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1.
Comput Struct Biotechnol J ; 23: 1562-1571, 2024 Dec.
Article in English | MEDLINE | ID: mdl-38650588

ABSTRACT

Human leukocyte antigen (HLA) genes play pivotal roles in numerous immunological applications. Given the immense number of polymorphisms, achieving accurate high-throughput HLA typing remains challenging. This study aimed to harness the human pan-genome reference consortium (HPRC) resources as a potential benchmark for HLA reference materials. We meticulously annotated specific four field-resolution alleles for 11 HLA genes (HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4 and -DRB5) from 44 high-quality HPRC personal genome assemblies. For sequencing, we crafted HLA-specific probes and conducted capture-based targeted sequencing of the genomic DNA of the HPRC cohort, ensuring focused and comprehensive coverage of the HLA region of interest. We used publicly available short-read whole-genome sequencing (WGS) data from identical samples to offer a comparative perspective. To decipher the vast amount of sequencing data, we employed seven distinct software tools: OptiType, HLA-VBseq, HISAT genotype, SpecHLA, T1K, QzType, and DRAGEN. Each tool offers unique capabilities and algorithms for HLA genotyping, allowing comprehensive analysis and validation of the results. We then compared these results with benchmarks derived from personal genome assemblies. Our findings present a comprehensive four-field-resolution HLA allele annotation for 44 HPRC samples. Significantly, our innovative targeted next-generation sequencing (NGS) approach for HLA genes showed superior accuracy compared with conventional short-read WGS. An integrated analysis involving QzType, T1K, and DRAGEN was developed, achieving 100% accuracy for all 11 HLA genes. In conclusion, our study highlighted the combination of targeted short-read sequencing and astute computational analysis as a robust approach for HLA genotyping. Furthermore, the HPRC cohort has emerged as a valuable assembly-based reference in this realm.

2.
PLoS Negl Trop Dis ; 18(2): e0011928, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38315729

ABSTRACT

BACKGROUND: Although Japan is a major endemic area for human T-lymphotropic virus type 1 (HTLV-1) and the virus has been well-studied in this region, there is limited research on HTLV-1 in surrounding regions. In this study, we determined the complete genome sequences of HTLV-1 strains isolated from Taiwan and Japan and investigated the geographic characteristics of molecular subgroups and substitution mutations to understand the spread of HTLV-1 and its correlation with human migration. METHODOLOGY/PRINCIPAL FINDINGS: The complete genome sequences of 26 HTLV-1 isolates from Taiwan were determined using next-generation sequencing and were compared with those of 211 isolates from Japan in terms of subgroup and genetic mutations. In total, 15/26 (58%) isolates from Taiwan belonged to the transcontinental subgroup and 11/26 (42%) isolates belonged to the Japanese subgroup. The transcontinental subgroup was significantly more prevalent among Taiwanese isolates than Japanese isolates (58% vs 18%, P < 0.0001). The mutation rate for the complete HTLV-1 sequence was as low as 0.2%. On examining individual base substitutions, the G-to-A mutation was predominant. Bayesian phylogenetic tree analysis estimated the time to the most recent common ancestor for the transcontinental and Japanese subgroups to be 28447 years. The transcontinental subgroups from Taiwan and Japan appeared to form clusters according to their respective regions. CONCLUSIONS/SIGNIFICANCE: The transcontinental subgroup of HTLV-1 is predominant in Taiwan, while the Japanese subgroup is common in Japan. The difference in subgroup distribution may be attributed to the initial spread of the transcontinental subgroup in East Asia, followed by the influx of the Japanese subgroup.


Subject(s)
HTLV-I Infections , Human T-lymphotropic virus 1 , Humans , Japan/epidemiology , HTLV-I Infections/epidemiology , Taiwan/epidemiology , Phylogeny , Bayes Theorem , Sequence Analysis, DNA , Asia, Eastern/epidemiology , Whole Genome Sequencing
3.
J Vis Exp ; (182)2022 04 22.
Article in English | MEDLINE | ID: mdl-35532273

ABSTRACT

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.


