OBJECTIVE: To characterize the mutational profile of patients with core-binding factor acute myeloid leukemia (CBF-AML). METHODS: A total of 81 acute myeloid leukemia patients were recruited, which included 36 cases of CBF-AML and 45 cases of cytogenetically normal acute myeloid leukemia (CN-AML) . Mutations of FLT3-ITD, FLT3-TKD, NPM1, c-KIT, NRAS, KRAS, TET2, IDH1/2, RUNX1, DNMT3A, GATA2, ASjXL1, TP53, PTPN11, JAK2V617F, SETBP1 and CEBPA genes were simultaneously detected by DNA-based PCR and Sanger sequencing. RESULTS: Over all, mutations were detected in 68 patients (83.9%), with the most common ones including double CEBPA mutations (n=17), followed by NPM1 (n=15), c-KIT (n=11), NRAS (n=10), TET2 (n=9), FLT3-TKD (n=9), FLT3-ITD (n=8), IDH1 (n=7), RUNX1 (n=7), KRAS (n=7), DNMT3A (n=6), IDH2 (n=4), and GATA2 (n=4) mutations. AML1-ETO and CBFβ-MYH11 fusions were present in 21 and 15 patients, respectively. Coexistence of ≥2 mutations was more common in CN-AML comparing with CBF-AML. The mutation rate of NPM1, FLT3-ITD, DNMT3A, IDH1 and CEBPA double mutations were higher in patients with CN-AML. NRAS, c-KIT and KRAS mutations were identified more frequently in patients with CBF-AML (P<0.05). Based on the function, aberration of genes involved in DNA methylation, NPM1 proteins and transcription predominated in CN-AML, while tyrosine kinase receptor signaling and RAS pathways have predominated in CBF-AML. CONCLUSION The genomic landscape of CBF-AML patients has differed from that of CN-AML patients. Synergy of fusion genes with particular mutations may impact the clinical phenotype and prognosis of patients.
Core Binding Factors ; genetics ; DNA Mutational Analysis ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation ; Prognosis

OBJECTIVEIn order to elucidate role of RUNX3 gene in hepatocarcinogenesis, we detected genetic and epigenetic alteration of RUNX3 gene in hepatocellular carcinoma (HCC).

METHODSPCR-SSCP, analysis of loss of heterozygosity (LOH), sequencing and methylation-specific PCR (MSP) were used to detect mutation, LOH and DNA methylation of RUNX3 gene in 90 HCCs.

RESULTSNo mutation was found, but three single-nucleotide polymorphisms (SNP) were found and distributed over exon1 and exon4. 30.6% (11/36) of cases showed LOH; 54.4% (49/90) of cases was in hypermethylation. There is a significant correlation between LOH and major portal vein invasive or micro vessel invasion or intrahepatic metastasis.

CONCLUSIONHigh frequent hypermethylation and LOH of RUNX3 gene were found in HCC. Aberrant RUNX3 gene may play an important role in the development of HCC.

Carcinoma, Hepatocellular ; genetics ; Core Binding Factor Alpha 3 Subunit ; DNA Methylation ; DNA-Binding Proteins ; genetics ; Female ; Humans ; Liver Neoplasms ; genetics ; Loss of Heterozygosity ; Male ; Middle Aged ; Transcription Factors ; genetics

OBJECTIVETo investigate the effects of AML1B and AML1/ETO fusion gene on the transcription activity of TSC1 and TSC2 promotor and to explore its role in leukemogenesis.

METHODSThe luciferase reporter plasmids of TSCs gene promoter containing a AML1 binding site were constructed, and cotransfected into CV-1 cells with AML1B or AML1/ETO expression plasmids separately. The transactivity of TSCs gene promoter was assayed by luminometer.

RESULTSAML1B exhibited a transactivity to TSC2 gene promoter in a dosage-dependant manner, but showed no significant transactivity to TSC1's. The transactivity to TSC2 gene promoter showed 8.55 fold increase as companed with control group at 75 ng of pCMV5-AML/B. AML1/ETO showed a significant transactivity to TSC1, but no transactivity to TSC2's. However, AML1/ETO antagonized the effect of AMLlB to TSC2 gene promoter.

CONCLUSIONAML1B and AML1/ETO can regulate the transcription of TSC genes.

Binding Sites ; Core Binding Factor Alpha 2 Subunit ; genetics ; Humans ; Oncogene Proteins, Fusion ; genetics ; Plasmids ; Promoter Regions, Genetic ; Transcription Factors ; genetics ; Transcriptional Activation

OBJECTIVETo investigate the osteoblast-like characteristics of human periodontal ligament cells affected by elastic stress in vitro, and the role of corebinding factor a 1 (cbfa1) in alveolar bone formation during orthodontic tooth movement.

