Characterization of growth performance of F1 hybrid CB6 F1 C57-ras transgenic mouse model / 中国比较医学杂志

Objective To obtain the basic growth parameters of a self-established F1 hybrid CB6F1 C57-ras transgenic mouse model, and to provide basic information for commercialization of this mouse model. Methods F1 hybrid mice (CB6F1) were produced by crossing C57-ras heterozygous transgenic (c-Ha-ras+/-) male mice and wild-type BALB/cJ female mice.The average litter size, weaning rate, sex ratio, growth performance and C57-ras transgenic positive rate were recorded and analyzed.Results The average litter size was eight, weaning rate was 90%, and sex ratio was approximately in accordance with prediction.The average body weight of newborn mice was 1.73 ±0.05 g.The average body weight of 10-week old c-Ha-ras transgenic female and male mice in CB6F1 background was 24.38 ±1.74 g and 29.42 ±1.72g, respectively, which had a significant difference (P<0.01).The c-Ha-ras transgenic positive rate was 46.9%. which was in accordance with genetic rules.Conclusions The F1 hybrid mice (CB6F1) produced by crossing C57-ras heterozygous transgenic ( c-Ha-ras +/-) male mice and wild-type BALB/cJ female mice show normal growth performance and development characteristics, and it can be used for large-scale commercial supply.

The Difference in Biological Properties between Parental and v-Ha-ras Transformed NIH3T3 Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We performed experiments to investigate the change in cellular signaling that occurs during the transformation of a normal cell to a cell capable of cancerous growth, and we did so by using the NIH 3T3 cells that were transformed by transfection with the v-Ha-ras oncogene. MATERIALS AND METHODS: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems. The siRNA transfections were performed using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. Immunoblot analysis was performed using the ECL-Plus chemiluminescent system and a KODAK Image Station 4000R. RESULTS: The v-Ha-ras-transformed cells were found to be significantly more resistant to PP2 treatment, which is a potent inhibitor of the Src family tyrosine kinases, than were the parental cells at earlier times after treatment. However, PP2 induced growth arrest and the senescence-like phenotypes in both cell lines after longer treatment. Furthermore, the Raf-1 kinase of the v-Ha-ras-transformed cells was not affected by the expressed level of Sprouty proteins, which are negative regulators of the MAPK pathway, as evidenced by the failure of siRNA-mediated knockdown of Spry4 to activate Raf-1 kinase. Dephostatin (a tyrosine phosphatase inhibitor) effectively inhibited the proliferation of the v-Ha-ras transformed cells, whereas dephostatin had only a small effect on the parental cells' proliferation. This implied an inhibitory role for tyrosine phosphatase that is specific to the signaling pathway in the v-Ha-ras transformed cells. CONCLUSION: Taken together, our results show that the sustained activation of the oncogenic pathways through their resistance to negative feedback regulation might contribute to the transformation of NIH 3T3 cells.

Merlin Represses Ras-Induced Cyclin D1 Transcription through the Cyclic AMP-Responsive Element

Mutations in the NF2 tumor suppressor gene cause neurofibromatosis type 2, an autosomal dominant inherited syndrome predisposed to the multiple tumors of the nervous system. Merlin, the NF2 gene product was reported to block Ras-mediated cell transformation and represses Ras-induced expression of cyclin D1. However, the potential mechanism underlying the anti-Ras function of merlin on the cyclin D1 is still unclear. In this study, we investigated whether merlin decreases Ha-ras-induced accumulation of cyclin D1 at the transcriptional level, and demonstrated that merlin suppressed Ras-induced cyclin D1 promoter activity mediated by the cyclic AMP-responsive element (CRE) in SK-N-BE (2) C neuroblastoma cells. Furthermore, we found that merlin attenuated active Ras and forskolin-induced CRE-driven promoter activity. These results suggest that the transcriptional repression of the cyclin D1 expression by merlin may contribute to the inhibition of Ras-induced cell proliferation

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%.

