Although a loss of heterozygosity (LOH) is commonly observed using microsatellite markers in a cell-proliferating malignant disorder, controversial findings of psoriasis, a keratinocyte-outgrowth disease, remain to be explained. It was hypothesized that unstable natures of the microsatellite markers for the polymerase chain reaction (PCR) might give a rise to either a false-positive or -negative LOH. Twentyone frozen skin tissues and 33 formalin-fixed paraffin-embedded archives were obtained from patients with psoriatic plaques and colorectal cancers, respectively. In the frozen psoriatic skin, two of the 17 microsatellite markers selected from 11 chromosomal arms were associated with artifact LOHs that were not reproduced in repeated PCRs. The remaining 15 stable microsatellite markers with few PCR artifacts demonstrated a borderline-level LOH in cases with an ambiguous heterozygosity such as a juxtaposed allelic band. Infrequent LOHs (3 out of 242 heterozygous markers, 1.2%) were detected in psoriatic cases with two separate alleles. In colorectal cancers, a set of the 15 stable microsatellite markers identified a minimal borderline-level LOH at the cut-off point that was same with that of psoriasis. These results indicate that the selection of reproducible microsatellite sequences and the cautious criteria for informative heterozygosity are required to obtain the reliable LOH results from variable genomic DNAs, and that psoriatic lesions harbor few LOH.
Alleles ; Colorectal Neoplasms/*genetics ; DNA/genetics/isolation & purification ; DNA, Neoplasm/genetics/isolation & purification ; Female ; Humans ; *Loss of Heterozygosity ; Male ; *Microsatellite Repeats ; Polymerase Chain Reaction ; Psoriasis/*genetics ; Reproducibility of Results

OBJECTIVETo identify and clone the gene encoding human M96 gene and study its expression spectrum in several blood cell lines.

METHODSAccording to the sequence of human EST which was highly homologous to the mouse M96 gene, primers used for library screening were synthesized, then the human adult testis and fetal brain cDNA library were screened. The gene was analyzed by making use of BLAST and CLUSTAL W, and its expression spectrum was studied by multiple-cell lines Northern blot analysis. The expression change of M961 in cell differentiation was observed by use of K562 cell line induced by hemin.

RESULTSTwo cDNA clones encoding human M96 gene were isolated, identified and named as M961, and M962. They were found to be isoforms of each other. Northern, blot showed that M961 gene was expressed highly in CEM, Hel, Dami and K562 cell lines. However, during K562 cell line differentiation, process the expression of M961 elevated only slightly.

CONCLUSIONSM961 gene was expressed highly in pluripotent cell lines with erythrocytic and megakaryocytic potentials.

Alternative Splicing ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Neoplasm ; genetics ; isolation & purification ; Hemin ; pharmacology ; Humans ; K562 Cells ; Molecular Sequence Data ; Protein Isoforms ; genetics ; isolation & purification ; Protein Splicing ; Zinc Fingers ; genetics

Transitional-CpG methylation between unmethylated promoters and nearby methylated retroelements plays a role in the establishment of tissue-specific transcription. This study examined whether chromosomal losses reducing the active genes in cancers can change transitional-CpG methylation and the transcription activity in a cancer-type-dependent manner. The transitional-CpG sites at the CpG-island margins of nine genes and the non-island-CpG sites round the transcription start sites of six genes lacking CpG islands were examined by methylation-specific polymerase chain reaction (PCR) analysis. The number of active genes in normal and cancerous tissues of the stomach, colon, breast, and nasopharynx were analyzed using the public data in silico. The CpG-island margins and non-island CpG sites tended to be hypermethylated and hypomethylated in all cancer types, respectively. The CpG-island margins were hypermethylated and a low number of genes were active in the normal stomach compared with other normal tissues. In gastric cancers, the CpG-island margins and non-island-CpG sites were hypomethylated in association with high-level chromosomal losses, and the number of active genes increased. Colon, breast, and nasopharyngeal cancers showed no significant association between the chromosomal losses and methylation changes. These findings suggest that chromosomal losses in gastric cancers are associated with the hypomethylation of the gene-control regions and the increased number of active genes.
Alu Elements/genetics ; *Chromosome Deletion ; CpG Islands/*genetics ; *DNA Methylation ; DNA, Neoplasm/chemistry/isolation & purification ; Gene Expression Profiling ; *Genes, Neoplasm ; Humans ; Long Interspersed Nucleotide Elements/genetics ; Polymerase Chain Reaction ; *Promoter Regions, Genetic ; Stomach Neoplasms/*genetics

OBJECTIVETo screen the differentially methylated DNA sequences between mucosa adjacent to colorectal cancer (MACC) and normal colonic mucosa.

METHODSThe methylated DNA sequences were enriched by methylation CpG islands amplification (MCA), and the differentially methylated DNA sequences between MACC and normal colonic mucosa were isolated by representational difference analysis (RDA). Similarities between the separated fragments and the human genomic DNA were analyzed with BLAST program system in GenBank. With the separated fragment 1A12 as probe, dot blot was used to study its distribution between RDA products (No. 1-4 rounds), MACC (tester) and normal colonic mucosa(driver).

