Ras oncogene and Hypoxia-inducible factor-1 alpha (hif-1α) expression in the Amazon fish Colossoma macropomum (Cuvier, 1818) exposed to benzo[a]pyrene

Abstract Benzo[a]pyrene (B[a]P) is a petroleum derivative capable of inducing cancer in human and animals. In this work, under laboratory conditions, we analyzed the responses of Colossoma macropomum to B[a]P acute exposure through intraperitoneal injection of four different B[a]P concentrations (4, 8, 16 and 32 μmol/kg) or corn oil (control group). We analyzed expression of the ras oncogene and the Hypoxia-inducible factor-1 alpha (hif-1α) gene using quantitative real-time PCR. Additionally, liver histopathological changes and genotoxic effects were evaluated through the comet assay. Ras oncogene was overexpressed in fish exposed to 4, 8 of 16 μmol/kg B[a]P, showing 4.96, 7.10 and 6.78-fold increases, respectively. Overexpression also occurred in hif-1α in fish injected with 4 and 8 μmol/kg B[a]P, showing 8.82 and 4.64-fold increases, respectively. Histopathological damage in fish liver was classified as irreparable in fish exposed to 8, 16 and 32 μmol/kg μM B[a]P. The genotoxic damage increased in fish injected with 8 and 16 μmol/kg in comparison with the control group. Acute exposure of B[a]P was capable to interrupt the expression of ras oncogene and hif-1α, and increase DNA breaks due to tissue damage.

Bioinformatic analysis on the Rap2B-like gene from Clonorchis sinnensis and its cloning and expression / 中国人兽共患病学报

To analyze the structural characteristics of the Rap2B gene from Clonorchis sinensis by bioinformatics and to clone and express this gene through genetic engineering methods in order to obtain the corresponding recombinant protein.The structural and functional characteristics of the Rap2B protein were analyzed by using the related bioinformatic softwares. Using the full-length cDNA plasmid clone (Noc009e03) as template , the coding region of Rap2B gene sequence was amplified with PCR and cloned into the prokaryotic expression vector pET-28a(+) and then expressed in E.coli BL21 through IPIG induction. The recombinant protein expressed was purified by Ni-IDA affinity chromatography and detected by SDS-PAGE and Western blotting. It was demonstrated that the size of this gene was 847 bp in length, encoding 141 amino acids and its theoretical molecular weight was 15851.1 daltons. As demonstrated by PCR, double enzyme digestion and DNA sequencing, the recombinant expression plasmid pET-28a(+)-Rap2B was successively constructed and the Rap2B-like gene was highly expressed in E.coli BL21. The recombinant protein was obtained through purification with His affinity chromatography and could react specifically with the serum of rats infected with C.sinensis. Through these investigations, the Rap2B protein may be predicted as a member of the Ras oncogene family protein and is potential in the carcinogenic process of C.sinensis.

Mutation of Ha-ras Oncogene in Rat Salivary Gland Tumors Induced by DMBA / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The incidence of salivary gland tumor is approximately 2% among all head and neck tumors, of which malignant cases account for only about 5%. Much research has been performed in order to clarify the mechanism of oncogene activation, however salivary gland tumors remain understudied. We performed this study in order to characterize the ras gene in these tumors. MATERIALS AND METHODS: We treated white rats with 7, 12-dimethylbenz[a]anthracene (DMBA) and confirmed the occurrence of salivary gland tumors after ten to thirty weeks. Isolated genomic DNAs from tumor tissues were added to NIH 3T3 cells. In order to detect Ha-ras mutations, we performed a two-step PCR-RFLP and 7analyzed the mutated sequences. RESULTS: We induced salivary gland tumors by DMBA treatment in white rats. Isolated DNAs from the tumor tissues transformed the NIH 3T3 cells. Point mutations were observed in codons 12 and 61 of the Ha-ras oncogene. The total frequency of point mutations was 13.9% in DMBA-induced salivary gland tumors in rats. CONCLUSION: Our results demonstrate that a variety of cancers ras oncogene mutations were also found in salivary gland tumors. We confirmed that a point mutation of the Ha-ras oncogene in a DMBA-induced salivary gland tumor occurs at a frequency of 13.9%.