Subject(s)
DNA Repair , Uracil-DNA Glycosidase , DNA/metabolism , Kinetics , Lasers , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Uracil/metabolism , Uracil-DNA Glycosidase/metabolism
4.
Cancer Lett ; 499: 290-300, 2021 02 28.
Article in English | MEDLINE | ID: mdl-33271263

ABSTRACT

Protocadherin 10 (PCDH10) is identified as a tumor suppressor in multiple cancers. The molecular mechanisms that mediate the functions of PCDH10 have yet to be fully elucidated. Here, we demonstrated that ectopic expression of PCDH10 in colorectal cancer (CRC) cells induced cell cycle retardation and increased apoptosis through regulation of the p53/p21/Rb axis and Bcl-2 expression. Overexpression of PCDH10 reversed the epithelial-mesenchymal transition (EMT) process with morphological changes and EMT marker alterations. Mechanistic study revealed that PCDH10 inhibited AKT/GSK3ß signaling pathway which in turn reduced ß-catenin activity and thus attenuated Snail and Twist1 expression. Furthermore, PCDH10 inhibited the stemness of CRC cells, including spheroid formation and stem cell markers. A proteomics approach revealed that PCDH10 could interact with EGFR, which was further verified by co-immunoprecipitation. Moreover, restoration of PCDH10 expression reduced EGFR phosphorylation. Accordingly, our work proposes a novel pathway by which PCDH10 directly engages in the negative regulation of EGFR/AKT/ß-catenin signaling pathway, resulting in tumor suppression.


Subject(s)
Cadherins/metabolism , Colorectal Neoplasms/pathology , Tumor Suppressor Proteins/metabolism , Apoptosis , Cadherins/genetics , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/genetics , Epithelial-Mesenchymal Transition , ErbB Receptors/metabolism , G1 Phase Cell Cycle Checkpoints , Gene Knockdown Techniques , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Protocadherins , Spheroids, Cellular , Tumor Suppressor Proteins/genetics , Wnt Signaling Pathway
5.
DNA Repair (Amst) ; 97: 103028, 2021 01.
Article in English | MEDLINE | ID: mdl-33254084

ABSTRACT

Uracil-DNA glycosylase (UDG) is a highly conserved DNA repair enzyme that acts as a key component in the base excision repair pathway to correct hydrolytic deamination of cytosine making it critical to genome integrity in living organisms. We report here a non-labeled, non-radio-isotopic and very specific method to measure UDG activity. Oligodeoxyribonucleotide duplex containing a site-specific G:U mismatch that is hydrolyzed by UDG then subjected to Matrix Assisted Laser Desorption/Ionization time-of-flight mass spectrometry analysis. A protocol was developed to maintain the AP product in DNA without strand break then the cleavage of uracil was identified by the mass change from uracil substrate to AP product. From UDG kinetic analysis, for G:U substrate the Km is 50 nM, Vmax is 0.98 nM/s and Kcat = 9.31 s-1. The method was applied to uracil glycosylase inhibitor measurement with an IC50 value of 7.6 pM. Single-stranded and double-stranded DNAs with uracil at various positions of the substrates were also tested for UDG activity albeit with different efficiencies. The simple, rapid, quantifiable, scalable and versatile method has potential to be the reference method for monofunctional glycosylase measurement, and can also be used as a tool for glycosylase inhibitors screening.


Subject(s)
DNA Repair , DNA, Bacterial/metabolism , Escherichia coli/enzymology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Uracil-DNA Glycosidase/metabolism , Uracil/analysis , DNA Damage , Escherichia coli/genetics , Kinetics , Uracil/metabolism
6.
Hepatology ; 69(4): 1412-1425, 2019 04.
Article in English | MEDLINE | ID: mdl-30382583

