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1.  Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells 
Yonsei Medical Journal  2014;55(5):1206-1213.
Purpose
Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties. However, the precise regulatory mechanisms controlling metastasis genes and their role in metastatic transformation are largely unknown. To address epigenetically-regulated gene products involved in ovarian cancer metastasis, we examined the mechanisms regulating mucin 13 (MUC13) expression and its influence on aggressive behaviors of ovarian malignancies.
Materials and Methods
We injected SK-OV-3 ovarian cancer cells peritoneally into nude mice to mimic human ovarian tumor metastasis. Overexpression of MUC13 mRNA was detected in metastatic implants from the xenografts by expression microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The DNA methylation status within the MUC13 promoter region was determined using bisulfite sequencing PCR and quantitative methylation-specific PCR. We evaluated the effects of exogenous MUC13 on cell invasion and migration using in vitro transwell assays.
Results
MUC13 mRNA expression was up-regulated, and methylation of specific CpG sites within the promoter was reduced in the metastatic implants relative to those in wild-type SK-OV-3 cells. Addition of a DNA methyltransferase inhibitor to SK-OV-3 cells induced MUC13 expression, thereby implying epigenetic regulation of MUC13 by promoter methylation. MUC13 overexpression increased migration and invasiveness, compared to control cells, suggesting aberrant up-regulation of MUC13 is strongly associated with progression of aggressive behaviors in ovarian cancer.
Conclusion
We provide novel evidence for epigenetic regulation of MUC13 in ovarian cancer. We suggest that the DNA methylation status within the MUC13 promoter region may be a potential biomarker of aggressive behavior in ovarian cancer.
doi:10.3349/ymj.2014.55.5.1206
PMCID: PMC4108803  PMID: 25048476
Ovarian cancer; mouse xenograft; MUC13; DNA methylation
2.  Crystallization and preliminary X-ray crystallographic studies of a new class of enoyl-(acyl-carrier protein) reductase, FabV, from Vibrio fischeri  
An orthorhombic crystal of an enoyl-(acyl-carrier protein) reductase from V. fischeri was obtained and diffraction data were collected to 2.7 Å resolution.
Enoyl-(acyl-carrier protein) reductase (ENR) catalyzes the last step of the fatty-acid elongation cycle of the bacterial fatty-acid biosynthesis (FAS II) pathway. Recently, a new class of ENR has been identified from Vibrio cholerae and was named FabV. In order to understand the molecular mechanism of the new class of ENR at the structural level, FabV from V. fischeri was overexpressed, purified and crystallized. Diffraction data were collected to 2.7 Å resolution from a native crystal. The crystal belonged to the orthorhombic space group P21212, with unit-cell parameters a = 123.53, b = 164.14, c = 97.07 Å. The presence of four molecules of FabV in the asymmetric unit gave a V M value of 2.81 Å3 Da−1, with a corresponding solvent content of 54.5%.
doi:10.1107/S1744309111049426
PMCID: PMC3253841  PMID: 22232178
FAS II pathway; enoyl-(acyl-carrier protein) reductase; Vibrio fischeri; FabV
3.  Crystallization and preliminary X-ray crystallographic studies of the ice-binding protein from the Antarctic yeast Leucosporidium sp. AY30 
The ice-binding protein from Leucosporidium sp. AY30 was cloned, expressed, purified and crystallized. A complete data set was collected to 1.5 Å resolution.
Freezing is dangerous to cellular organisms because it causes an increase in the concentration of ions and other solutes in the plasma, denatures biomolecules and ruptures cell membranes. Some cold-adapted organisms can survive at subzero temperatures by producing proteins that bind to and inhibit the growth of ice crystals. To better understand the structure and function of these proteins, the ice-binding protein from Leucosporidium sp. AY30 (LeIBP) was overexpressed, purified and crystallized. The native crystal belonged to space group P43212, with unit-cell parameters a = b = 98.05, c = 106.13 Å. Since LeIBP lacks any cysteine or methionine residues, two leucine residues (Leu69 and Leu155) were substituted by methionine residues in order to obtain selenomethionine-substituted LeIBP for use in multiple-wavelength anomalous diffraction (MAD) phasing. The selenomethionine-substituted mutant crystallized in the same space group as the native protein.
doi:10.1107/S1744309111018446
PMCID: PMC3144800  PMID: 21795798
freezing; ice-binding proteins; cold-adaptation; Antarctic yeast
4.  Crystallization and preliminary X-ray crystallographic studies of enoyl-acyl carrier protein reductase (FabI) from Psuedomonas aeruginosa  
Enoyl-acyl carrier protein reductase (FabI) from P. aeruginosa was purified and crystallized. FabI was also cocrystallized with the inhibitor triclosan and the cofactor NADH. Crystals of native and complexed FabI diffracted to resolutions of 2.6 and 1.8 Å, respectively.