Study on mutations of K-ras gene in pancreatic diseases / 中华消化杂志

Objective To investigate the clinical significance of K-ras mutations in pancreatic diseases, specifically in the diagnosis of pancreatic carcinoma. Methods One hundred and seventeen surgical-resected pancreatic specimens, including 24 pancreatic ductal adenocarcinoma, 19 peritumoral ductal atypical hyperplasia, 58 peritumoral ductal hyperplasia, 19 normal duct at the tumor-free margin and 24 ductal lesion from chronic pancreatitis were obtained, and DNA was extracted from the specimens. Codon 12 K-ras mutations were examined using two-step polymerase chain reaction (PCR) combined with restriction enzyme digestion, non radioisotopic single-strand conformation polymorphism (SSCP) analysis and automated DNA sequencing. Results K-ras mutation rate in the lesion of pancreatic carcinoma was 79.2%(19/24), which was significantly higher than 33.3%(8/24) in the lesion of chronic pancreatitis( P

Identification of mutagenic site of c-H-ras oncogene damaged by N-acetoxyacetylaminofluorene(AAAF)

A molecularly cloned human cellular H-ras (c-H-ras) oncogene(pbc N1 plasmid) was treated with N-acetoxyacetylaminofluorene (AAAF) in vitro and subcloned into E.coli. This was done to identify the mutational changes at specific codons of the gene. Guanine nucleotides were identified as the major AAAF binding site of the DNA adduct formed. Base changes in codons 12 and 61 were determined by the analysis of restriction fragment length polymorphism (RFLP) and site specific oligonucleotide hybridization. RFLP was observed due to the loss of the Hpall recognition site at codon 11 and 12 of AAAF-treated c-H-ras gene. Hybridization of AAAF treated c-H-ras with 32P-labeled oligonucleotide probes for the mutant alleles of codon 61 showed no substitutions at codon 61. From these results, it is assumed that AAAF treatment in vitro caused mutation at codon 12 but not at codon 61 of the c-H-ras oncogene and that codon 12 is the primary target of mutation by AAAF

Immunohistochemical Study on the Ha-ras p21 Expression in the Gastric Carcinoma

We have investigated an immunohistochemical expression of the Human-Ha-ras oncogene product p21 in tumor cells of the primary mass and metastatic lymph nodes with different histological features of gastric cancer by using avidinbiotin complex immunoperoxidase method in formalin-fixed tissue sections from 73 cases of primary tumor mass and 23 cases of metastatic lymph node. Histologic type of the gastric cancer was classification. The results obtained were as follows: 1) Expression of Ha-ras p21 was consistantly increased in the well differentiated tubular adenocarcinoma as compared with poorly differentiated tubular adenocarcinoma (p<0.01), and was substantially decreased in mucinous carcinoma and signet ring cell carcinoma. 2) Signet ring cell carcinoma showed that positive immunoperoxidase reaction for Ha-ras p21 exhibited in the majority of immature signet ring cell with scant cytoplasm rather than in the mature signet ring cells which have abundant cytoplasm filled with mucin. This findings indicate that mucin production from the tumor cell was not correlated with activation of ras gene in the tumor tissue of gastric carcinoma. 3) In general Ha-ras p21 expression was enhanced in the metastatic tumor cells of the regional lymph node compared with primary tumor, especially it was consistantly increase in the well differentiated tubular adenocarcinoma.

Expression of ras Oncogene in Tumor Cells of Epidermis / 대한피부과학회지

DNA isolated from human tumor cells can induce malignant transfarmation of tissue culture cells. The DNA is then called an oncogene. Its protein produets have been detected in animal and human tumors and are considered to play a significant role in carcinogenesis. In order to evaluate whether the oncogenes are involved in development of tumors of epidermis and whether they could be used as tumor markers, immunoperoxidase staining was performed for the ras product in sections of squamous cell carcinoma, Bowen's disease, actinic keratosis, keratoacanthoma and seborrheic keratosis. Three cases of sgamous cell carcinoma showed 10~20 positive cells per high power field(HPF). Three cases of Bowen's disease revealed 1-9 positive cells per HPF, whereas the actinic keratosis 1~9 or no positive cells per 10 HPF in all three cases. The keratoacanthoma and seborrheic keratosis showed 1~9 or no positive cells in all observed cases. The positive staining was observed in the cytoplasm. The increasing positivity in parallel with the increase of malignant potential strongly suggests that the ras oncogene is closely related to development of epidermal malignancy and also point out the possibility of ras as a cancer marker.