Fabrication of a microarray using a combination of the large circular sense and antisense DNA.

In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.

Synthesis of novel cholesterol-based cationic lipids for gene delivery.

The new cholesterol-based cationic lipids B, C, and D with an ether linked spacer were synthesized by using aminopropyl chain extension with acrylonitrile. The cholesterol-based cationic lipid A with carbamoyl linkage were also synthesized in order to compare the difference in transfection efficiency of the two linkage types. To this end, GFP expression of these cationic lipids was confirmed respectively.

Sanguinarine induces apoptosis in A549 human lung cancer cells primarily via cellular glutathione depletion.

Sanguinarine is a plant-derived benzophenanthridine alkaloid and has been shown to possess anti-tumor activities against various cancer cells. In this study, we investigated whether sanguinarine induces apoptosis in A549 human lung cancer cells. Treatment of A549 cells with sanguinarine induced apoptosis in a dose- and time-dependent manner. Treatment with sanguinarine led to activation of caspases and MAPKs as well as increased MKP-1 expression. Importantly, pretreatment with z-VAD-fmk, a pan caspase inhibitor suppressed the sanguinarine-induced apoptosis in A549 cells. Moreover, pretreatment with NAC, a sulfhydryl group-containing reducing agent strongly suppressed the apoptotic response and caspase activation to sanguinarine. However, the sanguinarine-mediated cytotoxicity in A549 cells was not protected by pharmacological inhibition of MAPKs or MKP-1 siRNA-mediated knockdown of MKP-1. These results collectively suggest that sanguinarine induces apoptosis in A549 cells through cellular glutathione depletion and the subsequent caspase activation.

Cadmium specifically induces MKP-1 expression via the glutathione depletion-mediated p38 MAPK activation in C6 glioma cells.

Cadmium is a toxic heavy metal and an environmental pollutant. Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a negative regulator of the family of MAPK. In this study, we investigated the effect of heavy metals on MKP-1 expression in C6 rat glioma cells. Cadmium treatment induced MKP-1 at both protein and mRNA levels while cobalt or manganese treatment did not, suggesting the specificity. Cadmium treatment also depleted intracellular GSH and activated p38 MAPK, JNKs, and AKT. Profoundly, pretreatment with thiol-containing compounds NAC or GSH, but not vitamin E, blocked GSH depletion, 38 MAPK activation and MKP-1 expression by cadmium. Moreover, pharmacological inhibition of p38 MAPK by SB203580 suppressed the cadmium-induced MKP-1. Collectively, these results demonstrate that cadmium specifically induces MKP-1 by transcriptional up-regulation in C6 cells in a mechanism associated with the glutathione depletion-dependent p38 MAPK activation.

Prevention of CCl4-induced liver cirrhosis by ribbon antisense to transforming growth factor-beta1.

Transforming growth factor-beta1 (TGF-beta1) is an important mediator of tissue fibrosis, including liver cirrhosis. Ribbon-type antisense oligonucleotide to TGF-beta1 (TGF-beta1 RiAS) was designed and combined with cationic peptide derived from the nuclear localization signal of human immunodeficiency virus-1 Tat protein for enhanced cellular uptake. When Hepa1c1c7 cells were transfected with TGF-beta1 RiAS, the level of TGF-beta1 mRNA was reduced by >70%. TGF-beta1 RiAS, mismatched RiAS, and normal saline were each injected into mice via the tail vein, beginning the week after intraperitoneal CCl4 injection and continuing for 7 weeks, in order to determine whether TGF-beta1 RiAS prevents the fibrotic changes induced by the CCl4 injection. After 8 weeks of the experiment, all of the mice treated with TGF-beta1 RiAS survived, compared to 50% of the control group and 65% of the mismatched RiAS-treated group. Upon examining the biochemical effects on the liver, TGF-beta1 mRNA levels were reduced significantly only in the TGF-beta1 RiAS-treated group. Immunohistochemical studies showed a reduced accumulation of collagen and alpha-smooth muscle actin. Our experimental results suggest that ribbon antisense to TGF-beta1, with efficient uptake, effectively blocks the expression of TGF-beta1 and prevents fibrosis of the liver.

Marked transfection enhancement by the DPL (DNA/peptide/lipid) complex.

