cyclin-dependent kinases 1 and 2. The combination with higher additive effect resulted in higher percentage of cells in G2-phase, showing that G2-phase arrest is associated with cytotoxicity. Moreover, hesperetin induced cytostatic effects. In conclusion, our results suggest that G2-phase arrest is an important step for hesperetin-induced cytotoxicity in U2OS cells. Hesperetin shows potential cytotoxicity when combined with etoposide, which may have implications on therapeutic developments for osteosarcoma.     

Effect of cytochalasins on F-actin and morphology of Ehrlich ascites tumor cells

Cytochalasins have been used extensively to probe the role of F-actin in different aspects of cellular function. Most of the data obtained are interpreted on the basis of the well-established depolymerizing effects of cytochalasins on F-actin preparations in vitro. However, some evidence indicates that, in intact cells, different cytochalasins can have varying effects on cell morphology and F-actin content and organization. To examine this problem in more detail, we analyzed the effects of cytochalasins on the cell morphology of and F-actin content and organization in Ehrlich ascites tumor (EAT) cells. After a 3-min exposure to 0.5 microM cytochalasin D, B, or E, F-actin content was equally reduced in all cases and this correlated with a reduction in the amount of cortical F-actin associated with the EAT cell membrane. However, only with CE was cell morphology markedly altered, with the appearance of numerous blebs. At 10 microM, blebbing was present in all conditions and the organization of cortical F-actin was disrupted. F-actin content, however, was not further reduced by this higher concentration and in CD it was identical to control levels. Exposure of EAT cells to similar concentrations of cheatoglobosin C, an analog of the cytochalasins that has little to no affinity for F-actin, resulted in a loss of F-actin content, a reduction in F-actin fluorescence, but no change in cell morphology, including a complete lack of bleb formation. Myosin II immunoreactivity, concentrated in the cortical cytoplasm colocalized with F-actin and in an area associated with the Golgi, was reduced by the high-dose cytochalasin. These results demonstrate that caution must be exercised in the use of cytochalasins to probe the role of F-actin in cellular function and that several parameters must be analyzed to obtain an accurate assessment of the effect of cytochalasin on the actin filament system.     

Knockdown of hypoxia-inducible factor-1alpha in breast carcinoma MCF-7 cells results in reduced tumor growth and increased sensitivity to methotrexate

The hypoxia-inducible factor (HIF) 1alpha is a key regulator of the cellular response to oxygen deprivation. Specific disruption of the HIF-1 pathway is important for exploring its role in tumor biology and developing more efficient weapons to treat cancer. In this study, we stably transfected human breast tumor MCF-7 cells with short hairpin RNA expression vectors targeting HIF-1alpha. After knockdown of HIF-1alpha, hypoxia-induced expression of its target genes such as vascular endothelial growth factor, Glut-1, phosphoglycerate kinase, and P-glycoprotein were markedly attenuated. Moreover, HIF-1alpha knockdown was found to suppress the shift from S-phase to G(1) induced by hypoxia and increase drug sensitivity to methotrexate. The growth rates of HIF1alpha-knockdown tumors were drastically retarded in both subcutaneous and orthotopic xenograft models, which were accompanied by decreased angiogenesis and reduced expression of glucose transporter in tissue sections. These data demonstrate that HIF-1alpha knockdown reduces tumorigenicity of MCF-7 cells and suggest a promising combination of both anti-HIF-1 strategy and traditional chemotherapy to improve cancer treatment.     

Apoptosis induced by adenovirus-mediated p14ARF expression in U2OS osteosarcoma cells is associated with increased Fas expression

