ABSTRACT
Peritoneal metastasis is one of the major patterns of unresectability in colorectal cancer (CRC) and a cause of death in advanced CRC. Identification of distinct gene expressions between primary CRC and peritoneal seeding metastasis is to predict the metastatic potential of primary human CRC. Three pairs of primary CRC (SNU-2335A, SNU-2404A, and SNU-2414A) and corresponding peritoneal seeding (SNU-2335D, SNU-2404B, and SNU-2414B) cell lines were established to determine the different gene expressions and resulting aberrated signaling pathways in peritoneal metastasis tumor using whole exome sequencing and microarray. Whole exome sequencing detected that mutation in CYP2A7 was exclusively shared in peritoneal seeding cell lines. Microarray identified that there were five upregulated genes (CNN3, SORBS1, BST2, EPSTI1, and KLHL5) and two downregulated genes (TRY6 and STYL5) in the peritoneal metastatic cell lines. CNN3 expression was highly augmented in both mRNA and protein levels in peritoneal metastasis cells. Knockdown of Calponin 3 resulted in augmented level of E-cadherin in peritoneal metastasis cells, and migration and invasiveness decreased accordingly. We suggest that CNN3 takes part in cell projection and movement, and the detection and distribution of CNN3 may render prognostic information for predicting peritoneal seeding metastasis from primary colorectal cancer.
ABSTRACT
BACKGROUND: Resistance to preoperative radiotherapy is a major clinical problem in the treatment for locally advanced rectal cancer. The role of NDRG1 in resistance to ionizing radiation in rectal cancer has not been fully elucidated. This study aimed to investigate the effect of the reduced intracellular NDRG1 expression on radio-sensitivity of human rectal cancer cells for exploring novel approaches for treatment of rectal cancer. METHODS: Three radio-resistant human rectal cancer cell lines (SNU-61R80Gy, SNU-283R80Gy, and SNU-503R80Gy) were established from human rectal cancer cell lines (SNU-61, SNU-283, and SNU-503) using total 80 Gy of fractionated irradiation. Microarray analysis was performed to identify differently expressed genes in newly established radio-resistant human rectal cancer cells compared to parental rectal cancer cells. RESULTS: A microarray analysis indicated the RNA expression of five genes (NDRG1, ERRFI1, H19, MPZL3, and UCA1) was highly increased in radio-resistant rectal cancer cell lines. Short hairpin RNA-mediated silencing of NDRG1 sensitized rectal cancer cell lines to clinically relevant doses of radiation by causing more DNA double strand breakages to rectal cancer cells when exposed to radiation. CONCLUSIONS: Targeting NDRG1 represents a promising strategy to increase response to radiotherapy in human rectal cancer.
Subject(s)
Cell Cycle Proteins/metabolism , Gene Expression Regulation, Neoplastic/radiation effects , Intracellular Signaling Peptides and Proteins/metabolism , Radiation Tolerance , Rectal Neoplasms/radiotherapy , Cell Cycle Proteins/genetics , Cell Line, Tumor , DNA Breaks, Double-Stranded/radiation effects , Dose Fractionation, Radiation , Down-Regulation , Gene Expression Profiling , Humans , Intracellular Signaling Peptides and Proteins/genetics , Oligonucleotide Array Sequence Analysis , RNA Interference , RNA, Small Interfering/metabolism , Radiation, Ionizing , Rectal Neoplasms/genetics , Treatment OutcomeABSTRACT
Clinical detection of ovarian clear cell carcinomas is important because of the poor prognosis. To identify microRNA profiles specific for clear cell carcinomas, microRNA expression profiles were compared between clear cell carcinomas and serous carcinomas of the ovary using microRNA microarray. In parallel, clear cell carcinomas were compared with germ cell tumors of the ovary. Six microRNAs differentially expressed between ovarian clear cell and serous carcinomas distinguished uterine clear cell carcinomas from endometrioid carcinomas. MiR-449 was underexpressed in both ovarian and uterine clear cell carcinomas. When germ cell tumors were compared with clear cell carcinomas of the ovary, miR-302d was the most significantly overexpressed microRNA in germ cell tumors. Thus, here we describe microRNA profiles characteristic for clear cell carcinomas of the ovary and uterus.