A short peptide, corresponding to the nuclear localization signal of the human immunodeficiency virus-1 Tat protein, Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, was modified by adding a cysteine residue at the COOH terminus. The peptide was mixed with a reporter plasmid, and then with cationic lipids, to form a tripartite complex, DNA/peptide/lipid (DPL). Various cell lines were treated with the DPL complex and compared for transfection efficiency with those of the conventional DNA/lipid (DL) complex. With the simple inclusion of the peptide, the DPL complex showed much enhanced transfection. Meanwhile, the plasmid DNA mixed only with the peptide exhibited some improvement but with much lower transfection than the DPL complex. When the DPL complex was formed with various cationic lipids, the DOSPA/DOPE exhibited superior transfection efficiency than the other cationic lipids tested at the optimal ratio of 1:3:5 (w:w:w) in many cell types. At the optimal ratio of the DPL components, transfection efficiency was routinely shown to be approximately 10-fold higher for reporter gene expression than that of the conventional DL complex. Furthermore, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with antisense oligos, k-ras-RiAS, delivered as a DPL complex, tumor growth was markedly suppressed. This study shows that the DPL complex, which is easy to formulate by ordered mixing, can be employed for a much enhanced cellular uptake of a transgene both in vitro and in vivo.

Glucosamine hydrochloride specifically inhibits COX-2 by preventing COX-2 N-glycosylation and by increasing COX-2 protein turnover in a proteasome-dependent manner.

COX-2 and its products, including prostaglandin E(2), are involved in many inflammatory processes. Glucosamine (GS) is an amino monosaccharide and has been widely used for alternative regimen of (osteo) arthritis. However, the mechanism of action of GS on COX-2 expression remains unclear. Here we describe a new action mechanism of glucosamine hydrochloride (GS-HCl) to tackle endogenous and agonist-driven COX-2 at protein level. GS-HCl (but not GS sulfate, N-acetyl GS, or galactosamine HCl) resulted in a shift in the molecular mass of COX-2 from 72-74 to 66-70 kDa and concomitant inhibition of prostaglandin E(2) production in a concentration-dependent manner in interleukin (IL)-1beta-treated A549 human lung epithelial cells. Remarkably, GS-HCl-mediated decrease in COX-2 molecular mass was associated with inhibition of COX-2 N-glycosylation during translation, as assessed by the effect of tunicamycin, the protein N-glycosylation inhibitor, or of cycloheximide, the translation inhibitor, on COX-2 modification. Specifically, the effect of low concentration of GS-HCl (1 mM) or of tunicamycin (0.1 microg/ml) to produce the aglycosylated COX-2 was rescued by the proteasomal inhibitor MG132 but not by the lysosomal or caspase inhibitors. However, the proteasomal inhibitors did not show an effect at 5 mM GS-HCl, which produced the aglycosylated or completely deglycosylated form of COX-2. Notably, GS-HCl (5 mM) also facilitated degradation of the higher molecular species of COX-2 in IL-1beta-treated A549 cells that was retarded by MG132. GS-HCl (5 mM) was also able to decrease the molecular mass of endogenous and IL-1beta- or tumor necrosis factor-alpha-driven COX-2 in different human cell lines, including Hep2 (bronchial) and H292 (laryngeal). However, GS-HCl did not affect COX-1 protein expression. These results demonstrate for the first time that GS-HCl inhibits COX-2 activity by preventing COX-2 co-translational N-glycosylation and by facilitating COX-2 protein turnover during translation in a proteasome-dependent manner.

Dexamethasone suppresses interleukin-1beta-induced human beta-defensin 2 mRNA expression: involvement of p38 MAPK, JNK, MKP-1, and NF-kappaB transcriptional factor in A549 cells.

Human beta-defensin (HBD)-2 is an inducible antimicrobial peptide that plays an important role in innate immunity. Glucocorticoids, on the other hand, exert immunosuppressive and anti-inflammatory actions. We have previously reported that interleukin (IL)-1beta induces HBD-2 mRNA expression through the activation of nuclear factor-kappaB (NF-kappaB) transcriptional factor, as well as p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), or phosphatidylinositol-3-kinase/AKT in A549 cells. In this study, we further investigated whether dexamethasone (Dex) controls IL-1beta-induced HBD-2 mRNA expression in A549 cells and the molecular mechanism associated with it. Dex suppressed IL-1beta-induced HBD-2 mRNA expression, which is mediated by a glucocorticoid receptor, at the transcriptional level. Interestingly, Dex attenuated IL-1beta-mediated activation of p38 MAPK and JNK, but not of AKT. Dex increased the expression of MAPK phosphatase (MKP)-1, which dephosphorylated p38 MAPK, but not JNK, by IL-1beta. However, although Dex did not inhibit the nuclear translocation of p65 NF-kappaB in response to IL-1beta, it profoundly inhibited NF-kappaB promoter- and HBD-2 promoter-driven luciferase activities. These results suggest that Dex acts to inhibit IL-1beta-induced HBD-2 mRNA expression through blockage of the nuclear transcriptional activation of p65 NF-kappaB as well as through inactivation of p38 MAPK and JNK. Specifically, Dex-induced MKP-1 expression is responsible for the inactivation of p38 MAPK, but not JNK, in response to IL-1beta in A549 cells.