The Expression of c-met, c-Ha-ras and c-myc Oncogene in Thyroid Papillary Cancer and Prognostic Factor

BACKGROUND: Many oncogenes have been recently identified in human thyroid carcinomas, but the molecular mechanisms that lead to thyroid neoplasia are not well understood. To assess whether oncogene- encoded proteins can be regarded as useful prognostic indicators, we have evaluated the expressions of c-met, c-Ha-ras, c-myc oncogenes in patients with papillary thyroid cancer (PTC) in relation to the prog nostic factors. METHODS: We used immunohistochemistry to examine the expressions of c-met, c-Ha-ras, and c-myc oncogenes in 54 paraffin-embedded PTC specimens. We measured both the proportion (scale of 0-3) and the intensity (scale of 0-3) of the stained cells and then calculated the staining index (scale of 0-9) by multiplying the proportion and the intensity scores. The staining index was thus categorized as negative/low (staining index 5). The considered prognostic factors were age (over 45), tumor size (over 1.5 cm), lymph node metastasis, capsular invasion, vascular invasion, and distant metastasis. RESULTS: 1) The rates of expression of c-met, c-Ha-ras, c-myc oncogenes were 100%, 81.5%, and 70.3% in papillary thyroid cancer and 100%, 30%, and 10% in benign tumors and 60%, 10%, and 0% in normal thyroid tissue, respectively. The expression of c-met oncogene was restricted to the membrane the expression of c-Ha-ras was stromal in 95.5% of the specimens, and that of c-myc was stromal in 94.7%. 2) High expression (staining index >5) of c-met, c-Ha-ras and c-myc were not associated with the prognostic factors such as age, tumor size, lymph node metastasis, capsular invasion, vascular invasion and distant metastasis. CONCLUSION: Although the rates of expression of c-met, c-Ha-ras, and c-myc oncogenes were high in papillary carcinomas (100%, 81.5%, and 70.3%, respectively), there was no relationship between the high expression rates of the oncogenes and prognostic factors.

Morphologic Changes and Ha - ras Mutation in DMBA - treated Rat Mammary Tissues / 대한암학회지

PURPOSE: To understand the morphologic and molecular changes in carcinogen-induced breast tissues, DMBA (10-dimethy1-1,2 benzanthracene) was administrated in Sprague- Dawley female rats. MATERIALS AND METHODS: At 50 days of age, all experimental rats were given 20 mg DMBA by gastric intubation. Until the seventh week after DMBA administration, six rats were sacrificed every week, thereafter all tumors found during 20 weeks were removed every week. The morphologic changes were evaluated in routinely processed sections stained with H-E and with anti-smooth muscle actin antibody. Mutation of Ha-ras codons 12 and 61 was examined by ARMS (amplification refractory mutation system) method in frozen tissues. RESULTS: The epithelial cell proliferation of terminal end buds began 2 weeks after DMBA treatment and progressed to the 6th week, resulting in microscopic malignant tumor in one of the 7th weeks rats. The tumors were developed in 43 of 62 rats (69.4%); 8 benign lesions in 4 rats and 72 malignant tumors in 39 rats. Mutations in the 12th and 61th codon of Ha-ras gene were respectively found in 29.7% and 2.7% of preneoplastic breasts, 25% in benign lesions, 2.6% and 31.6% of malignant tumors. CONCLUSION: DMBA treatment in rats induced epithelial proliferation, then benign and malignant tumors through Ha-ras gene mutation, especially in codon 61 leading to cancer.

Expression of Cellular Oncogenes in Colorectal Cancer : c-myc, c-Ha-ras and c-erbB-2

Although causative factors are not completely defined, carcinogenesis of colorectal cancer is attributed to multiple genetic alterations. The abnormal expressions of oncogenes are regarded to be responsible for the production of malignant phenotype, subsequent invasion and metastasis. From 63 surgically resectable colorectal adenocarcinoma patients, expression of oncogenes in colorectal cancer tissue was evaluated with immunohistochemical staining methods using monoclonal antibodies to products of the oncogenes. To evaluate the possibility of oncogenes as a prognostic factor, we studied the relationship between the expression of oncogenes and the clinicopathologic findings which are well known prognostic factors. Rates of expression in colorectal cancer tissue were 27% for c-myc, 74.6% for c-Ha-ras and 77.8% for c-erbB-2 oncogenes. The positive rate of c-erbB-2 oncogene was higher in the well differentiated group than in the poorly differentiated group. The rates of expression of c-myc and c-Ha-ras oncogenes were significantly correlated each other. Expression of these oncogenes in colorectal cancer were not correlated with the pathologic stage, location of cancer, DNA ploidy pattern and histologic differentiation except between c-erbB-2 and histologic differentiation. In conclusion, there seems to be a possibility that c-erbB-2 could be used as a prognostic factor of colorectal cancer. However, further and more intensive study seems to be required.