Subject(s)
Adenocarcinoma, Clear Cell/genetics , Biomarkers, Tumor/genetics , Carcinoma, Endometrioid/genetics , Cystadenocarcinoma, Serous/genetics , MicroRNAs/genetics , Neoplasms, Germ Cell and Embryonal/genetics , Ovarian Neoplasms/genetics , Adenocarcinoma, Clear Cell/pathology , Adenocarcinoma, Clear Cell/surgery , Adolescent , Adult , Aged , Carcinoma, Endometrioid/pathology , Carcinoma, Endometrioid/surgery , Child , Cystadenocarcinoma, Serous/pathology , Cystadenocarcinoma, Serous/surgery , Female , Follow-Up Studies , Humans , Middle Aged , Neoplasm Grading , Neoplasm Staging , Neoplasms, Germ Cell and Embryonal/pathology , Neoplasms, Germ Cell and Embryonal/surgery , Ovarian Neoplasms/pathology , Ovarian Neoplasms/surgery , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Young AdultABSTRACT
BACKGROUND/AIM: The molecular mechanism for aggressive clinical behaviour related to v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2) amplification is not fully-understood. In particular, little is known about microRNAs in the human epidermal growth factor receptor 2 (HER2) signaling network. PATIENTS AND METHODS: Using microRNA microarray, the microRNA profiles of 16 HER2-positive breast carcinomas were compared with those of five luminal-type breast carcinomas. Additionally, two frozen, ERBB2-amplified gastric carcinomas were compared with their adjacent normal tissue samples. MicroRNAs that were differentially expressed according to the HER2 status in breast and gastric carcinomas were identified as the HER2 microRNA signature. RESULTS: MiR-337 and miR-302f were commonly overexpressed in HER2-postive breast and gastric cancer. MiR-139 and miR-129 were commonly underexpressed in HER2-positive breast and gastric cancer. A concordant pattern of microRNA expression was noted between discovery sets and the majority of candidate microRNAs (two out of three) in three validation sets. CONCLUSION: Our study identified novel microRNAs that were differentially expressed according to the HER2 status across different tumor types.
Subject(s)
Breast Neoplasms/enzymology , Breast Neoplasms/genetics , MicroRNAs/genetics , Receptor, ErbB-2/genetics , Stomach Neoplasms/enzymology , Stomach Neoplasms/genetics , Adult , Female , Humans , Male , MicroRNAs/biosynthesis , Middle Aged , Principal Component Analysis , Receptor, ErbB-2/metabolism , Signal TransductionABSTRACT
BACKGROUND/AIM: Distinguishing between primary and metastatic adenocarcinomas in the lung may sometimes be difficult by conventional histopathological methods. In addition, novel biomarkers are needed for the more accurate subtyping of primary lung carcinomas. MATERIALS AND METHODS: MicroRNA microarrays were performed on 26 primary lung adenocarcinomas, 3 squamous cell carcinomas, 6 small cell lung cancers (SCLCs), and 2 colorectal cancer metastases in the lung. RESULTS: Forty-four microRNAs differentially expressed between three histological subtypes at p<10(-6) predicted histology with 100% accuracy in 100 randomly drawn datasets. Prominent among differentially expressed genes were miR-375, miR-217 and miR-216a, which were found overexpressed in SCLC compared to lung adenocarcinomas. Lung adenocarcinomas overexpressed miR-29b-1, miR-375, miR-2110, miR-29c-star, 199b-5p, and 146b-3p and underexpressed miR-617, miR-205-star, and miR-1246 compared to squamous cell carcinomas. In primary vs. metastatic lung adenocarcinomas, miR-552 and miR-592 were differentially expressed at p<10(-6); the level of expression of miR-552 in colorectal cancer metastases was 39-times higher and that of miR-592 was six-times higher. Furthermore, microRNA profiles of primary colorectal cancer in our database indicated that these two microRNAs were overexpressed in primary colorectal cancer relative to primary lung adenocarcinomas. CONCLUSION: MicroRNA profiles predict the histology of primary lung carcinomas, and differentiate between primary lung adenocarcinomas and colorectal cancer metastases.