Triptolide suppresses interleukin-1beta-induced human beta-defensin-2 mRNA expression through inhibition of transcriptional activation of NF-kappaB in A549 cells.

The immunosuppressive effect of triptolide has been associated with suppression of T-cell activation. However, the immunosuppressive effects of triptolide on innate immunity in the epithelial barrier remain to be elucidated. Human beta-defensin (HBD)-2 is an inducible antimicrobial peptide and plays an important role in the innate immunity. We have previously demonstrated that IL-1beta induced HBD-2 mRNA expression in A549 cells through activation of nuclear factor-kappaB (NF-kappaB) transcriptional factor as well as p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), or phosphatidylinositol-3-kinase (PI3K). In this study, we investigated effects of triptolide on IL-1beta-induced HBD-2 mRNA expression in A549 cells. Triptolide inhibited IL-1beta-induced HBD-2 mRNA expression in a dose-dependent manner. Addition of triptolide did not suppress activation of p38 MAPK, JNK, or PI3K in response to IL-1beta. Triptolide inhibited IL-1beta-induced MAPK phosphatase-1 expression at the transcriptional level and resulted in sustained phosphorylation of JNK or p38 MAPK, explaining the little effect of triptolide on IL-1beta-induced phosphorylation of these kinases. Although triptolide partially suppressed IL-1beta-mediated degradation of IkappaB-alpha and nuclear translocation of p65 NF-kappaB, triptolide potently inhibited NF-kappaB promoter-driven luciferase activity in A549 cells. These results collectively suggest that the inhibitory effect of triptolide on IL-1beta-induced HBD-2 mRNA expression in A549 cells seems to be at least in part mediated through nuclear inhibition of NF-kappaB transcriptional activity, but not inhibition of p38 MAPK, JNK, or PI3K. This inhibition may explain the ability of triptolide to diminish innate immune response.

Leptomycin B, a metabolite of Streptomyces, inhibits the expression of inducible nitric oxide synthase in BV2 microglial cells.

Overexpression of inducible nitric oxide synthase (iNOS) and the resultant overproduction of NO has been implicated in neuronal inflammatory diseases. Leptomycin B (LMB), a metabolite of Streptomyces, has been identified as a specific inhibitor of CRM1 nuclear export receptor. In this study, we evaluated the effect of LMB on lipopolysaccharide (LPS)-induced iNOS expression in BV2 cells, a murine microglial cells and the associated mechanisms. LMB strongly inhibited LPS-induced iNOS protein and mRNA expressions in BV2 cells in which 10 ng/ml of LMB (18 nM) was sufficient to greatly down-regulate iNOS by LPS, suggesting the potency of LMB to inhibit iNOS. The data of iNOS promoter-driven luciferase assay further suggested that the LMB inhibitory effect was in part due to inhibition of iNOS transcription. However, LPS-induced activation of various intracellular signaling proteins, such as nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated kinases (ERKs), p38s, and c-Jun N-terminal kinases (JNKs), whose activations are known to be important for iNOS expression by LPS in BV2 cells, were not affected in the presence of LMB. Together, these results suggest that LMB inhibits iNOS expression in response to LPS in BV2 microglia, and the inhibition seems to be associated with blockage of CRM1-mediated iNOS mRNA nuclear export and also in part transcriptional down-regulation of iNOS, but not through modulation of NF-kappaB and the mitogen-activated protein kinase signaling pathways.