METHODSRat dig-labeled cbfa1 cDNA probe was prepared from SD rat osteoblasts cultured in vitro. Human periodontal ligament cells were cultured on the elastic bottom plate and stimulated by elastic stress using mechanical loading system for cultured cells in vitro. The expression of cbfa1 mRNA was detected by in situ hybridization method.

RESULTSCbfa1 mRNA express in human periodontal ligament cells stimulated by elastic stress and did not express in normal human periodontal ligament cells.

CONCLUSIONIt is suggested that elastic stress plays a role in the differentiation process from human periodontal ligament cells to osteoblast-like cells. Cbfa1 is a transcription factor in alveolar bone remodeling during orthodontic tooth movement.

Adolescent ; Animals ; Cells, Cultured ; Child ; Core Binding Factor alpha Subunits ; DNA-Binding Proteins ; genetics ; Elasticity ; Humans ; Periodontal Ligament ; cytology ; metabolism ; RNA, Messenger ; analysis ; Rats ; Transcription Factors ; genetics

OBJECTIVE: To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. METHODS: The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. RESULTS: We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. CONCLUSION pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.
Core Binding Factor Alpha 1 Subunit/genetics* ; Humans ; Lipopolysaccharides/pharmacology* ; Luciferases/genetics* ; Metformin/pharmacology* ; NFATC Transcription Factors/genetics* ; Promoter Regions, Genetic ; T-Lymphocytes ; Transcription, Genetic/drug effects* ; Transfection

The purpose of this study was to evaluate the clinical value of interphase fluorescence in situ hybridization (I-FISH) in diagnosis of core-binding factor acute myelocytic leukemia (CBF AML). The cytogenetic characteristics in leukemia cells from 82 cases of AML-M(2) and 43 cases of AML-M(4)/M(5) were detected by using I-FISH with AML1-ETO double color double fusion probe and double color break point isolated gene probe CBFβ-MYH11, and the detected results were compared with results detected by conventional cytogenetic R banding technique (CC). The results indicated that AML1-ETO fusion gene was detected in 30.5% cases (25/82) by FISH, and t(8;21)(q22;q22) karyotypic aberrations was found in 28.0% cases (23/82) by CC method. Among 25 FISH positive cases, typical FISH positive signal pattern (1R1G2F) was displayed in 22 cases and atypical signal pattern (1R2G1F and 2R1G2F) was found in the other 3 cases. Among all 43 AML-M(4)/M(5) cases, the CBFβ-MYH11 fusion gene was detected in 23.3% cases (10/43) by FISH, which sensitivity was significant higher than that by CC method (2/43) (p < 0.05). It is concluded that some insufficiency of CC technique can be compensated by FISH, and combination of I-FISH with CC technique play a crucial role in diagnosis of CBF AML and in monitoring of minimal residual disease.
Adolescent ; Adult ; Child ; Core Binding Factors ; genetics ; Cytogenetic Analysis ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotyping ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Male ; Middle Aged ; Young Adult

OBJECTIVETo set up a method of analyzing gene expression profile from mouse whole embryos.

METHODSMouse whole mount RNA in situ hybridization(WM-ISH) of E10.5-E14 embryos was carried out by using digoxigenin-labeled Runx1 and Runx3 RNA probes and their expression profile was observed by detecting the existence and status of corresponding mRNAs in the embryonic tissues.

RESULTSClear hybridization signals were observed in different tissues and organs hybridized by Runx1 or Runx3 RNA probe. Different probes and ages of embryos had need of their own optimal proteinase K treatment conditions.

CONCLUSIONMouse whole mount RNA in situ hybridization is an effective method of analyzing gene expression. It is useful for revealing whole gene expression profile and has a great potentiality in the era of functional genomics. It provides an alternative method of studies on gene expression which is at least as good as LacZ staining and immunohistochemistry. The key factor of the success to mouse whole mount RNA in situ hybridization is whether the proteinase K treatment conditions are optimal or not.

Animals ; Core Binding Factor Alpha 2 Subunit ; Core Binding Factor Alpha 3 Subunit ; DNA-Binding Proteins ; genetics ; Embryo, Mammalian ; metabolism ; Gene Expression Profiling ; methods ; Gene Expression Regulation, Developmental ; In Situ Hybridization ; methods ; Mice ; Proto-Oncogene Proteins ; genetics ; RNA ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Sensitivity and Specificity ; Transcription Factors ; genetics

OBJECTIVETo discuss the clinical and cytogenetic features of core binding factor (CBF) acute myeloid leukemia (AML) patients and the main factors that influence the prognosis.