Subject(s)
Adenocarcinoma/diagnosis , Adenocarcinoma/secondary , Biomarkers, Tumor/genetics , Colorectal Neoplasms/diagnosis , Lung Neoplasms/diagnosis , MicroRNAs/genetics , Adenocarcinoma/genetics , Aged , Colorectal Neoplasms/genetics , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/secondary , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Principal Component Analysis , TranscriptomeABSTRACT
To elucidate the molecular basis of early gastric cancer (EGC), the genome-wide expression pattern of cancer and normal tissues from 27 patients were analyzed by a microarray-based method. Using an integrative systematic bioinformatics approach, we classified the differentially expressed genes in EGC. Interestingly, the more highly expressed genes in EGC exhibited the most significant correlation with cell migration and metastasis. This implies that, even at the early stage of gastric cancer, the molecular properties usually observed in late-stage cancer are already present. Furthermore, we have found a novel association between the expression pattern and molecular pathways of EGC and estrogen receptor α (ERα)-negative breast cancer through cross-experimental analysis. These results provide new insights into the biological properties of EGC, as well as yielding useful basic data for the study of molecular mechanisms of EGC carcinogenesis.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Adult , Aged , Cluster Analysis , Computational Biology/methods , Female , Genome-Wide Association Study , Humans , Male , Matrix Metalloproteinases/metabolism , Middle Aged , Neoplasm Staging , Reproducibility of Results , Signal TransductionABSTRACT
OBJECTIVES: Many efforts have shown multi-oncologic roles of galectin-3 for cell proliferation, angiogenesis, and apoptosis. However, the mechanisms by which galectin-3 is involved in cell proliferation are not yet fully understood, especially in human colon cancer cells. METHODS: To cluster genes showing positively or negatively correlated expression with galectin-3, we employed human colon cancer cell lines, SNU-61, SNU-81, SNU-769B, SNU-C4 and SNU-C5 in high-throughput gene expression profiling. Gene and protein expression levels were determined by using real-time quantitative polymerase chain reaction (PCR) and western blot analysis, respectively. The proliferation rate of human colon cancer cells was measured by using a 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Expression of γ-aminobutyric acid B receptor 1 (GABABR1) showed a positive correlation with galectin-3 at both the transcriptional and the translational levels. Downregulation of galectin-3 decreased not only GABABR1 expression but also the proliferation rate of human colon cancer cells. However, Korean herbal extract, HangAmDan-B (HAD-B), decreased expression of GABABR1 without any expressional change of galectin-3, and offset γ-aminobutyric acid (GABA)-enhanced human colon cancer cell proliferation. CONCLUSIONS: Our present study confirmed that GABABR1 expression was regulated by galectin-3. HAD-B induced galectin-3-independent down-regulation of GABABR1, which resulted in a decreased proliferation of human colon cancer cells. The therapeutic effect of HAD-B for the treatment of human colon cancer needs to be further validated.
ABSTRACT
BACKGROUND: Non-Hodgkin lymphoma (NHL) is a hematologic malignancy for which good diagnostic markers are lacking. Despite continued improvement in our understanding of NHL, efforts to identify diagnostic markers have yielded dismal results. Here, we translated low-mass-ion information in urine samples from patients with NHL into a diagnostic marker. METHODS: To minimize experimental error, we tested variable parameters before MALDI-TOF analysis of low-mass ions in urine. Urine from 30 controls and 30 NHL patients was analyzed as a training set for NHL prediction. All individual peak areas were normalized to total area up to 1000 m/z. The training set analysis was repeated four times. Low-mass peaks that were not affected by changes in experimental conditions were collected using MarkerView software. Human Metabolome Database (HMDB) searches and ESI LC-MS/MS analyses were used to identify low-mass ions that exhibited differential patterns in control and NHL urines. Identified low-mass ions were validated in a blinded fashion in 95 controls and 66 NHL urines to determine their ability to discriminate NHL patients from controls. RESULTS: The 30 highest-ranking low-mass-ion peaks were selected from the 60-urine training set, and three low-mass-ion peaks with high intensity were selected for identification. Of these, a 137.08-m/z ion showed lower mass-peak intensity in urines of NHL patients, a result that was validated in a 161-urine blind validation set (95 controls and 66 NHL urines). The 130.08-m/z ion was identified from HMDB searches and ESI LC-MS/MS analyses as hypoxanthine (HX). The HX concentration in urines of NHL patients was significantly decreased (P < 0.001) and was correlated with the mass-peak area of the 137.08-m/z ion. At an HX concentration cutoff of 17.4 microM, sensitivity and specificity were 79.2% and 78.4%, respectively. CONCLUSIONS: The present study represents a good example of low-mass-ion profiling in the setting of disease screening using urine. This technique can be a powerful non-invasive diagnostic tool with high sensitivity and specificity for NHL screening. Furthermore, HX identified in the study may be a useful single urine marker for NHL screening.