ABSTRACT

Smoking interacts with hepatitis B virus (HBV) to increase the risk of hepatocellular carcinoma (HCC), which might be explained by its role in antiviral immunity. We evaluated the potential mediating role of viral load and/or alanine aminotransferase (ALT) in the relation of smoking with HBV-associated HCC risk. Using multiple mediation analyses to analyze data from 209 HCC cases and 1,256 controls nested within a cohort of 4,841 male HBV carriers, we found that the effect of smoking on the risk of subsequent HCC was substantially mediated through viral load (percent mediated, 31.7%; P = 0.0054), and a significant mediation effect by both viral load and ALT was also evidenced. Among the 1,143 subjects with repeated measures of viral load and ALT over periods of up to 16 years, we further observed that a higher number of pack-years of smoking was associated with higher viral load, maintenance of a high viral load (>4.39 log copies/mL), more severe hepatotoxicity grade, and increased likelihood of ALT ≥80 U/L (odds ratio, 3.14; 95% confidence interval, 1.03-9.64; odds ratio, 6.06; 95% confidence interval, 1.10-33.25, respectively, for 10-19 and ≥20 pack-years versus nonsmokers) during follow-up. Furthermore, plasma interferon-γ levels were reduced in smokers compared with nonsmokers (interferon-γ-positive rate, 14.9% versus 28.7%; P < 0.0001) at baseline. Smoking was also associated with a reduced natural killer (NK) cell frequency in peripheral blood, characterized by reduced NK function through a systems immunology approach, after long-term follow-up in a subsample (n = 171). The combination of smoking and reduced NK cell frequency further increased viral load and the likelihood of ALT ≥80 U/L. Conclusion: The data highlight a role of smoking in HBV viral load, underlining the importance of smoking prevention and cessation in hepatitis B management.


Subject(s)
Alanine Transaminase/blood , Carcinoma, Hepatocellular/virology , Hepatitis B/complications , Liver Neoplasms/virology , Smoking/adverse effects , Adult , Carcinoma, Hepatocellular/blood , Case-Control Studies , Cohort Studies , Humans , Killer Cells, Natural , Liver Neoplasms/blood , Male , Middle Aged , Smoking/immunology , Viral Load
7.
J Vis Exp ; (136)2018 06 19.
Article in English | MEDLINE | ID: mdl-29985320

ABSTRACT

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.


Subject(s)
DNA Repair/genetics , DNA Replication/genetics , Nucleotides/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Humans
8.
DNA Repair (Amst) ; 64: 59-67, 2018 04.
Article in English | MEDLINE | ID: mdl-29522920

ABSTRACT

Deamination of adenine can occur spontaneously under physiological conditions to generate the highly mutagenic lesion, deoxyinosine (hypoxanthine deoxyribonucleotide, dI). In DNA, dI preferably pairs with cytosine rather than thymine and results in A:T to G:C transition mutations after DNA replication. The deamination of adenine is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. In Escherichia coli, dI repair is initiated by endonuclease V (endo V; nfi gene product) nicking but a complete repair mechanism has yet to be elucidated. Using in vitro minimum component reconstitution assays, we previously showed that endo V, DNA polymerase I (pol I), and E. coli DNA ligase were sufficient to repair this dI lesions efficiently and that the 3'-5' exonuclease of pol I is essential. Here we employed a phagemid-based T-I substrate mimicking adenine deamination product to demonstrate pol I proofreading exonuclease is required by the endo V repair pathway both in vitro and in vivo. In vivo we found that the repair level of an nfi mutant (11%) was almost 8-fold lower than the wild type (87%). while the polA-D424A strain, a pol I mutant defective in 3'-5' exonuclease, showed a high repair level similar to wild type (both more than 80%). Using additional C-C mismatch as strand discrimination marker we found that the high level of dI removal in polA-D424A was due to strand loss (more than 60%) associated with incomplete repair. Thus, pol I proofreading exonuclease is the major function responsible for dI lesion removal after endoV nicking both in vitro and in vivo. Finally, using MALDI-TOF to analyze single-nucleotide extension product we show that the pol I proofreading exonuclease excises only 2-nt 5' upstream of endo V incision site further honing the role of pol I in the endoV dI dependent repair pathway.


Subject(s)
DNA Damage , DNA Polymerase I/metabolism , DNA Repair , DNA-Directed DNA Polymerase/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Inosine/analogs & derivatives , DNA/metabolism , DNA Ligases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Inosine/metabolism
9.
DNA Repair (Amst) ; 61: 63-75, 2018 01.
Article in English | MEDLINE | ID: mdl-29223016