RESULTSTwenty-five differentially methylated DNA sequences were obtained. Preliminary studies indicated that 1A01 fragment was concerned with two different genes (LOC256866 and CECR7), it was located in the first exon of CECR7. 1A12 fragment was located in 5 flanking region of GR6 gene. By dot blot with 1A12 probe, hybridized signals were detected in MCA product of MACC and RDA products of No. 1-4 rounds, respectively. No signal was detected in MCA product of normal colonic mucosa.

CONCLUSIONThe differentially methylated DNA sequences can be isolated effectively between two different tissues with MCA coupled with RDA. Different methylated DNA fragments exist between MACC and normal colonic mucosa and these fragments may be concerned with colorectal cancer.

Adult ; Base Sequence ; Colorectal Neoplasms ; diagnosis ; genetics ; CpG Islands ; genetics ; DNA Methylation ; DNA, Neoplasm ; chemistry ; isolation & purification ; metabolism ; Genetic Testing ; Humans ; Intestinal Mucosa ; metabolism ; Polymerase Chain Reaction ; Sequence Analysis, DNA

PURPOSE: Circulating free DNA (cfDNA) in plasma is promising to be a surrogate for tumor tissue DNA. However, not all epidermal growth factor receptor (EGFR) mutations in tumor tissue DNA has been detected in matched cfDNA, at least partly due to inefficient cfDNA extraction method. The purpose of this study was to establish an efficient plasma cfDNA extraction protocol. MATERIALS AND METHODS: The yield of plasma cfDNA extracted by our modified phenol-chloroform (MPC) method from non-small-cell lung cancer (NSCLC) patients was compared with that by QIAamp MinElute Virus Spin kit (Qiagen kit) as control, using the Wilcoxon rank-sum test. TaqMan quantitative polymerase chain reaction (qPCR) assays were used to quantify the plasma cfDNA extracted. Both Mutant-enriched PCR (ME-PCR) coupled sequencing and DxS EGFR mutation test kit were used to evaluate the impact of extraction method on EGFR mutation analysis. RESULTS: MPC method extracted more plasma cfDNA than Qiagen kit method (p=0.011). The proportion of longer fragment (> or =202 bp) in cfDNA extracted by MPC method was significantly higher than by Qiagen kit method (p=0.002). In the sequencing maps of ME-PCR products, a higher mutant peak was observed on plasma cfDNA extracted by MPC method than by Qiagen kit method. In DxS EGFR mutation test kit results, plasma cfDNA extracted by MPC method contained more tumor-origin DNA than by Qiagen kit method. CONCLUSION: An improved plasma cfDNA extraction method of MPC is provided, which will be beneficial for EGFR mutation analysis for patients with NSCLC.
Base Sequence ; Carcinoma, Non-Small-Cell Lung/*genetics ; Chloroform ; DNA Mutational Analysis/*methods ; DNA, Neoplasm/*blood/*isolation & purification ; Genetic Testing/methods ; Humans ; Lung Neoplasms/*genetics ; Molecular Sequence Data ; Phenol ; Polymerase Chain Reaction/methods ; Receptor, Epidermal Growth Factor/*genetics

The article submitted the new progress of the study in antitumor mechanism of bererine (Ber). Reports indicated that Ber suppressed growth of tumor cells through impacting tumor cells growth cycle, inhibiting synthesises of DNA and protein, and reducing the activity of topoisomerase. Ber improved tumor cells apoptosis through several ways such as regulating apoptotic gene expression, inducing the decline of transmembrane potential on mitochondria. And Ber still could inhibit tumor metastasis through suppressing the formation of tumor angiogenesis, blocking signal transduction pathway, antagonizing extralumen, et al. In addition, Ber could induce tumor cells to differentiate to antagonize tumor.
Animals ; Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Berberine ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA, Neoplasm ; biosynthesis ; Humans ; Neoplasm Proteins ; biosynthesis ; Neoplasms ; genetics ; metabolism ; pathology ; Plants, Medicinal ; chemistry

OBJECTIVETo analyze the DNA sequence characteristics of translocation t (X; 18) genomic breakpoints and to study the mechanism underlying chromosomal translocation t (X; 18) in synovial sarcoma.

METHODSTwo cases of synovial sarcoma were studied utilizing long-distance polymerase chain reaction (PCR) and sequence analysis to amplify the genomic DNA of translocation t (X; 18) breakpoints.