Subject(s)
Hypoxanthine/urine , Lymphoma, Non-Hodgkin/diagnosis , Lymphoma, Non-Hodgkin/urine , Aged , Case-Control Studies , Chemistry, Clinical/methods , Female , Humans , Male , Metabolomics/methods , Middle Aged , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
Promoter hypermethylation of the ADAM23 gene, which is normally involved in cell-to-cell and cell-to matrix adhesion, has been reported in pancreatic, breast and brain cancer, and recently the role of this gene was examined in gastric cancer. In this study, we analyzed ADAM23 expression in colorectal cancer cell lines and examined its methylation by methylation-specific PCR (MSP) and bisulfate-modified DNA sequencing analysis. Methylated cells were treated with 5-aza-2'-deoxycytidine to restore the ADAM23 expression. We then examined ADAM23 methylation status in colorectal cancer tissues and their corresponding normal tissues. We found that ADAM23 was aberrantly silenced or expressed at very low levels in 28 of the 32 (88%) colorectal cancer cell lines. MSP analysis showed that ADAM23 was methylated in 29 of 32 (91%) colorectal cancer cell lines and attenuated expression of ADAM23 was found to be related to hypermethylation in its promoter region. Moreover, the CpG dinucleotide methylation threshold of 70-90% was found to be required for complete silencing. In addition, when some cell lines without ADAM23 expression were treated with 5-aza-2'-deoxycytidine, ADAM23 was reexpressed. In colorectal cancer tissues, the promoter region of ADAM23 was hypermethylated in 36 of 76 (47%). These results demonstrated that ADAM23 may be down-regulated by aberrant promoter hypermethylation during the progression of colorectal cancer.
Subject(s)
ADAM Proteins/genetics , Colorectal Neoplasms/genetics , Promoter Regions, Genetic , Base Sequence , Cell Line, Tumor , DNA Methylation , DNA Primers , DNA, Neoplasm/genetics , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Stomach Neoplasms/geneticsABSTRACT
BACKGROUND: Elucidating the molecular mechanisms by which tumors become resistant to Herceptin is critical for the treatment of Her2-overexpressed metastatic breast cancer. METHODS: To further understand Herceptin resistance mechanisms at the molecular level, we used comparative proteome approaches to analyze two human breast cancer cell lines; Her2-positive SK-BR-3 cells and its Herceptin-resistant SK-BR-3 (SK-BR-3 HR) cells. RESULTS: Heat-shock protein 27 (HSP27) expression was shown to be upregulated in SK-BR-3 HR cells. Suppression of HSP27 by specific siRNA transfection increased the susceptibility of SK-BR-3 HR cells to Herceptin. In the presence of Herceptin, Her2 was downregulated in both cell lines. However, Her2 expression was reduced by a greater amount in SK-BR-3 parent cells than in SK-BR-3 HR cells. Interestingly, co-immunoprecipitation analysis showed that HSP27 can bind to Her2. In the absence of Herceptin, HSP27 expression is suppressed and Her2 expression is reduced, indicating that downregulation of Her2 by Herceptin can be obstructed by the formation of a Her2-HSP27 complex. CONCLUSION: Our present study demonstrates that upregulated HSP27 in human breast cancer cells can reduce Herceptin susceptibility by increasing Her2 protein stability.
Subject(s)
Antibodies, Monoclonal/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Heat-Shock Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Receptor, ErbB-2/metabolism , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm , Enzyme Stability , HSP27 Heat-Shock Proteins , Heat-Shock Proteins/genetics , Humans , Molecular Chaperones , Neoplasm Metastasis , Neoplasm Proteins/genetics , Trastuzumab , Up-RegulationABSTRACT
The adenomatous polyposis coli (APC), which is the susceptible gene for familial adenomatous polyposis (FAP) and sporadic colorectal cancer, spans 15 exons. The open reading frame of APC is 8529 bp, which encodes 2843 amino acids. Conventional genetic screening involves extensive time as well as high cost and labor. Thus, we developed a novel APC ready-to-use plate for high-throughput mutational analysis by denaturing high performance liquid chromatography (DHPLC). To prepare the ready-to-use APC plate, all 38 primer pairs and PCR mixtures were aliquoted into individual wells of a 96-well plate, and frozen at -20 degrees C until use. All 38 PCR primers were designed to be amplified at the same temperature (52 degrees C). We examined a total of 27 FAP patient samples with APC germline mutations (17 for multiple bp deletions, 1 for 1 bp deletion, 9 for nonsense mutations) and 50 APC-negative noncarriers. All 17 multiple bp deletion mutations were detected during the initial 50 degrees C running analysis and thus ruled out for further analyses. All other mutations were clearly detected under specific optimized conditions. More than 50% of the APC germline mutations were multiple base pair deletions and efficiently selected by omitting time-consuming partial denaturing conditions.