Inhibitory modulation of ATP-sensitive potassium channels by gallate-ester moiety of (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenolic substance found in green tea, is well recognized to be beneficial for human health. However, it is still controversial as to what dose of this compound is indeed good for human health. Though some recent studies have interestingly reported various beneficial effects of EGCG in cell culture system, however, plasma levels of EGCG attainable by oral regular intake in humans are normally in nanomolar range. However, potential side effects of EGCG when administered parenterally at higher concentration have not been thoroughly tested. Here, we evaluated the effect of EGCG on ATP-sensitive potassium (K(ATP)) channels expressed in Xenopus oocytes. EGCG inhibited the activity of the Kir6.2/SUR1 and Kir6.2DeltaC36 channels with IC(50) of 142+/-37 and 19.9+/-1.7microM, respectively. Inhibition of EGCG was also observed in Kir6.2/SUR2A or Kir6.2/SUR2B channels. Notably, (-)-epicatechin-3-gallate (ECG), another major polyphenolic substance in green tea, was found to reduce the channel activity with greater potency than EGCG. In contrast to EGCG and ECG, which have the gallic acid-ester moiety in their own structures, (-)-epigallocatechin and (-)-epicatechin exhibited very weak inhibition of the K(ATP) channel. Collectively, these results suggest that the gallate-ester moiety of epicatechins may be critical for inhibiting the K(ATP) channel activity via the pore-forming subunit Kir6.2 and this may be a possible mechanism by which green tea extracts or EGCG may cause unexpected side effects at micromolar plasma level.

Rapid blockade of telomerase activity and tumor cell growth by the DPL lipofection of ribbon antisense to hTR.

Ribbon antisense (RiAS) to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. The antisense molecule, hTR-RiAS, combined with enhanced cellular uptake was shown to effectively inhibit telomerase activity and cause rapid cell death in various cancer cell lines. When cancer cells were treated with hTR-RiAS, the level of hTR RNA was reduced by more than 90% accompanied with reduction in telomerase activity. When checked for cancer cell viability, cancer cell lines treated with hTR-RiAS using DNA+Peptide+Lipid complex showed 70-80% growth inhibition in 3 days. The reduced cell viability was due to apoptosis as the percentage of cells exhibiting the sub-G0 arrest and DNA fragmentation increased after antisense treatment. Further, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with hTR-RiAS, tumor growth was markedly suppressed with almost total ablation of hTR RNA in the tumor tissue. Cells in the tumor tissue were also found to undergo apoptosis after hTR-RiAS treatment. These results suggest that hTR-RiAS is an effective anticancer reagent, with a potential for broad efficacy to diverse malignant tumors.

Gene knockdown by large circular antisense for high-throughput functional genomics.

Single-stranded genomic DNA of recombinant M13 phages was tested as an antisense molecule and examined for its usefulness in high-throughput functional genomics. cDNA fragments of various genes (TNF-alpha, c-myc, c-myb, cdk2 and cdk4) were independently cloned into phagemid vectors. Using the life cycle of M13 bacteriophages, large circular (LC)-molecules, antisense to their respective genes, were prepared from the culture supernatant of bacterial transformants. LC-antisense molecules exhibited enhanced stability, target specificity and no need for target-site searches. High-throughput functional genomics was then attempted with an LC-antisense library, which was generated by using a phagemid vector that incorporated a unidirectional subtracted cDNA library derived from liver cancer tissue. We identified 56 genes involved in the growth of these cells. These results indicate that an antisense sequence as a part of single-stranded LC-genomic DNA of recombinant M13 phages exhibits effective antisense activity, and may have potential for high-throughput functional genomics.

Catalase induced expression of inflammatory mediators via activation of NF-kappaB, PI3K/AKT, p70S6K, and JNKs in BV2 microglia.

Catalase induces COX-2 or iNOS expression in some type of cells, but the mechanism remains unclear. Here we investigated the effect of catalase on COX-2 and iNOS expression in BV2 microglia and the inductive mechanism associated. Exposure of catalase to BV2 microglia induced expression of COX-2 and iNOS that was related with transcriptional up-regulation. Importantly, catalase-induced COX-2 and iNOS expression needed activations of NF-kappaB, PI3K/AKT, and JNKs, which were important for the transcriptional up-regulation of COX-2 and iNOS. Notably, rapamycin inhibition of p70S6K led to down-regulation of COX-2 and iNOS protein expression, but not steady-state mRNA expression and transcription, induced by catalase, suggesting that p70S6K is involved in increased COX-2 and iNOS mRNA translation by catalase. Interestingly, there was PI3K-dependent activation of AKT, p70S6K, JNKs, and NF-kappaB in response to catalase. These data collectively suggest catalase-induced COX-2 and iNOS expression in BV2 microglia is, in part at least, mediated through activation of multiple signaling proteins.