During fatty-acid biosynthesis, enoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the reduction of trans-2-enoyl-ACP to fully saturated acyl-ACP via the ubiquitous fatty-acid synthase system. NADH-dependent enoyl-ACP reductase (FabI) from Pseudomonas aeruginosa has been purified and crystallized as an apoenzyme and in a complex form with NADH and triclosan. Triclosan is an inhibitor of FabI and forms a stable ternary complex in the presence of NADH. The crystals of native and complexed FabI diffracted to resolutions of 2.6 and 1.8 Å, respectively. The crystals both belonged to space group P21, with unit-cell parameters a = 117.32, b = 155.844, c = 129.448 Å, β = 111.061° for the native enzyme and a = 64.784, b = 107.573, c = 73.517 Å, β = 116.162° for the complex. Preliminary molecular replacement further confirmed the presence of four tetramers of native FabI and one tetramer of the complex in the asymmetric unit, corresponding to Matthews coefficients (V M) of 2.46 and 2.05 Å3 Da−1 and solvent contents of 50.1 and 40.1%, respectively.
doi:10.1107/S1744309110048827
PMCID: PMC3034610  PMID: 21301088
enoyl-acyl carrier protein reductases; FabI; Pseudomonas aeruginosa; fatty-acid synthesis; triclosan
5.  Prognostic classification of pediatric medulloblastoma based on chromosome 17p loss, expression of MYCC and MYCN, and Wnt pathway activation 
Neuro-Oncology  2011;14(2):203-214.
Pediatric medulloblastoma is considered a highly heterogeneous disease and a new strategy of risk stratification to optimize therapeutic outcomes is required. We aimed to investigate a new risk-stratification approach based on expression profiles of medulloblastoma cohorts. We analyzed gene expression profiles of 30 primary medulloblastomas and detected strong evidence that poor survival outcome was significantly associated with mRNA expression profiles of 17p loss. However, it was not supported in independent cohorts from previously published data (n = 100). We speculated that this discrepancy might come from complex conditions of two important prognostic determinants: loss of tumor suppressors (chromosome 17p) and high expression of oncogenes c-myc (MYCC) or N-myc (MYCN). When patients were stratified into 5 or 7 subgroups based on simultaneous consideration of these 2 factors while defining the Wnt group as independent, obviously different survival expectancies were detected between the subgroups. For instance, predicted 5-year survival probabilities ranged from 19% to 81% in the 5 subgroups. We also found that age became a significant prognostic marker after adjusting for 17p, MYCC, and MYCN status. Diminished survival in age <3 years was more substantial in subgroups with high expression of MYCC, MYCN, or 17p loss but not in other subgroups, indicating that poor survival outcome might be synergistically affected by these 3 factors. Here we suggest a more tailored subgrouping system based on expression profiles of chromosome 17p, MYCC, and MYCN, which could provide the basis for a novel risk-stratification strategy in pediatric medulloblastoma.
doi:10.1093/neuonc/nor196
PMCID: PMC3266382  PMID: 22090452
chromosome 17p; medulloblastoma; MYC; MYCN; prognosis; Wnt
6.  A statin-regulated microRNA represses human c-Myc expression and function 
EMBO Molecular Medicine  2012;4(9):896-909.
c-Myc dysregulation is one of the most common abnormalities found in human cancer. MicroRNAs (miRNAs) are functionally intertwined with the c-Myc network as multiple miRNAs are regulated by c-Myc, while others directly suppress c-Myc expression. In this work, we identified miR-33b as a primate-specific negative regulator of c-Myc. The human miR-33b gene is located at 17p11.2, a genomic locus frequently lost in medulloblastomas, of which a subset displays c-Myc overproduction. Through a small-scale screening with drugs approved by the US Food and Drug Administration (FDA), we found that lovastatin upregulated miR-33b expression, reduced cell proliferation and impaired c-Myc expression and function in miR-33b-positive medulloblastoma cells. In addition, a low dose of lovastatin treatment at a level comparable to approved human oral use reduced tumour growth in mice orthotopically xenografted with cells carrying miR-33b, but not with cells lacking miR-33b. This work presents a highly promising therapeutic option, using drug repurposing and a miRNA as a biomarker, against cancers that overexpress c-Myc.
doi:10.1002/emmm.201101045
PMCID: PMC3491823  PMID: 22887866
c-Myc; lovastatin; medulloblastoma; microRNA; miR-33b
7.  Transglutaminase 2 facilitates the distant hematogenous metastasis of breast cancer by modulating interleukin-6 in cancer cells 
Introduction
Inflammation has been implicated in cancer aggressiveness. As transglutaminase 2 (TG2), which has been associated with inflammatory signaling, has been suggested to play a role in tumor behavior, we propose that TG2 may be an important linker inducing interleukin (IL)-6-mediated cancer-cell aggressiveness, including distant hematogenous metastasis.