Subject(s)
Adenomatous Polyposis Coli Protein/genetics , Adenomatous Polyposis Coli/genetics , Base Pairing/genetics , Chromatography, High Pressure Liquid/methods , Gene Deletion , Mutation , DNA Mutational Analysis , Exons , Genetic Testing/methods , HumansABSTRACT
CHFR was recently identified as an early mitotic checkpoint that delays transition to metaphase in response to mitotic stress. Although studies have shown that CHFR is relevant to tumorigenesis, no previous report has investigated whether polymorphisms in the CHFR gene are associated with the risk of cancer development. Here, we genotyped polymorphisms in the CHFR gene and analyzed the possible associations of single polymorphisms and haplotypes with the risk and clinicopathological characteristics of colorectal cancer. Six coding SNPs in the CHFR gene were genotyped in 462 colorectal cancer patients and 245 healthy normal controls, using either the TaqMan assay or direct sequencing. Our results revealed that the V539M polymorphism was significantly associated with a lower risk of colorectal cancer (P=0.03; OR, 0.533; 95% CI, 0.302-0.94), and significantly correlated with no distant metastasis (M0 stage), different TNM stage, and microsatellite instability (MSI) among the colorectal cancer patients. Among the five tested haplotypes, hap 10 (TGACTA) was significantly associated with a lower risk of colorectal cancer (P=0.017; OR, 0.496; 95% CI, 0.279-0.883), and colorectal cancer patients carrying this haplotype showed no distant metastasis, different TNM stage, and microsatellite instability at a significantly higher frequency. These results reveal for the first time that polymorphisms in the CHFR gene are associated with colorectal cancer susceptibility.
Subject(s)
Cell Cycle Proteins/genetics , Colorectal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Mitosis/genetics , Neoplasm Proteins/genetics , Polymorphism, Single Nucleotide , Adult , Aged , Case-Control Studies , Colorectal Neoplasms/pathology , Genetic Predisposition to Disease , Haplotypes , Humans , Microsatellite Instability , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , Odds Ratio , Phenotype , Poly-ADP-Ribose Binding Proteins , Risk Assessment , Risk Factors , Ubiquitin-Protein LigasesABSTRACT
BACKGROUND: Little is known about the genetic risk factors associated with colorectal cancer. Although the Ser326Cys polymorphism of 8-oxoguanine DNA glycosylase (hOGG1) is consistently associated with a range of cancers, there is no consensus regarding this polymorphism and colorectal cancer risk. METHODS: In the present study, conducted in a Korean population, we used the TaqMan assay to investigate whether the hOGG1 Ser326Cys polymorphism was associated with colorectal cancer in 439 colorectal cancer patients and 676 healthy normal controls. We also examined whether the hOGG1 Ser326Cys polymorphism is associated with tumor location, microsatellite instability (MSI) status and tumor-node-metastasis (TNM) stage in colorectal cancer. RESULTS: We found no significant difference between the cancer and control populations in terms of genotype distribution (CC, CG and GG). In addition, we found no association between the hOGG1 Ser326Cys polymorphism and cancer risk, MSI status, TNM stage or tumor location in colorectal cancer patients. CONCLUSIONS: These results suggest that unlike for other cancer types, the hOGG1 Ser326Cys polymorphism is not a major genetic risk factor for colorectal cancer.