The INK4A/ARF locus on chromosome 9 is a tumor suppressor gene frequently mutated in human cancers. In order to study the effects of p14ARF expression in tumor cells, we constructed a recombinant adenovirus containing p14ARF cDNA (Adp14ARF). Adp14ARF infection of U2OS osteosarcoma cells which has wild type p53 and mutant p14ARF revealed high levels of p14 (ARF) expression within 24h. In addition, Adp14ARF-mediated expressing of p14 (ARF) was associated with increased levels of p53, p21, and mdm2 protein. Growth inhibition assays following Adp14ARF infection demonstrated that the growth of U2OS cells was inhibited relative to infection with control virus. Furthermore, TUNEL analysis as well as PARP cleavage assays demonstrated that Adp14ARF infection was associated with increased apoptosis in U2OS cell line and that it was associated with Adp14ARF induced overexpression of Fas and Fas-L. Addition of Fas-L neutralizing antibody NOK-1 decreased Adp14-mediated cell death, indicating that p14 (ARF) induction of the Fas pathway is associated with increased apoptosis. The finding that Adp14ARF infection did not induce Fas expression in U2OS/E6 and MCF/E6 cells suggests that wild type p53 expression may be necessary for Adp14ARF-mediated induction of Fas. The observation that overexpression of p53 by Adp53 infection in MCF-7 does not induce increased Fas protein levels nor apoptotic cell death suggests that p53 overexpression is required but not sufficient enough for apoptosis. These studies suggest there are other mechanisms other than induction of p53 in ARF-mediated apoptosis and gene therapy using Adp14ARF may be a promising treatment option for human cancers containing wild type p53 and mutant or deleted p14 expression.     

U2OS cells lacking Chk1 undergo aberrant mitosis and fail to activate the spindle checkpoint

Chk1 is a conserved protein kinase originally identified in fission yeast, required to delay entry of cells with damaged or unreplicated DNA into mitosis. The requirement of Chk1 for both S and G2/M checkpoints has been elucidated while only few studies have connected Chk1 to the mitotic spindle checkpoint. We used a small interference RNA strategy to investigate the role of Chk1 in unstressed conditions. Chk1 depletion in U2OS human osteosarcoma cells inhibited cell proliferation and raised the percentage of cells with a 4N DNA content, which correlated with accumulation of giant polynucleated cells morphologically distinct from apoptotic cells, while no increased number of cells in G2 or mitosis could be detected. Down-regulation of Chk1 also caused accumulation of cells in the last step of cytokinesis, and of tetraploid cells in G1 phase, which coincided with activation of p53 and increased levels of p21. In addition, Chk1-depleted U2OS cells failed to arrest in mitosis after spindle disruption by nocodazole and showed decreased protein levels of Mad2 and BubR1. These studies show that U2OS cells lacking Chk1 undergo abnormal mitosis and fail to activate the spindle checkpoint, suggesting a role of Chk1 in this checkpoint.     

Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells

Interleukin-24 (mda-7/IL-24) is a cytokine in the IL-10 family that has received a great deal of attention for its properties as a tumor suppressor and as a potential treatment for cancer. In this study, we have identified and characterized five alternatively spliced isoforms of this gene. Several, but not all of these isoforms induce apoptosis in the osteosarcoma cell line U2OS, while none affect the survival of the non-cancerous NOK cell line. One of these isoforms, lacking three exons and encoding the N-terminal end of the mda-7/IL-24 protein sequence, caused levels of apoptosis that were higher than those caused by the full-length mda-7/IL-24 variant. Additionally, we found that the ratio of isoform expression can be modified by the splice factor SRp55. This regulation suggests that alternative splicing of mda-7/IL-24 is under tight control in the cell, and can be modified under various cellular conditions, such as DNA damage. In addition to providing new insights into the function of an important tumor suppressor gene, these findings may also point toward new avenues for cancer treatment.     

Increase of cytosolic calcium results in formation of F-actin aggregates in endothelial cells

In XTH-2 cells, a line derived from tadpole heart endothelia, changes of the F-actin pattern have been followed after injection of calcium and after permeabilization of the cells to calcium using the ionophore A 23187 and an external pCa of 6.0. On the dorsal surface numerous microvilli appear, the stress fibres show an interrupted pattern and F-actin accumulates at the cell periphery. These reactions are interpreted to result from contractions. Finally most of the F-actin becomes depolymerized (in about 30 min). In addition actin containing accumulations are developed in the perinuclear area. In G1 cells stress fibres are less sensitive to enhanced calcium, they persist for more than 30 min in pCa 6.0. Taking into account a relatively high cytosolic calcium concentration in many malignantly transformed cells, their more rounded appearance and lack of an ordered array of actin fibrils could be closely related phenomena.