Methods
To investigate the role for TG2 and IL-6, TG2-knocked-down and IL-6-knocked-down cancer cells were generated by using shRNA. Human breast cancer cell xenograft model in highly immunocompromised mice and human advanced breast cancer primary tumor tissue microarrays were used in this study.
Results
IL-6 production in human breast cancer cells was dependent on their TG2 expression level. In vitro tumor-sphere formation was dependent on TG2 and downstream IL-6 production from cancer cells. Primary tumor growth in the mammary fat pads and distant hematogenous metastasis into the lung was also dependent on TG2 and downstream IL-6 expression levels. The effect of TG2 expression on human breast cancer distant metastasis was investigated by analyzing a tissue microarray of primary tumors from 412 patients with their clinical data after 7 years. TG2 expression in primary tumor tissue was inversely correlated with recurrence-free survival (P = 0.019) and distant metastasis-free survival (DMFS) (P = 0.006) in patients with advanced breast cancer. Furthermore, by using public datasets that included a total of 684 breast cancer patients, we found that the combined high expression of TG2 and IL-6 was associated with shorter DMFS, compared with the high expression of IL-6 only (P = 0.013).
Conclusions
We provide evidence that TG2 is an important link in IL-6-mediated tumor aggressiveness, and that TG2 could be an important mediator of distant metastasis, both in a xenograft animal model and in patients with advanced breast cancer.
doi:10.1186/bcr3034
PMCID: PMC3262209  PMID: 21967801
8.  Coordinated Regulation of ATF2 by miR-26b in γ-Irradiated Lung Cancer Cells 
PLoS ONE  2011;6(8):e23802.
MicroRNA regulates cellular responses to ionizing radiation (IR) through translational control of target genes. We analyzed time-series changes in microRNA expression following γ-irradiation in H1299 lung cancer cells using microarray analysis. Significantly changed IR-responsive microRNAs were selected based on analysis of variance analysis, and predicted target mRNAs were enriched in mitogen-activated protein kinase (MAPK) signaling. Concurrent analysis of time-series mRNA and microRNA profiles uncovered that expression of miR-26b was down regulated, and its target activating transcription factor 2 (ATF2) mRNA was up regulated in γ-irradiated H1299 cells. IR in miR-26b overexpressed H1299 cells could not induce expression of ATF2. When c-Jun N-terminal kinase activity was inhibited using SP600125, expression of miR-26b was induced following γ-irradiation in H1299 cells. From these results, we concluded that IR-induced up-regulation of ATF2 was coordinately enhanced by suppression of miR-26b in lung cancer cells, which may enhance the effect of IR in the MAPK signaling pathway.
doi:10.1371/journal.pone.0023802
PMCID: PMC3161999  PMID: 21901137
9.  Crystallization and preliminary X-ray crystallographic studies of the ice-binding protein from the Antarctic yeast Leucosporidium sp. AY30. Corrigendum 
A correction to the article by Park et al. [(2011). Acta Cryst. F67, 800–802].
A correction is made to the title of the article by Park et al. [(2011). Acta Cryst. F67, 800–802].
doi:10.1107/S1744309111027060
PMCID: PMC3151143
freezing; ice-binding proteins; cold-adaptation; Arctic yeast; corrigendum
10.  Crystallization and preliminary X-ray crystallographic studies of DesR, a thermosensing response regulator in a two-component signalling system from Streptococcus pneumoniae  
The response regulator DesR from S. pneumoniae was cloned, expressed, purified and crystallized. A complete data set was collected to 1.7 Å resolution.
The response regulator DesR, which activates the transcription of the des gene by binding to a regulatory region, is essential for controlling the fluidity of membrane phospholipids. DesR from Streptococcus pneumoniae was overexpressed in Escherichia coli. The protein was purified and crystallized for structural analysis. Diffraction data were collected to 1.7 Å resolution using synchrotron radiation and the crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 46.91, b = 71.38, c = 117.73 Å. Assuming the presence of a dimer in the asymmetric unit, this corresponds to a V M of 2.21 Å3 Da−1.
doi:10.1107/S1744309109023082
PMCID: PMC2705646  PMID: 19574651
two-component systems; fatty-acid desaturation; response regulators
11.  Crystallization and preliminary X-ray crystallographic studies of the ρ-class glutathione S-transferase from the Antarctic clam Laternula elliptica  
Monoclinic crystals of the ρ-class glutathione S-transferase from L. elliptica were obtained in both glutathione- and CDNB-complexed forms.
Glutathione S-transferases are involved in phase II detoxification processes and catalyze the nucleophilic attack of the tripeptide glutathione on a wide range of endobiotic and xenobiotic electrophilic substrates. The ρ-class glutathione S-­transferase from Laternula elliptica was overexpressed in Escherichia coli, purified and crystallized with two substrates: glutathione and 1-chloro-2,4-dinitrobenzene (CDNB). Diffraction data were collected to 2.20 Å resolution for the glutathione-complex crystals and to 2.00 Å resolution for the CDNB-complex crystals using a synchrotron-radiation source. Both crystals belonged to the C-centred monoclinic space group C2. The unit-cell parameters for the CDNB-complex crystals were a = 89.66, b = 59.27, c = 55.45 Å, β = 124.52°. The asymmetric unit contained one molecule, with a corresponding V M of 2.36 Å3 Da−1 and a solvent content of 47.8%.
doi:10.1107/S1744309108034003
PMCID: PMC2593695  PMID: 19052367
glutathione S-transferases; glutathione; CDNB; ρ class
12.  Polymorphisms in innate immunity genes and risk of childhood leukemia 
Human immunology  2010;71(7):727-730.
Objectives
To evaluate whether candidate genes in innate immunity are associated with childhood leukemia, we conducted an association study with the 1,536 SNPs in 203 genes related to innate immunity.
Methods
Incident childhood leukemia cases (n=136) aged from 0 to 18 were recruited from three teaching hospitals in Seoul between 2003 and 2006. Non-cancer controls (n=140) were frequency-matched to cases by age and gender. The information on the characteristics of children and their parents were collected by trained interviewers using structured questionnaire. Candidate genes were selected based on SNP databases (CGAP and SNP500 database), and genotype assay was performed using GoldenGate (Illumina) oligonucleotide pool assay (OPA). False discovery rate (FDR), permutation test, and haplotype analyses were used to identify the SNP with significant association with childhood leukemia. Childhood leukemia risk was estimated as ORs and 95% CIs adjusted for age, gender and birth weight.
Results
Fourteen SNPs in 13 genes (LMAN1, TLR4, STAT4, CCR9, MBP, ZP1, C8B, XDH, C7, C1QG, FGF2, LOC390183, and STAT6) were significantly associated with childhood leukemia risk (FDR p-values <0.05). In particular, LMAN1 rs1127220, TLR4 rs11536897, STAT4 rs13020076, CCR9 rs1471962, and MBP rs10514234 were significant in 5,000 permutation tests (Permutation p-value <0.05). The most significant association with childhood leukemia risk was for the LMAN1 rs1127220 that is in the protein-coding region, this finding was also supported by haplotype analysis.
Conclusions
A number of innate immunity related genes are associated with childhood leukemia, suggesting possible links between the innate immunity system and development of the childhood leukemia.
doi:10.1016/j.humimm.2010.04.004
PMCID: PMC2967770  PMID: 20438785
Childhood Leukemia; Innate Immunity; Single Nucleotide Polymorphism
13.  Crystallization and preliminary X-ray crystallographic studies of a PduO-type ATP:cob(I)alamin adenosyltransferase from Bacillus cereus  
Orthorhombic crystals of a PduO-type ATP:cob(I)alamin adenosyltransferase from B. cereus were obtained both as an apoenzyme and in the presence of Mg2+ and ATP.
Cobalamin adenosyltransferases transfer a 5′-deoxyadenosyl moiety from ATP and covalently attach it to the cobalt(I) ion of the corrin ring of cobalamin to generate adenosylcobalamin. The PduO-type adenosyltransferase from Bacillus cereus was overexpressed in Escherichia coli, purified and crystallized as the apoenzyme as well as in complex with Mg2+ and ATP (MgATP). Diffraction data were collected to 1.9 Å resolution for the native crystals and 2.0 Å resolution for the complexed crystals. Both crystals belonged to the orthorhombic space group C2221; the native crystals have unit-cell parameters a = 64.93, b = 137.08, c = 158.55 Å. The asymmetric unit contained one trimer, with a corresponding V M of 2.69 Å3 Da−1.
doi:10.1107/S1744309108016552
PMCID: PMC2443973  PMID: 18607099
adenosyltransferases; cobalamins; adenosylcobalamin; MgATP

Results 1-13 (13)