Subject(s)
Colorectal Neoplasms/genetics , DNA Glycosylases/genetics , Colorectal Neoplasms/pathology , Female , Genotype , Humans , Korea/epidemiology , Male , Middle Aged , Neoplasm Metastasis , Polymorphism, Single Nucleotide , Risk FactorsABSTRACT
PURPOSE: Preoperative chemoradiotherapy is widely used to improve local control and sphincter preservation in patients with locally advanced rectal cancer. In the present study, we investigated whether microarray gene expression analysis could predict complete response to preoperative chemoradiotherapy in rectal cancer. METHODS: Tumor tissues were obtained from 46 patients with rectal cancer (31 for training and 15 for validation testing). All patients underwent preoperative chemoradiotherapy involving 50.4 gray radiotherapy, followed by surgical excision 6 weeks later. Response to chemoradiotherapy was evaluated according to Dworak's tumor regression grade. Tumor regression Grades 1, 2, and 3 were considered partial responses, and tumor regression Grade 4 was considered a complete response. By using the 31 training samples, genes differentially expressed between partial response and complete response were identified, and clustering analysis was performed. Prediction analysis of response to chemoradiotherapy was performed on the 31 training samples by using a selected set of 95 "predictor" genes. Those findings were validated by independent analysis of the 15 test samples. RESULTS: The 31 training samples comprised 20 partial response and 11 complete response cases. A primary set of 261 genes was identified as differentiating between partial response and complete response. By supervised clustering using these 261 genes, 30 of 31 training samples were clustered correctly according to tumor response. A gene set comprising the top-ranked 95 genes displaying differential expression between partial response and complete response was applied to predict response to chemoradiotherapy. Complete response and partial response were accurately predicted in 84 percent (26/31) of training samples and 87 percent (13/15) of validation samples. CONCLUSIONS: Microarray gene expression analysis was successfully used to predict complete responses to preoperative chemoradiotherapy in patients with advanced rectal cancer.
Subject(s)
DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Oligonucleotide Array Sequence Analysis/methods , Rectal Neoplasms/therapy , Adult , Aged , Colectomy , Female , Follow-Up Studies , Gene Expression Profiling , Humans , Male , Middle Aged , Predictive Value of Tests , Rectal Neoplasms/drug therapy , Rectal Neoplasms/radiotherapy , Rectal Neoplasms/surgery , Reproducibility of Results , Treatment OutcomeABSTRACT
BACKGROUND: Most investigations on MutY human homolog (MYH)-associated polyposis (MAP) have been conducted in Western countries. Limited data on MAP in Asia are currently available. The present study investigated germline mutations of the MYH gene among patients with 10 to 99 adenomatous colorectal polyps and familial adenomatous polyposis (FAP) without adenomatous polyposis coli (APC) germline mutations in Korea. MATERIALS AND METHODS: The study population included 46 patients with 10 to 99 adenomatous polyps in the colorectum and 16 FAP patients with no identified APC germline mutations. Subjects were screened for MYH germline mutations, and we additionally screened for MYH mutations in 96 normal control individuals. RESULTS: Two of 46 (4.3%) patients with multiple polyps displayed heterozygous biallelic germline mutations of the MYH gene. A 39-year-old male patient with biallelic MYH mutations (p.G272E and p.A359V) received total proctocolectomy for rectal cancer and 36 colorectal polyps. A 58-year-old female patient with biallelic MYH mutations (p.Q253X and p.Q440P) received right hemicolectomy for ascending colon cancer and 16 colonic polyps. The frequency of biallelic MYH mutation in 14 of 46 multiple-polyp patients, who had 15 to 99 polyps, was 14.3% (2 of 14). No biallelic MYH mutations were detected in the 32 patients with 10 to 14 colorectal polyps, 16 FAP patients, or 96 normal controls. CONCLUSION: We identified biallelic MYH germline mutations in 2 of 14 (14.3%) Korean patients with 15 to 99 colorectal polyps. In this study, there was no Y165C or G382D hot-spot mutation, which had been reported most frequently in previous studies.