METHODTotally 130 CBF AML patients were followed up and their clinical features, immunophenotype, chromosome karyotype, treatment regimen, overall survival (OS), and relapse-free survival (RFS) were analyzed.

RESULTSThe overall complete remission (CR) rate was 96.1%, among which the CR rate after the first treatment course was 77.2%. The overall median OS was 51.64 (0.26-132.5) months, while the median RFS did not reach 1.18-96.62 months. The 3-year OS was 50% and the 5-year OS was 41%; the 3-year RFS was 59% and the 5-year RFS was 54%. Patients who were over 45 years and those with chromosome karyotype of 9q- tended to have poorer prognosis. During the consolidating chemotherapy, patients who had received two or more courses of intermediate-dose Ara-C therapy had better prognosis and longer survival. AML patients with inv (16) /t (16; 16) had a significantly higher OS than those with t (8; 21) (P = 0.046), while the RFS showed an opposite finding (P = 0.038).

CONCLUSIONSAge, chromosomal karyotype, and consolidating chemotherapy are the main factors that influence the survival and prognosis of CBF AML patients. Two or more courses of intermediate-dose Ara-C during consolidating chemotherapy can obviously prolong the OS and RFS of CBF AML patients. AML patients with a chromosomal karyotype of inv (16) /t (16; 16) have longer OS and better prognosis than those with t (8; 21).

Adolescent ; Adult ; Aged ; Core Binding Factors ; Female ; Follow-Up Studies ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute ; genetics ; therapy ; Male ; Middle Aged ; Prognosis ; Survival Rate ; Young Adult

Tetraploidy or near-tetraploidy is a rare cytogenetic abnormality found in AML, and is divided into primary and secondary forms. The secondary tetraploidy or near-tetraploidy found in AML is known to be specifically associated with t(8;21). In this case report, FISH analysis detected RUNX1-RUNX1T1 gene rearrangement in the absence of cytogenetic abnormality of t(8;21), which suggests the presence of unvailed t(8;21). This is the first case report of tetraploidy or near-tetraploidy AML with cryptic RUNX1/RUNX1T1 in Korea. Although the prognosis of tetraploidy or near- tetraploidy with t(8;21) is known to be poor, this patient shows a relatively good clinical course compared to other reported cases.
Adult ; Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 8 ; Core Binding Factor Alpha 2 Subunit/*genetics ; Female ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Leukemia, Myeloid, Acute/*genetics ; *Polyploidy ; Proto-Oncogene Proteins/*genetics ; Transcription Factors/*genetics ; Translocation, Genetic

OBJECTIVE: To determine the relation between the promoter methylation status of RUNX3 gene and clinicopathological parameters, prognosis of non-small cell lung cancer (NSCLC). METHODS: We collected 80 formalin-fixed paraffin-embedded lung cancer tissue samples from NSCLC patients who received postoperative adjuvant chemotherapy with cisplatin. Genomic DNA was extracted through phenol/chloroform extraction. The methylation status of RUNX3 was determined by nested methylation-specific PCR (nMSP). We investigated the pathological and prognostic characteristics of NSCLC stratified by methylation status. RESULTS: The RUNX3 promoter methylation was observed in 20 of the 80 NSCLC samples (25.0%). Methylation of RUNX3 was more frequent in adenocarcinomas (36%) than in squamous cell carcinomas (11%) (P=0.020). In multivariate Logistic regression, positive RUNX3 methylation status (P=0.011) was found to be independent disease-free survival factor as was N stage (P<0.001). Kaplan-Meier curves showed patients with RUNX3 methylation had a significantly poorer overall survival than those without methylation (P=0.003; log-rank test). In multivariate Cox proportional hazards regression analysis, RUNX3 methylation (RR:2.345, 95% CI:1.30-4.865, P=0.022) was a significant independent prognostic factor for the overall survival. CONCLUSION RUNX3 methylation is a significant independent prognostic factor for disease-free survival and overall survival.
Aged ; Carcinoma, Non-Small-Cell Lung ; genetics ; surgery ; Core Binding Factor Alpha 3 Subunit ; genetics ; DNA Methylation ; Female ; Humans ; Lung Neoplasms ; genetics ; surgery ; Male ; Middle Aged ; Prognosis ; Promoter Regions, Genetic ; genetics ; Proportional Hazards Models ; Time Factors