ABSTRACT

Proofreading and DNA repair are important factors in maintaining the high fidelity of genetic information during DNA replication. Herein, we designed a non-labeled and non-radio-isotopic simple method to measure proofreading. An oligonucleotide primer is annealed to a template DNA forming a mismatched site and is proofread by Klenow fragment of Escherichia coli DNA polymerase I (pol I) in the presence of all four dideoxyribonucleotide triphosphates. The proofreading excision products and re-synthesis products of single nucleotide extension are subjected to MALDI-TOF mass spectrometry (MS). The proofreading at the mismatched site is identified by the mass change of the primer. We examined proofreading of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus can be proofread efficiently. Internal single mismatches can also be proofread at different efficiencies, with the best correction for mismatches located 2-4-nucleotides from the primer terminus. For mismatches located 5-nucleotides from the primer terminus there was partial correction and extension. No significant proofreading was observed for mismatches located 6-9-nucleotides from the primer terminus. We also subjected primers containing 3' penultimate deoxyinosine (dI) lesions, which mimic endonuclease V nicked repair intermediates, to pol I repair assay. The results showed that T-I was a better substrate than G-I and A-I, however C-I was refractory to repair. The high resolution of MS results clearly demonstrated that all the penultimate T-I, G-I and A-I substrates had been excised last 2 dI-containing nucleotides by pol I before adding a correct ddNMP, however, pol I proofreading exonuclease tolerated the penultimate C-I mismatch allowing the primer to be extended by polymerase activity.


Subject(s)
DNA Repair , DNA Replication , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , DNA Polymerase I/metabolism , Templates, Genetic
10.
Oncotarget ; 7(51): 84938-84950, 2016 Dec 20.
Article in English | MEDLINE | ID: mdl-27793051

ABSTRACT

Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) are the first enzymes that mediate the initiation of heparan sulfate sulfation. We previously identified NDST4 as a putative tumor suppressor in human colorectal cancer. In the study, we generated an Ndst4 knockout (Ndst4-/-) mouse strain and explored its phenotypic characteristics, particularly in the development of colonic epithelial homeostasis. The Ndst4-deficient mice were viable and fertile, and their life spans were similar to those of wild-type littermates. No gross behavioral or morphological differences were observed between the Ndst4-/- and wild-type mice, and no significant changes were determined in the hematological or serum biochemical parameters of the Ndst4-/- mice. Ndst4 RNA transcripts were expressed in the brain, lung, gastrointestinal tract, pancreas, and ovary. However, Ndst4-null mice exhibited no gross or histological abnormalities in the studied organs, except for the colon. Although no alterations were observed in the crypt length or number of proliferating cells, the Ndst4-/- mice exhibited an increased number of goblet cells and a decreased number of colonocytes in the proximal colon compared with the wild-type mice. Moreover, Ndst4 deficiency increased the basal level of apoptosis in the colonic epithelium. Taken together, we established, for the first time, an Ndst4-/- mouse strain and revealed the involvement of Ndst4 in the development and homeostasis of colonic epithelium. Accordingly, NDST4 in human colon might direct the biosynthesis of specific heparan sulfate proteoglycans that are essential for the maintenance of colonic epithelial homeostasis. Thus, the loss of its function may result in the tumorigenesis and progression of colorectal cancer.


Subject(s)
Colon/pathology , Colorectal Neoplasms/metabolism , Epithelial Cells/physiology , Goblet Cells/physiology , Sulfotransferases/metabolism , Animals , Apoptosis , Carcinogenesis , Cells, Cultured , Colon/physiology , Colorectal Neoplasms/genetics , Heparan Sulfate Proteoglycans/metabolism , Homeostasis , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Sulfotransferases/genetics
11.
Cell Biosci ; 5: 52, 2015.
Article in English | MEDLINE | ID: mdl-26357532

ABSTRACT

BACKGROUND: Deamination of adenine can occur spontaneously under physiological conditions generating the highly mutagenic lesion, hypoxanthine. This process is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. Hypoxanthine in DNA can pair with cytosine which results in A:T to G:C transition mutations after DNA replication. In Escherichia coli, deoxyinosine (hypoxanthine deoxyribonucleotide, dI) is removed through an alternative excision repair pathway initiated by endonuclease V. However, the correction of dI in mammalian cells appears more complex and was not fully understood. RESULTS: All four possible dI-containing heteroduplex DNAs, including A-I, C-I, G-I, and T-I were introduced to repair reactions containing extracts from human cells. The repair reaction requires magnesium, dNTPs, and ATP as cofactors. We found G-I was the best substrate followed by T-I, A-I and C-I, respectively. Moreover, judging from the repair requirements and sensitivity to specific polymerase inhibitors, there were overlapping repair activities in processing of dI in DNA. Indeed, a hereditable non-polyposis colorectal cancer cell line (HCT116) demonstrated lower dI repair activity that was partially attributed to lack of mismatch repair. CONCLUSIONS: A plasmid-based convenient and non-radioisotopic method was created to study dI repair in human cells. Mutagenic dI lesions processed in vitro can be scored by restriction enzyme cleavage to evaluate the repair. The repair assay described in this study provides a good platform for further investigation of human repair pathways involved in dI processing and their biological significance in mutation prevention.