Subject(s)
Adenomatous Polyposis Coli/genetics , Colonic Polyps/genetics , DNA Glycosylases/genetics , Mutation/genetics , Adult , Aged , Aged, 80 and over , Asian People , Base Sequence , Female , Genetic Predisposition to Disease/genetics , Humans , Male , Middle AgedSubject(s)
Colorectal Neoplasms/genetics , Dyneins/genetics , Mutation , Signal Transduction/genetics , Stomach Neoplasms/genetics , Transforming Growth Factor beta/genetics , Cell Line, Tumor , Colorectal Neoplasms/enzymology , Cytoplasmic Dyneins , Dyneins/physiology , Humans , Stomach Neoplasms/enzymology , Transforming Growth Factor beta/physiologyABSTRACT
We herein describe the development of a sensitive microarray hybridization method called competitive DNA hybridization (CDH) and its use for analysis of BRAF somatic mutations. These mutations have been identified in many human cancers, and fast, reliable BRAF mutation detection may one day facilitate directed therapy of BRAF-mutated tumors. Our fast, reliable mutation detection by CDH is based on the principle that competition among multiple fluorescent-labeled samples for binding to shared wild-type sequences should reduce nonspecific results and increase the positive signals of unshared mutated sequences. The positive signals can then be discriminated based on the labeling of each sample (ie, with Cy3, Cy5, or Alexa-594). For testing of this method, we developed a BRAF oligonucleotide microarray containing 65 mutation types (more than 95% of the known BRAF mutations) and validated this microarray with 20 colorectal cancer tissues/cancer cell lines with BRAF mutations and 60 BRAF-negative samples. In sum, we were able to screen up to nine cancer samples on a single BRAF microarray (three per CDH on three regions per slide), indicating that this method may dramatically decrease the experimental time, cost, and effort of mutation detection in BRAF and other genes amenable to microarray analysis.
Subject(s)
Colorectal Neoplasms/genetics , Molecular Diagnostic Techniques/methods , Mutation/genetics , Oligonucleotide Array Sequence Analysis/methods , Proto-Oncogene Proteins B-raf/genetics , Humans , Nucleic Acid Hybridization/methods , Oligonucleotides/genetics , Sequence Analysis, DNAABSTRACT
BACKGROUND: Glutathione S-transferases are a group of enzymes that participate in detoxification and defense mechanisms against toxic carcinogens and other compounds. These enzymes play an important role in human carcinogenesis. In the present study, we sought to determine whether GSTT2 promoter single nucleotide polymorphisms (SNPs) are associated with colorectal cancer risk. METHODS: A total of 436 colorectal cancer patients and 568 healthy controls were genotyped for three GSTT2 promoter SNPs (-537G>A, -277T>C and -158G>A), using real-time TaqMan assay and direct sequencing. An electrophoretic mobility shift assay (EMSA) was performed to determine the effects of polymorphisms on protein binding to the GSTT2 promoter. RESULTS: The -537A allele (-537G/A or A/A) was significantly associated with colorectal cancer risk (OR = 1.373, p = 0.025), while the -158A allele (-158G/A or A/A) was involved in protection against colorectal cancer (OR = 0.539, p = 0.032). Haplotype 2 (-537A, -277T, -158G) was significantly associated with colorectal cancer risk (OR = 1.386, p = 0.021), while haplotype 4 (-537G, -277C, -158A) protected against colorectal cancer (OR = 0.539, p = 0.032). EMSA data revealed lower promoter binding activity in the -537A allele than its -537G counterpart. CONCLUSION: Our results collectively suggest that SNPs and haplotypes of the GSTT2 promoter region are associated with colorectal cancer risk in the Korean population.
Subject(s)
Colorectal Neoplasms/genetics , Genetic Predisposition to Disease/genetics , Glutathione Transferase/genetics , Polymorphism, Genetic/genetics , Promoter Regions, Genetic/genetics , Genotype , HeLa Cells , HumansABSTRACT
MDK is a heparin-binding growth factor associated with cancer development. Here, we sought to examine the association of MDK expression with resistance and sensitivity to different chemotherapeutic agents. We established stable HeLa cell transfectants (HeLa-MDK) and tested for decreased sensitivity to chemotherapeutic agents (5-FU, doxorubicin, and cisplatin). In addition, we used siRNA to block MDK expression in SNU-638 human gastric cancer cells and examined the chemosensitizing effect. HeLa-MDK cells treated with 5-FU, doxorubicin, and cisplatin showed a fold increase in the average IC(50) and an increased cell survival. siRNA-based knockdown of MDK expression in SNU-638 cells decreased the average IC(50) by 18-44% in cells treated with three drugs. Further investigations on the molecular mechanism should be clarified, but these results indicate that MDK up- and down-regulation appears to be capable of changing the chemosensitivities of cancer cells and MDK may have possible importance as a candidate therapeutic molecule.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Neoplasms/drug therapy , Nerve Growth Factors/physiology , Cell Line, Tumor , Cisplatin/pharmacology , Doxorubicin/pharmacology , Drug Resistance, Multiple , Fluorouracil/pharmacology , Humans , Midkine , Neoplasms/metabolism , RNA, Small Interfering/pharmacology , TransfectionABSTRACT
Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p = 0.033) and distal CRC (p = 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC.