In Young Park

Functional and Clinical Evidence for NDRG2 as a Candidate Suppressor of Liver Cancer Metastasis

We searched for potential suppressors of tumor metastasis by identifying the genes that are frequently down-regulated in hepatocellular carcinomas (HCC) while being negatively correlated with clinical parameters relevant to tumor metastasis, and we report here on the identification of N-myc downstream regulated gene 2 (NDRG2) as a promising candidate. NDRG2 expression was significantly reduced in HCC compared with nontumor or normal liver tissues [87.5% (35 of 40) and 62% (62 of 100) at RNA and protein levels, respectively]. Reduction of NDRG2 expression was intimately associated with promoter hypermethylation because its promoter region was found to carry extensively methylated CpG sites in HCC cell lines and primary tumors. Immunohistochemical analysis of NDRG2 protein in 100 HCC patient tissues indicated that NDRG2 expression loss is significantly correlated with aggressive tumor behaviors such as late tumor-node-metastasis (TNM) stage (P = 0.012), differentiation grade (P = 0.024), portal vein thrombi (P = 0.011), infiltrative growth pattern (P = 0.015), nodal/distant metastasis (P = 0.027), and recurrent tumor (P = 0.021), as well as shorter patient survival rates. Ectopically expressed NDRG2 suppressed invasion and migration of a highly invasive cell line, SK-Hep-1, and experimental tumor metastasis in vivo, whereas small interfering RNA-mediated knockdown resulted in increased invasion and migration of a weakly invasive cell line, PLC/PRF/5. In addition, NDRG2 could antagonize transforming growth factor beta1-mediated tumor cell invasion by specifically down-regulating the expression of matrix metalloproteinase 2 and laminin 332 pathway components, with concomitant suppression of Rho GTPase activity. These results suggest that NDRG2 can inhibit extracellular matrix-based, Rho-driven tumor cell invasion and migration and thereby play important roles in suppressing tumor metastasis in HCC.

In Vitro Development of Reconstructed Porcine Oocytes after Somatic Cell Nuclear Transfer

Abstract This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 ± 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 ± 2.3%) (P < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 ± 2.9, 6.6 ± 2.3, and 8.1 ± 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 ± 10.4 > 24.6 ± 10.1 > 16.5 ± 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P < 0.05). Nuclear-transferred embryos (9.4 ± 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 ± 1.9%) or parthenogenetic embryos (22.4 ± 7.2%) (P < 0.01). The mean cell number (28.9 ± 11.4) of NT-derived blastocysts was smaller than that (38.6 ± 10.4) of IVF-derived blastocysts (P < 0.05) and was similar to that (29.9 ± 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.

Functional Switching of TGF-β1 Signaling in Liver Cancer via Epigenetic Modulation of a Single CpG Site in TTP Promoter

BACKGROUND & AIMS Acquisition of resistance to the antiproliferative effect of transforming growth factor (TGF)-beta1 is crucial for the malignant progression of cancers. In this study, we sought to determine whether deregulated expression of tristetrapolin (TTP), a negative posttranscriptional regulator of c-Myc, confers resistance to the antiproliferative effects of TGF-beta1 on liver cancer cells. METHODS The epigenetics of TTP promoter regulation and its effects on TGF-beta1 signaling were examined in hepatocellular carcinoma (HCC) cell lines and patient tissues. RESULTS TTP was down-regulated in HCC cell lines (10/11), compared with normal liver, as well as in tumor tissues (19/24) from paired HCC specimens. Methylation of a specific single CpG site located within the TGF-beta1-responsive region (TRR) of the TTP promoter was significantly associated with TTP down-regulation in both HCC cell lines and tumor tissues (r = -0.606383, P < .001). The singly methylated CpG site was specifically bound by a transcriptional repressor complex consisting of MECP2/c-Ski/DNMT3A and abolished the TGF-beta1-induced as well as basal-level expression of TTP. The epigenetic inactivation of TTP led to an increased half-life of c-Myc mRNA and blocked the cytostatic effect of TGF-beta1. Statistically significant correlations were observed between the single CpG site methylation and expression levels of TTP or c-Myc in clinical samples of HCC. CONCLUSIONS Abrogation of the post-transcriptional regulation of c-Myc via methylation of a specific single CpG site in the TTP promoter presents a novel mechanism for the gain of selective resistance to the antiproliferative signaling of TGF-beta1 in HCC.

Hepatitis B virus-X protein recruits histone deacetylase 1 to repress insulin-like growth factor binding protein 3 transcription

Hepatitis B virus (HBV), a major causative agent of hepatocelluar carcinoma (HCC), encodes an oncogenic X-protein (HBx) which has been known as a transcriptional transactivator on multiple viral and celluar promoters. In the report, we verified that HBx transcriptionally repress insulin-like growth factor binding protein-3 (IGFBP-3) by promoting HBx/histone deacetylase 1 (HDAC1) complex formation. HBx recruited HDAC1 forms complex with Sp1 in a p53-independent manner) and deacetylates Sp1 which resulted in the diminished binding of Sp1 on targeted DNA during transcriptional repression. Deacetylation of Sp1 by HBx recruited HDAC1 likely to be a part of the mechanism that controls HBx induced IGFBP-3 repression and the modification of chromatin structure.