12.
Ann Surg Oncol ; 22 Suppl 3: S1481-9, 2015 Dec.
Article in English | MEDLINE | ID: mdl-25968618

ABSTRACT

BACKGROUND: Shisa3 is a novel tumor suppressor identified in lung cancer. However, its antitumor activity in other human cancers and the mechanism of gene inactivation remain unknown. METHODS: SHISA3 expression was measured by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). DNA methylation was determined by bisulfite sequencing and pyrosequencing. RESULTS: Down-regulation of SHISA3 expression was observed in all of 11 colorectal cancer (CRC) cell lines and was further confirmed in 34 (65.4 %) of 52 colorectal carcinomas by RT-qPCR. Four of six CRC cell lines could restore SHISA3 expression after treatment with 5-aza-2'-deoxycytidine. Tumor-specific methylation of five CpG sites in the first intron of SHISA3 was identified by bisulfite sequencing, and their methylation levels were quantified in 127 pairs of primary CRC tissues by bisulfite pyrosequencing. The methylation levels of SHISA3 in tumors were noticeably higher than that in their matched normal mucosae. In addition, SHISA3 hypermethylation was significantly associated with an increased risk of disease recurrence in patients with stage II and III disease (P = 0.007) and was an independent predictor of poor overall survival [hazard ratio (HR) 2.9, 95 % confidence interval (CI) 1.5-5.8; P = 0.002] and disease-free survival (HR 4.0, 95 % CI 1.6-10.2; P = 0.003) of CRC patients. CONCLUSIONS: SHISA3 gene is epigenetically inactivated in a substantial fraction of CRC, and its hypermethylation is of prognostic significance in predicting clinical outcome. The quantitative bisulfite pyrosequencing assay established could be a cost-effective tool for providing a potential biomarker of adverse prognosis in CRC.


Subject(s)
Adenocarcinoma/genetics , Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , DNA Methylation , Membrane Proteins/genetics , Neoplasm Recurrence, Local/genetics , Adenocarcinoma/secondary , Adult , Aged , Aged, 80 and over , Case-Control Studies , Colorectal Neoplasms/pathology , CpG Islands , Female , Follow-Up Studies , Humans , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Prognosis , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sulfites , Survival Rate , Tumor Cells, Cultured
13.
Am J Respir Crit Care Med ; 190(4): 433-44, 2014 Aug 15.
Article in English | MEDLINE | ID: mdl-25036006

ABSTRACT

RATIONALE: Despite advances in treatment and prognosis of non-small cell lung cancer (NSCLC), patient outcomes are still unsatisfactory. OBJECTIVES: To reduce the morbidity and mortality of patients with NSCLC, a more comprehensive understanding of mechanisms involved in cancer progression is urgently needed. METHODS: By comparison of gene expression profiles in the cell line pair with differential invasion ability, CL1-0 and CL1-5, we found that Shisa3 was highly expressed in the low invasive cells. The effect of Shisa3 on invasion, migration, proliferation, apoptosis, epithelial-mesenchymal transition, and anchorage-independent growth activities in vitro and on tumor growth and metastasis in mice models were examined. The underlying mechanism of Shisa3 was explored by microarray and pathway analysis. Finally, the correlation of Shisa3 expression and clinical outcome was also calculated. MEASUREMENTS AND MAIN RESULTS: We identified Shisa3 as a novel tumor suppressor, which induces ß-catenin degradation resulting in suppression of tumorigenesis and invasion in vitro. Shisa3 decreased the tumor growth in mice with subcutaneous implantation and reduced the number of metastatic nodules in mice with tail vein injection and orthotopic implantation. Shisa3 performs the tumor suppression activity through WNT signaling predicted by microarray analysis. Our data found that Shisa3 accelerates ß-catenin degradation and was positively associated with overall survival and progression-free survival of NSCLC. CONCLUSIONS: Our results reveal that Shisa3 acts as a tumor suppressor by acceleration of ß-catenin degradation and provide new insight for cancer prognosis and therapy.


Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Lung Neoplasms/metabolism , Membrane Proteins/metabolism , beta Catenin/metabolism , Aged , Animals , Apoptosis/genetics , Blotting, Western/methods , Carcinoma, Non-Small-Cell Lung/genetics , Cell Movement/genetics , Cell Proliferation , Cell Survival/genetics , Disease Models, Animal , Humans , In Vitro Techniques , Lung Neoplasms/genetics , Membrane Proteins/genetics , Mice , Mice, SCID , Microarray Analysis/methods , Polymerase Chain Reaction/methods , Signal Transduction/genetics , Taiwan , Tumor Cells, Cultured , beta Catenin/genetics
14.
Int J Cancer ; 135(11): 2593-603, 2014 Dec 01.
Article in English | MEDLINE | ID: mdl-24740680

ABSTRACT

Protocadherin 10 (PCDH10), a novel tumor suppressor gene in human cancers, is located in a common deleted region at chromosome 4q28 in colorectal cancer (CRC). This study aimed to ascertain the genetic loss of PCDH10 and its clinical relevance in CRC and to explore the tumor suppressor function of PCDH10. The genetic deletion of PCDH10 was determined in 171 pairs of primary tumors and corresponding normal mucosae by loss of heterozygosity study. In total, 53 carcinomas were positive for allelic loss of PCDH10. The genetic aberration was significantly associated with tumor progression and distant metastasis (p = 0.021 and p = 0.018, respectively) and was an independent predictor of poor survival for CRC patients (p = 0.005). Expression of PCDH10 gene was silenced or markedly down-regulated in all of 12 CRC cell lines tested and in 41 of 53 colorectal carcinomas compared with their matched normal mucosae. Ectopic expression of PCDH10 suppressed cancer cell proliferation, anchorage-independent growth, migration and invasion in vitro. Subcutaneous injection of PCDH10-expressing CRC cells into SCID mice revealed the reduction of tumor growth compared with that observed in mock-inoculated mice. Furthermore, through intrasplenic implantation, the re-expression of PCDH10 in silenced cells restrained liver metastasis and improved survival in SCID mice. In conclusion, PCDH10 is a pivotal tumor suppressor in CRC, and the loss of its function promotes not only tumor progression but also liver metastasis. In addition, the genetic deletion of PCDH10 represents an adverse prognostic marker for the survival of patients with CRC.


Subject(s)
Cadherins/genetics , Cell Movement , Cell Proliferation , Colorectal Neoplasms/prevention & control , Liver Neoplasms/prevention & control , Loss of Heterozygosity , Neoplasm Recurrence, Local/prevention & control , Animals , Apoptosis , Blotting, Western , Cadherins/metabolism , Cell Adhesion , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Female , Flow Cytometry , Follow-Up Studies , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Immunoenzyme Techniques , Liver Neoplasms/genetics , Liver Neoplasms/mortality , Liver Neoplasms/secondary , Male , Mice , Mice, SCID , Middle Aged , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Prognosis , Protocadherins , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate , Tumor Cells, Cultured
15.
DNA Repair (Amst) ; 12(11): 899-911, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24012058

ABSTRACT

Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3'-5' exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3'-penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3' penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3'-5' exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3'-penultimate deoxyinosines derived from endo V nicking reaction.


Subject(s)
DNA Mismatch Repair , DNA Polymerase I/metabolism , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , DNA Breaks, Single-Stranded , DNA Ligases/metabolism , Deamination , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Inosine/analogs & derivatives , Inosine/metabolism , Mutation , Substrate Specificity
16.
Article in English | MEDLINE | ID: mdl-23970932

ABSTRACT

Colorectal cancer (CRC) is a serious public health problem that results due to changes of diet and various environmental stress factors in the world. Curcumin is a traditional medicine used for treatment of a wide variety of tumors. However, antimetastasis mechanism of curcumin on CRC has not yet been completely investigated. Here, we explored the underlying molecular mechanisms of curcumin on metastasis of CRC cells in vitro and in vivo. Curcumin significantly inhibits cell migration, invasion, and colony formation in vitro and reduces tumor growth and liver metastasis in vivo. We found that curcumin suppresses Sp-1 transcriptional activity and Sp-1 regulated genes including ADEM10, calmodulin, EPHB2, HDAC4, and SEPP1 in CRC cells. Curcumin inhibits focal adhesion kinase (FAK) phosphorylation and enhances the expressions of several extracellular matrix components which play a critical role in invasion and metastasis. Curcumin reduces CD24 expression in a dose-dependent manner in CRC cells. Moreover, E-cadherin expression is upregulated by curcumin and serves as an inhibitor of EMT. These results suggest that curcumin executes its antimetastasis function through downregulation of Sp-1, FAK, and CD24 and by promoting E-cadherin expression in CRC cells.