Extracellular Matrix, TGF - beta Gene and Ha-ras Oncogene Expression in Type I Neurofibromatosis / 대한피부과학회지

BACKGROUND: Neurofibroma, the hallmark of neurofibromatosis, is a cutaneous or subcutaneous lesion, with a variable clinical presentation. Histologically, neurofibroma consists of proliferation of nerve derived cellular elements, together with an abundant, collagenous extracellular matrix. Specifically, neurofibroma has been shown to contain 30-50% collagen in its matrix. Objective 5. METHODS: We examined the expression of extracellular matrix genes (collagen, fibronectin, laminin), TGF-b mRNA and Ha-ras oncogene mRNA by using Northern and slot-blot hybridization and immunoperoxidase stains. Result: In Northern blot analysis, Ha-ras and TGF-b genes revealed respectively, 8.8kb and 2. 5kb sized mRNA transcripts in neurofibroma. These parameters were normal in the control. The expression of these genes were 1.9, 2.0 fold increased in neurofibroma. In slot-blot analysis, expression of type I collagen showed fibronectin genes to be 2,401+210, 540+43, respectively, in neurofibroma. So there were 3.7 fold, 2.1 fold, differences respectively, compared to the normal control. However, there were no significant changes of type IV collagen and laminin Bl mRNA levels between neurofibroma and normal skin tissues. Irnmunoperoxidase staining by rnonoclonal anti type IV collagen antibody in neurofibroma showed type IV collagen to be diffusely and weakly stained in tissue. On staining by monoclonal anti-laminin antibody, laminin was stained in a matrix and around vessels. CONCLUSION: The increased expression of extracellular matrix genes may suggest that there is a subpopulation of fibroic cells in neurofibroma which are stimulated by TGF-b. Ha-ras genes which might have accumulated with the differentiation of neural tissue may be related to the pathogenesis of neurofibroma tissue formation. Further studies are needed to determine whether the other factors are related to the pathogenesis of neurofibroma.

Analysis and Clinical Implication of Ha-ras, p53 and RB Gene Mutations and Infection of Bladder Cancer with Papillomavirus / 中国肿瘤生物治疗杂志

To clarify the clinical significance of alterations of Ha-ras, p53 and RB gene as well as infection with HPV16,18 in human bladder cancer, we examined the state of Ha-ras, p53 and RB genes, sequences of HPV16/18, and their association with clinicopathological parameters in 39 cases of bladder cancer and 7 cases of normal tissue, using nonisotopic PCR-SSCP and dot blot. The overall incidences of Ha-ras, p53 and RB gene mutation and HPV infection in tumor were 61.5% ,36% ,30.8% and 15.4% , respectively. The HPV positive rate was negatively correlated with clinical stage and pathological classification. Rather, the mutations of Ha-ras and p53 gene were positively correlated with the above clinical parameters. The incidence of Ha-ras gene mutation in recurring tumors was significantly higher than that in primary ones. A negative correlation between HPV infection and p53 mutation was also found. The results suggest that the above molecular events and their interaction play important roles in the development of bladder cancer, and that they wonld be of practical assistance in the prognosis and monitoring of bladder cancer.

Expression of BrdU and C-Ha-ras in Experimentally Induced Enzyme Altered Foci of the Liver and Hepatocellular Carcinoma

For sequential phenotypic changes including enzyme altered hepatocytic foci, hyperplastic nodules, hepatocellular adenomas and carcinomas were produced in Sprague-Dawley rats by Solt-Farber method (administration of diethylnitrosamine and acetylaminofluorene (AAF), and partial hepatectomy). The immunohistochemical expressions of glutathione S transferase P (GST-P) and bromodeoxyuridine (BrdU) were assessed for selective proliferative activity in the enzyme altered foci and the subsequently developed lesions by double immunohistochemical staining technique. Immunoreactive areas against GSTP gradually increase from early period of carciogenesis. BrdU labeling in such areas remained high during the first week. but decreased thereafter. BrdU labeling index remained low in the GSTP negative area throughout the experimental period. This suggests that cells in the enzyme altered foci keep away from the suppressor effect of AAF in contrast to the normal cells in which their growth are inhibited by AAF. BrdU labeling index remained very low in both hyperplastic nodule and adenoma which were prevalent during the mid-experimental period, but increase markedly in carcinoma. The long period of low BrdU labeling index seems to correspond to the "slowly growing step of persistent nodule" during hepatocarcinogenesis. The differentiation index, a ratio of S phase fraction between GSTP positive and negative areas, was low in adenoma-developing period and high in carcinoma-developing period. C-Ha-ras p21 was not expressed in foci of enzyme altered hepatocyte and hyperplasia, but highly positive in carcinoma. This indicates that the c-Ha-ras may involve the late step of hepatocarcinogenesis.

Expression of c-myc and c-ha-ras oncogenes in human bladder cancer / 대한비뇨기과학회지

In order to elucidate the c-myc and c-Ha-ras oncogenes expressions in human bladder cancers. we examined twelve bladder cancer tissues. two normal bladder tissues and two blood specimens with Southern. Northern and Slot blot hybridizations. In Southern blot all of the specimens showed 14 kilobase(Kb) c-myc band and 6.6kb c-Ha-ras band. No significant amplifications were observed in the levels of c-myc and c-Ha-ras DNA in cancer tissues. In Northern and slot blot, four bladder cancer tissues showed increased expression of c-myc RNA. No significant differences were observed in the levels of c-Ha-ras RNA expression between cancer tissues and normals. In conclusion. these findings support that quantitative increase in c-myc expression plays its role in the development of bladder cancers. Further studies about c-Ha-ras point mutation analysis and P21 ras protein analysis should be done to identify the role or c-Ha-ras in bladder tumorigenesis.

Point mutation at codon 12 of the c-Ha-ras gene in human gastric cancers

The molecular mechanisms of the carcinogenic process of gastric cancer have not been fully understood yet. In order to know whether c-Ha-ras gene is being involved in the process of gastric carcinogenesis, 8 gastric cancer cell lines, 8 cases of gastric cancer and same number of adjacent dysplasia were analyzed for the presence of mutation at codon 12, 13 and 61 of the c-Ha-ras gene by using polymerase chain reaction (PCR) and mutant-specific oligonucleotide hybridization. Point mutations at codon 12 of the c-Ha-ras gene were found in 2 out of 8 gastric cancer and dysplasia samples in one case, but we found no mutation at codon 13 or 61 of the c-Ha-ras gene. These results suggest that the frequency of mutation of the c-Ha-ras gene detected by sensitive PCR technique is low indeed, however it would be notable that such a genetic change has been detected in the dysplastic lesion of the gastric cancer patient.

Expression of Cellular Oncogenes in Gastric Carcinoma Related with Its Histologic Subtype: Southern blot analysis and immunohistochemistry

To search biologic characteristics of gastric carcinoma, one of the most common cancer in Korea, the author examined the alterations in DNA level and the expression of Ha-ras gene and c-myc gene in 20 primary tumors. Amplification of c-Ha-ras DNA was detected in 4(40%) of 10 patients who showed histologic subtype of relatively differentiated adenocarcinoma, but rearrangement of c-Ha-ras DNA was absent. Neither augumentation nor deletion of the c-myc DNA was observed. Higher expression of the ras p21 in tumor cells was noted in more differentiated tumor cells rather than poorly differentiated cases. One mucinous carcinoma, two signet ring cell carcinomas and one papillary carcinoma did not disclose expression of p21. The expressions of c-myc oncogene product were variable and were not correspond to the expressions of ras p21. A tendency that poorly differentiated tumor cells had higher expression of c-myc oncogene was suggested.

Expression of Extracellular Matrix, c-Ha-ras and c-myc genes in Cultured Pterygial Fibroblasts

Pterygium, a disease of unknown origin and pathogennesis, is a chronic condition characterized by the encroachment of triangular portion of the bulbar conjunctiva onto the cornea. We have studied the expression of extracellular matrix genes and oncogenes in cultured pterygium using Northernm dot, and slot blot hybridizations. Northern hybridization with total RNA isolated from passaged (4-8 passages) cultures demonstrated expression of genes for alpha1(I) and alpha1(III) procollagen, fibronectin, and c-Ha-ras, but no expression of gene for c-myc was observed. The pterygium exhibited significantly increased expression of alpha1(I) and alphal(III) procollagen genes when compared with normal control cells(p<0.01). And We observed there were no differences between pterygium and normal control cells in the expression of genes for fibronectin and c-Ha-ras. According to these results we thought that the causes of pterygium may be related to the increased expression of alpha1(I) and alphal(III) procollagen genes but may not be related to c-Ha-ras, c-myc, and fibronectin genes.

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.