Catalase induces the expression of inducible nitric oxide synthase through activation of NF-kappaB and PI3K signaling pathway in Raw 264.7 cells.

It has been reported that macrophages produce substantial amounts of nitrite and nitrate after addition of catalase, but the mechanism associated remains unclear. In present study, we investigated whether catalase modulates the expression of inducible nitric oxide synthase (iNOS), an enzyme that produces nitric oxide. Exposure of Raw 264.7 macrophages (Raw cells) to catalase induced high expression of iNOS mRNA as well as protein with enzymatic activity. Data of mechanical analyses, such as iNOS promoter-driven luciferase assay and actinomycin D chase experiments demonstrated that the induction was due to increased iNOS transcription and post-transcriptional iNOS mRNA stability. Of interest, catalase-induced iNOS protein expression was abrogated through inactivation of NF-kappaB pathway by MG132 or BAY 11-7085 and PI3K pathway by LY294002 or wortmannin, respectively. In particular, blockage of PI3K pathway by LY294002 down-regulated iNOS transcription and steady-state iNOS mRNA levels as well as iNOS mRNA stability induced by catalase, suggesting regulation of PI3K pathway in catalase-induced iNOS expression at the levels of iNOS transcription, steady-state mRNA status, and mRNA stability. Additional cell culture works in different types of cells indicated that iNOS expression by catalase might be cell type-specific, based on the facts that catalase induced iNOS expression in BV2 microglial macrophage-like cells, but not in HT-29 or A549, human colon or lung cancer epithelial-like cells. Together, these results demonstrate for the first time that catalase induces iNOS expression in Raw cells, which seems to be associated with the increase of iNOS transcription and mRNA stability as well as the activation of NF-kappaB and PI3K signaling pathways.

Leptomycin B-induced apoptosis is mediated through caspase activation and down-regulation of Mcl-1 and XIAP expression, but not through the generation of ROS in U937 leukemia cells.

Leptomycin B (LMB), which is originally isolated from Streptomyces, possesses anti-tumor properties in vivo and in vitro. Though it was previously reported that LMB induces cell cycle arrest and p53-mediated apoptosis in certain cancer cells, however, the mechanism by which LMB induces apoptosis remains poorly understood. Here, we investigated the mechanisms of apoptosis induced by LMB in U937 cells. Treatment with LMB concentration-dependently induced cytotoxicity and apoptosis in U937 cells that correlated temporally with activation of caspases and down-regulation of Mcl-1 and XIAP. LMB did not change the expressions of Bcl-2 or Bax. A broad spectrum caspase inhibitor, z-VAD-fmk, blocked caspase-3 activation and elevated the survival in LMB-treated U937 cells, suggesting that caspase-3 activation is critical for LMB-induced apoptosis. Interestingly, Bcl-2 overexpression that blocked cytochrome c release by LMB effectively attenuated the apoptotic response to LMB, suggesting that LMB-induced apoptosis is mediated through the mitochondrial pathway. Antioxidants or antioxidant enzymes had no effects on LMB-induced apoptosis. Data of flow cytometry analysis using 2',7'-dichlorofluorescein-diacetate further revealed no reactive oxygen species (ROS) generation by LMB, indicating that apoptosis induced by LMB is ROS-independent. However, the apoptotic response to LMB was not shown in U937 cells pretreated with the sulfhydryl group-containing antioxidant N-acetylcysteine (NAC). Further analysis suggested that NAC directly binds LMB and abolishes the apoptotic effects of LMB. Collectively, these findings suggest that LMB potently induces apoptosis in U937 cells, and LMB-induced apoptosis in U937 cells is related with cytochrome c release, activation of caspases, and selective down-regulation of Mcl-1 and XIAP.

Induction of cyclooxygenase-2 in macrophages by catalase: role of NF-kappaB and PI3K signaling pathways.

Induction of COX-2 by catalase in smooth muscle cells, endothelial cells, and neuronal cells has been previously reported. However, the mechanism by which catalase up-regulates COX-2 remains poorly understood. In this study, we investigated the effect of catalase on induction of COX-2 in macrophages. The addition of catalase into Raw 264.7 macrophages induced COX-2 expression that was correlated with increased COX-2 transcription and mRNA stability. Catalase also induced activation of NF-kappaB, PI3K, ERKs, p38s, or JNKs. Catalase-induced COX-2 expression was abrogated by treatment of MG-132 (a NF-kappaB inhibitor) or LY294002 (a PI3K inhibitor), but not by treatment of PD98059 (an ERK inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor). Moreover, inhibition of PI3K by LY294002 caused partial decrease of catalase-induced COX-2 transcription and steady-state COX-2 transcript levels, but not COX-2 mRNA stability. Together, these results suggest that catalase induces the expression of COX-2 in Raw 264.7 macrophages, and the induction is related with activation of NF-kappaB transcription factor and PI3K signaling pathway.