17.
PLoS One ; 8(6): e67040, 2013.
Article in English | MEDLINE | ID: mdl-23825612

ABSTRACT

BACKGROUND: Genomic deletion at tumor suppressor loci is a common genetic aberration in human cancers. The study aimed to explore candidate tumor suppressor genes at chromosome 4q25-q28.2 and to delineate novel prognostic biomarkers associated with colorectal cancer (CRC). METHODS: Deletion mapping of chromosome 4q25-q28.2 was conducted in 114 sporadic CRC by loss of heterozygosity study with 11 microsatellite markers. A novel candidate tumor suppressor gene, namely NDST4, was identified at 4q26. Gene expression of NDST4 was investigated in 52 pairs of primary CRC tissues by quantitative reverse transcription-polymerase chain reaction. Allelic loss of NDST4 gene was further determined in 174 colorectal carcinomas by loss of heterozygosity analysis, and then was assessed for clinical relevance. RESULTS: One minimal deletion region was delineated between D4S2297 and D4S2303 loci at 4q26, where NDST4 was the only gene that had markedly been downregulated in CRC tumors. By laser capture microdissection, NDST4 RNA expression was demonstrated in colonic epithelial cells, but was undetectable in tumor cells. In total, 30 (57.7%) of 52 colorectal carcinomas showed a dramatic reduction in NDST4 gene expression compared with matched normal mucosae. The genetic loss of NDST4 was significantly associated with advanced pathological stage (P = 0.039) and poorer overall survival of patients (P = 0.036). CONCLUSIONS: NDST4 gene is a novel candidate tumor suppressor gene in human cancer, and the loss of its function might be involved in CRC progression. In addition, the loss of heterozygosity assay, which was established to determine the allelic loss of NDST4 gene, could be a cost-effective tool for providing a useful biomarker of adverse prognosis in CRC.


Subject(s)
Chromosomes, Human, Pair 4/genetics , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , Genes, Tumor Suppressor , Membrane Proteins/deficiency , Membrane Proteins/genetics , Sulfotransferases/deficiency , Sulfotransferases/genetics , Adult , Aged , Aged, 80 and over , Alleles , Colonic Polyps/genetics , Colorectal Neoplasms/pathology , Down-Regulation , Female , Gene Expression Regulation, Neoplastic/genetics , Genetic Loci/genetics , Humans , Intestinal Mucosa/metabolism , Male , Middle Aged , Neoplasm Staging , Prognosis
18.
Ann Surg Oncol ; 18(9): 2662-70, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21347784

ABSTRACT

PURPOSE: There is no detailed analysis of loss of heterozygosity (LOH) on chromosome 3 in colorectal cancer (CRC). Our aim was to define frequently deleted loci on chromosome 3 and to explore novel prognostic markers and the locations of candidate tumor suppressor genes associated with CRC. METHODS: LOH at 23 microsatellite markers spanning on chromosome 3 was determined in 112 sporadic CRC by automated fluorescence-based polymerase chain reaction. Genetic loss was assessed for the clinicopathological significance by univariate and multivariate analyses. RESULTS: Fifty-eight (51.8%) of 112 carcinomas exhibited LOH at one or more loci tested. Among seven loci with high LOH rates, allelic losses at D3S1297 and D3S1266 occurred more frequently in younger patients. A marked gender distortion for genetic deletion was observed at six loci, where LOH was identified more frequently in male cases. For clinical outcome, LOH solely at D3S1297 (3p26.3) was significantly associated with distant metastasis (P = 0.001) and was indicative of a shorter overall survival (P = 0.014). In addition, loss of one common deletion region at 3p25-pter was significantly correlated to distant metastasis (P = 0.009) and had an adverse effect on patients' overall survival in univariate and multivariate tests (P = 0.009 and 0.001, respectively). CONCLUSIONS: Loss of chromosome 3p25-pter could act as an independent predicator of poor prognosis in CRC, suggesting that microsatellite analysis is a useful means to stratify patients into different risk groups. In addition, inactivation of candidate tumor suppressor genes in this region might involve in CRC progression.