Deregulation of DNA methyltransferases and loss of parental methylation at the insulin-like growth factor II (Igf2)/H19 loci in p53 knockout mice prior to tumor development

To ascertain whether p53 deficiency in vivo leads to the deregulation of DNA methylation machinery prior to tumor development, we investigated the expression profile of DNA methyltransferases in the thymus and the liver of p53+/+, p53+/−, and p53−/− mice at 7 weeks of age before tumor development. The expression of DNA methyltransferases was examined in the thymus at 7 weeks of age, since the malignant T‐cell lymphoma develops most frequently in p53−/− mice around 20 weeks of age. Both mRNA and protein levels of Dnmt1 and Dnmt3b were increased in the thymus and the liver of p53‐deficient mice. The expression of Dnmt3a was also increased in the liver but not in the thymus of p53‐deficient mice. Dnmt3L expression was reduced in the thymus of p53+/− and p53−/− mice. The total 5‐methylcytosine (5‐MeC) in the genomic DNA of p53+/+, p53+/−, and p53−/− mice was quantitated by dot‐blot using antibody against 5‐MeC. Global methylation was increased in the thymus and the liver of p53‐deficient mice. To correlate the deregulated expression of DNA methyltransferases with the disturbance of the epigenetic integrity, we examined the DNA methylation of the imprinting control region (ICR) at the insulin‐like growth factor II (Igf2)/H19 loci in the thymus and the liver of p53+/+, p53+/−, and p53−/− mice. The region containing two CCCTC binding factor (CTCF) binding sites in the 5′‐ICR tended to be hypomethylated in the thymus of p53−/− mice, but not in the liver. The expression profile of Igf2 and H19 indicated that the thymus‐specific changes of Igf2 and H19 expression were coherent to the hypomethylation of the ICR in the thymus. Our results suggest that p53 is required for the maintenance of DNA methylation patterns in vivo.

Gene delivery using a derivative of the protein transduction domain peptide, K-Antp.

Due to their intracellular permeability, protein transduction domains (PTDs) have been widely used to deliver proteins and peptides to mammalian cells. However, their performance in gene delivery has been relatively poor. To improve the efficiency of PTD-mediated gene delivery, we synthesized a new peptide, KALA-Antp (K-Antp), which contains the sequences for PTD of the third alpha-helix of Antennapedia (Antp) homeodomain and the fusogenic peptide KALA. In this configuration, Antp is designed to provide the cell permeation capacity and nuclear localization signal, while the KALA moiety to promote cellular entry of the peptide-DNA complex. An optimal K-Antp/DNA formula was nearly 400-600 fold more efficient than Antp or poly-lysine-Antp (L-Antp) in gene delivery, and comparable or superior to a commercial liposome. The K-Antp-mediated plasmid DNA transfection not only exhibited temperature sensitivity, reflecting the involvement of an endocytosis-mediated gene transfer mechanism similar to other known PTDs, but also temperature insensitivity, suggesting the role of an energy-independent mechanism. Incorporation of an endosomolytic polymer polyethylenimine (PEI) into the system or treatment with chloroquine further increased the efficiency of K-Antp-mediated gene delivery. These results demonstrate the potential of the combinatorial use of KALA, Antp and PEI in the development of efficient PTD-derived gene carriers.

Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR

We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite-mediated sequencing) as a validation, deviations were within ±5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h.

Blastocyst viability and generation of transgenic cattle following freezing of in vitro produced, DNA-injected embryos.

This study examined whether the viability, determined in vitro, of DNA-injected bovine embryos produced in vitro was affected by freezing, and if the frozen embryos developed to term following transfer to recipients. In vitro fertilized zygotes were injected with the pBL1 gene and then co-cultured with mouse embryonic fibroblasts (MEF) in CR1aa medium. Embryos were prepared for cryopreservation by exposure to a 10% (v/v) glycerol solution, loaded into 0.25 ml straws and then frozen by conventional slow freezing. Thawing was by rapid warming in water (37 degrees C) and embryos were rehydrated in PBS diluents of 6%, 3% and 0% (v/v) glycerol supplemented with 0.25 M sucrose and 0.5% (w/v) BSA. In Experiment 1, blastocysts that developed from DNA-injected embryos were individually classified into three morphological groups and three stages of development prior to freezing. DNA-injected blastocysts of excellent quality at freezing showed a higher survival rate (78.8+/-10.6%) after thawing than those of good (60. 9+/-16.4%) or fair (12.5+/-5.9%) quality (P<0.05). Post-thaw survival rate, judged in vitro, increased with more advanced stage of blastocyst development at freezing (early 48.8+/-15.9%, mid 52. 1+/-12.6% and expanded 71.2+/-1.1; P<0.05). In Experiment 2, the frozen/thawed embryos were transferred to recipients to examine in vivo viability. Following transfer of one or two embryos per recipient, pregnancy rates at 60 days of gestation were 13.6% (13/96) for frozen embryos and 26.5% (43/162) for fresh embryos (P<0. 05). Of the 12 live calves born from the frozen/thawed embryos, two males (18.3%) were transgenic. None of the live-born calves derived from fresh embryos exhibited the transgene. One of transgenic bulls did not produce transgenic sperm. Three out of 23 calves (13.0%) produced from cows inseminated with semen of the other bull were transgenic, suggesting that this animal was a germ-line mosaic. These studies indicated that the viability of in vitro produced, DNA-injected bovine blastocysts was affected by freezing and by both the quality and stage of development of the embryo prior to freezing. The generation of transgenic cattle demonstrates that it is feasible to freeze DNA-injected, in vitro produced embryos.