RESULTSTranslocation t (X; 18) was detected in both cases, which generated SYT-SSX1 and SYT-SSX2 fusion gene respectively. Sequence analysis revealed that intron 10 of SYT was fused to the intron 4 of SSX1 or SSX2. Sequences highly homologous to consensus recognition motifs of translin were found adjacent to breakpoints in all three genes. Breakpoints in the three genes were close to or even at several palindromic oligomer sequences. The breaks in intron 4 of SSX1 and SSX2 were near an Alu sequence. No Alu or other repetitive sequences were found 500 bp upstream or downstream from the break in intron 10 of SYT. One topoisomerase II consensus site was found between the two breakpoints but with considerable distance from intron 10 of SYT.

CONCLUSIONSAll three genes involved in synovial sarcomas contain characteristic sequence motifs in the breakpoint regions which may play an important role in the genesis of chromosomal translocation in synovial sarcoma.

Base Sequence ; Chromosomes, Human, Pair 18 ; Chromosomes, Human, X ; Cloning, Molecular ; DNA, Neoplasm ; analysis ; Humans ; Molecular Sequence Data ; Oncogene Proteins, Fusion ; genetics ; isolation & purification ; Sarcoma, Synovial ; genetics ; Sequence Analysis, DNA ; Translocation, Genetic

OBJECTIVETo evaluate the expression of BRAF V600E mutation in 240 Chinese patients with thyroid lesions.

METHODSTwo hundred and forty Chinese patients with thyroid lesions, including 129 papillary thyroid carcinomas (PTC), 12 follicular carcinomas, 4 medullary carcinomas, 30 adenomas, 30 nodular goiters, and 35 papillary hyperplasia. DNA was extracted from thyroid biopsy and paraffin embedded thyroid tissues, and the expression of BRAF V600E mutation was detected by polymerase chain reaction and DNA sequencing assays.

RESULTSThe presence of BRAF V600E mutation was found in 61 of the total group of 240 cases (25.4%). It was only detected in PTC (47.3%), and not detected in other types of malignant and benign thyroid lesions. There was a statistically significant difference between the expression of BRAF V600E mutation in classic type PTC (49.6%) and in follicular type PTC (12.5%,P < 0.05), but statistical data did not show any correlation between BRAF V600E mutation and clinicopathologic parameters in PTC (P > 0.05).

CONCLUSIONSBRAF V600E mutation has a significant correlation with PTC and the detection of BRAF V600E mutation may be used as an important prognostic marker of PTC. Our new method of DNA extraction from paraffin embedded tissues is efficient and inexpensive.

Adenocarcinoma, Follicular ; genetics ; metabolism ; Adenoma ; genetics ; metabolism ; Adult ; Biomarkers, Tumor ; genetics ; Carcinoma, Papillary ; genetics ; metabolism ; Codon ; DNA Mutational Analysis ; DNA, Neoplasm ; genetics ; isolation & purification ; Female ; Goiter, Nodular ; genetics ; metabolism ; Humans ; Male ; Middle Aged ; Point Mutation ; Proto-Oncogene Proteins B-raf ; genetics ; metabolism ; Thyroid Neoplasms ; genetics ; metabolism

OBJECTIVETo investigate the clinicopathological significance of p16(INK4A) expression and DNA ploidy status in HPV-negative uterine cervical cancers and their precursors.

METHODSHPV-negative cervical lesions, including 20 cases of cervicitis, 20 cases of cervical intraepithelial neoplasm (CIN), 3 cases of cervical glandular intraepithelial neoplasm (CGIN), 38 cases of invasive squamous cell carcinoma (SCCs) and 15 cases of invasive adenocarcinoma were selected and subject to screening for HPV infection by PCR method. The p16(INK4A) protein expression and DNA ploidy status were studied by immunohistochemistry and flow cytometry respectively.

RESULTSSpecific expression of p16(INK4A) was seen in both the nucleus and cytoplasm of the dysplastic and malignant cells of CIN, CGIN, cervical SCC and adenocarcinoma. In contrast, no expression was present in normal and inflammatory squamous or glandular epithelium. DNA aneuploidy was significantly more frequent in invasive SCCs and adenocarcinomas than in CIN (P < 0.01). Aneuploid was also more frequent in the lymph node positive group than lymph node negative group, although no statistic significance was found. Among the 8 cases of p16(INK4A) negative SCCs, two showed DNA aneuploidy.

CONCLUSIONSImmunohistochemical detection for p16(INK4A) can be an early diagnostic marker for HPV-negative cervical SCC and adenocarcinoma. DNA ploidy analysis may further assist the diagnosis of cervical malignancies.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Aneuploidy ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; DNA, Neoplasm ; genetics ; Female ; Flow Cytometry ; Humans ; Immunohistochemistry ; Papillomaviridae ; genetics ; isolation & purification ; Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; genetics ; metabolism ; pathology ; Uterine Cervicitis ; genetics ; metabolism ; pathology

Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; DNA Mutational Analysis ; DNA, Neoplasm ; genetics ; isolation & purification ; Genes, erbB-1 ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Mutation ; Paraffin Embedding ; methods ; Polymerase Chain Reaction ; methods ; Real-Time Polymerase Chain Reaction ; methods ; Receptor, Epidermal Growth Factor ; genetics