Subject(s)
Chromosomes, Human, Pair 3/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Liver Neoplasms/genetics , Liver Neoplasms/mortality , Loss of Heterozygosity , Sequence Deletion , Adult , Aged , Aged, 80 and over , Chromosome Mapping , Colorectal Neoplasms/pathology , DNA, Neoplasm/genetics , Female , Follow-Up Studies , Humans , In Situ Hybridization, Fluorescence , Liver Neoplasms/secondary , Male , Microsatellite Repeats , Middle Aged , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Polymerase Chain Reaction , Prognosis , Survival Rate
19.
DNA Repair (Amst) ; 9(10): 1073-9, 2010 Oct 05.
Article in English | MEDLINE | ID: mdl-20696623

ABSTRACT

Deoxyinosine (dI) in DNA can arise from hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the nfi gene product, in Escherichia coli. Repair was studied in vitro using M13mp18 derived heteroduplexes containing a site-specific deoxyinosine. Unpaired dI/G mismatch resides within the recognition site for XhoI restriction endonucleases, permitting evaluation of repair occurring on deoxyinosine-containing DNA strand. Our results show that dI lesions were efficiently repaired in nfi(+)E. coli extracts but the repair level was much reduced in nfi mutant extracts. We subjected the deoxyinosine-containing heteroduplex to a purified system consisting of soluble endonuclease V fusion protein, DNA polymerase I, and DNA ligase, along with the four deoxynucleoside triphosphates. Interestingly we found these three proteins alone are sufficient to process the dI lesion efficiently. We also found that the 3'-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay.


Subject(s)
DNA Mismatch Repair , DNA Repair , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Inosine/analogs & derivatives , DNA Ligases/metabolism , DNA Polymerase I/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Deamination , Escherichia coli/genetics , Escherichia coli/metabolism , Inosine/metabolism
20.
Clin Chem Lab Med ; 48(9): 1287-93, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20509834

ABSTRACT

BACKGROUND: Minor histocompatibility antigens influence the occurrence of graft-vs.-host disease and graft-vs.-leukemia effects after hematopoietic stem cell transplantation (HSCT). We determined the population frequencies of HA-1 and HA-2 alleles in Taiwan and exploited their potential applications in allogeneic HSCT. METHODS: HA-1 and HA-2 were genotyped using polymerase chain reaction and restriction fragment length polymorphism in healthy controls (221 for HA-1 and 306 for HA-2) and HLA-matched donor-recipient sibling pairs with HSCT (92 for HA-1 and 38 for HA-2). The association of genetic polymorphisms with HSCT outcome was evaluated by univariate and multivariate analyses. RESULTS: The allele frequencies in controls were 35.3% and 64.7% for HA-1(H) and HA-1(R), and 89.0% and 11.0% for HA-2(V) and HA-2(M), respectively. HA-1 disparity was denoted in 16.3% of HLA-matched donor-recipient sibling pairs, while it was not associated with HSCT outcome. HA-2 disparity was not observed in the donor-recipient pairs studied. The possibilities of using HA-1 and HA-2 variabilities as molecular markers for hematopoietic chimerism after HSCT were 39.2% and 18.4%, respectively. CONCLUSIONS: Our data provide the information on allele and genotype frequencies of HA-1 and HA-2 in a Taiwanese population, and suggest that prospective genomic typing for HA-1 and HA-2 alleles of the donor and recipient could be a useful approach for molecular identification of hematopoietic chimerism after HSCT, rather than prognosis of clinical outcome.


Subject(s)
Asian People/genetics , Gene Frequency , Hematopoietic Stem Cell Transplantation , Minor Histocompatibility Antigens/genetics , Polymorphism, Genetic , Adolescent , Adult , Aged , Case-Control Studies , Child , Child, Preschool , Female , Genotype , Graft Rejection/genetics , Humans , Infant , Male , Middle Aged , Multivariate Analysis , Recurrence , Siblings , Taiwan , Tissue Donors , Treatment Outcome , Young Adult
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