Effective inhibition of cancer cell growth by a novel tripartite transfection complex containing ribbon antisense molecules to hTR.

PURPOSE: In the present study, ribbon antisense to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. MATERIALS AND METHODS: Ribbon antisense molecules to the human hTR gene (hTR-RiAS) were constructed and complexed with a short modified peptide and cationic liposomes to improve the cellular uptake of the antisense molecules. The DPL complexes containing hTR-RiAS were transfected into target cancer cells. Various assays were performed to confirm the effects of the hTR-RiAS on the gene expression and cell proliferation. RESULTS: When cancer cells were treated with hTR-RiAS, the cellular level of hTR mRNA was reduced by more than 95%, as shown by RT-PCR. Further, the telomerase acti vity was also affected by the antisense treatment. In contrast, both mismatched and scrambled oligonucleotides failed to reduce the levels of hTR mRNA and telomerase activity. When checked for cancer cell viability, hTR-RiAS inhibited cell growth by more than 70%, in a very rapid manner. The reduced cell viability was found to be due to apoptosis of cancer cells. CONCLUSION: These results show that hTR-RiAS is a powerful anticancer reagent, with the potential for broad efficacy to diverse malignant tumors.

Identification of genes involved in liver cancer cell growth using an antisense library of phage genomic DNA.

PURPOSE: Genes involved in liver cancer cell growth have been identified using an antisense library of large circular (LC-) genomic DNA of a recombinant M13 phage. MATERIALS AND METHODS: A subtracted cDNA library was constructed by combining procedures of suppression subtractive hybridization (SSH) and unidirectional cloning of the subtracted cDNA into an M13 phagemid vector. Utilizing the life cycle of M13 bacteriophages, LC-antisense molecules derived from 1,200 random cDNA clones selected by size were prepared from the culture supernatant of bacterial transformants. The antisense molecules were arrayed for transfection on 96-well plates preseeded with HepG2. RESULTS: When examined for growth inhibition after antisense transfection, 153 out of 1,200 LC-antisense molecules showed varying degrees of growth inhibitory effect to HepG2 cells. Sequence comparison of the 153 clones identified 58 unique genes. The observations were further extended by other cell-based assays. CONCLUSION: These results suggest that the LC-antisense library offers potential for unique high-throughput screening to find genes involved in a specific biological function, and may prove to be an effective target validation system for gene-based drug discovery.

Trichostatin A, a histone deacetylase inhibitor, activates the IGFBP-3 promoter by upregulating Sp1 activity in hepatoma cells: alteration of the Sp1/Sp3/HDAC1 multiprotein complex.

We determined the molecular mechanisms by which trichostatin A (TSA) induced insulin-like growth factor-binding protein 3 (IGFBP-3) gene expression in Hep3B cells, a p53-mutant human hepatocellular carcinoma (HCC) cell line. TSA induced the expressions of the IGFBP-3 mRNA and protein and the activation of its promoter. Using IGFBP-3 promoter deletion constructs, the TSA-responsive element was mapped to a region between -115 and -30, relative to the transcription start site. Promoter mutation analysis confirmed that the TSA-responsive element coincides with the Sp1/GC-rich region on the IGFBP-3 promoter. This transcriptional activation appears to be mediated by both the Sp1 and Sp3 transcription factors and, in particular, by the phosphorylation of Sp1, because treatment of Hep3B cells and Schneider (SL2) cells with TSA significantly activated phosphorylation of Sp1 in a dose-dependent manner. Consistent with the transcriptional activation of the IGFBP-3 promoter by TSA, TSA treatment led to the release of HDAC1 and Sp3 from the Sp1 transcriptional factor complex, indicating the involvement of multiprotein complexes containing Sp1, Sp3, p300, and HDAC-1 in IGFBP-3 activation by TSA. Taken together, these results show that Sp1 phosphorylation and the modulation of the Sp1/Sp3/HDAC1 multiprotein complex play a pivotal role in the transcriptional activation of the IGFBP-3 promoter through the Sp1/GC-rich site by TSA.