Molecules linked to Ras signaling as therapeutic targets in cardiac pathologies

The Ras family of small Guanosine Triphosphate (GTP)-binding proteins (G proteins) represents one of the main components of intracellular signal transduction required for normal cardiac growth, but is also critically involved in the development of cardiac hypertrophy and heart failure. The present review provides an update on the role of the H-, K- and N-Ras genes and their related pathways in cardiac diseases. We focus on cardiac hypertrophy and heart failure, where Ras has been studied the most. We also review other cardiac diseases, like genetic disorders related to Ras. The scope of the review extends from fundamental concepts to therapeutic applications. Although the three Ras genes have a nearly identical primary structure, there are important functional differences between them: H-Ras mainly regulates cardiomyocyte size, whereas K-Ras regulates cardiomyocyte proliferation. N-Ras is the least studied in cardiac cells and is less associated to cardiac defects. Clinically, oncogenic H-Ras causes Costello syndrome and facio-cutaneous-skeletal syndromes with hypertrophic cardiomyopathy and arrhythmias. On the other hand, oncogenic K-Ras and alterations of other genes of the Ras-Mitogen-Activated Protein Kinase (MAPK) pathway, like Raf, cause Noonan syndrome and cardio-facio-cutaneous syndromes characterized by cardiac hypertrophy and septal defects. We further review the modulation by Ras of key signaling pathways in the cardiomyocyte, including: (i) the classical Ras-Raf-MAPK pathway, which leads to a more physiological form of cardiac hypertrophy; as well as other pathways associated with pathological cardiac hypertrophy, like (ii) The SAPK (stress activated protein kinase) pathways p38 and JNK; and (iii) The alternative pathway Raf-Calcineurin-Nuclear Factor of Activated T cells (NFAT). Genetic alterations of Ras isoforms or of genes in the Ras-MAPK pathway result in Ras-opathies, conditions frequently associated with cardiac hypertrophy or septal defects among other cardiac diseases. Several studies underline the potential role of H- and K-Ras as a hinge between physiological and pathological cardiac hypertrophy, and as potential therapeutic targets in cardiac hypertrophy and failure. Highlights - The Ras (Rat Sarcoma) gene family is a group of small G proteins - Ras is regulated by growth factors and neurohormones affecting cardiomyocyte growth and hypertrophy - Ras directly affects cardiomyocyte physiological and pathological hypertrophy - Genetic alterations of Ras and its pathways result in various cardiac phenotypes? - Ras and its pathway are differentially regulated in acquired heart disease - Ras modulation is a promising therapeutic target in various cardiac conditions.

Relevant research of PD-1, PD-L1 protein expression and K-RAS gene mutations in lung adenocarcinoma / 临床与实验病理学杂志

Purpose To investigate the association of the PD-1 and PD-L1 protein expression with K-RAS gene mutations in lung adenocarcinoma.Methods The protein expression of PD-1 and PD-L1 was detected by immunohistochemical EnVision two-step staining,the K-RAS mutation was examined by realtime fluorescent quantitative PCR.Results The positive rate of PD-1 and PD-L1 was higher in lung adenocarcinoma than benign lung disease (P ＜ 0.01).There was no relationship between PD-1 and PD-L1 protein expression with the gender,age,smoking condition,differentiation,lymph node metastasis and TNM stages (P ＞ 0.05).The K-RAS gene mutations were detectable in 8 patients (22.2％) among 36 lung adenocarcinoma,there was no association between K-RAS gene mutation with the gender,age,smoking condition,differentiation,lymph node metastasis and TNM stages (P ＞ 0.05).The correction analysis showed that there was no relationship between PD-1 and PD-L1 protein expression with K-RAS mutation (P ＞ 0.05).Conclusion The positive rate of PD-1 and PD-L1 is higher in lung adenocarcinoma than benign lung disease,but there is no relationship among PD-1 and PD-L1 protein expression with its clinal pathological characteristics and K-RAS mutation in lung adenocarcinoma.

Significance of K-ras gene status and ras protein expression in immunophenotypic classification of gastric signet ring cell carcinoma / 重庆医学

Objective To investigate the significance of K-ras gene status and ras protein expression in immunophenotypic classification of gastric signet ring cell carcinoma.Methods The expression of ras protein in 180 cases of gastric signet-ring cell carcinoma was detected by tissue microarray immunohistochemistry.Meanwhile,the mutation in codon 12,13 of K-ras gene was determined by using PCR-based DNA direct sequencing analysis.Results The rate of ras protein expression was 27.8％.The rate of ras protein expression in intestinal phenotype was significantly higher than those in gastric and gastrointestinal phenotypes(P<0.05).The rate of ras protein expression in cases with lymph node metastasis was significantly higher than those in cases without nodal involvement(P<0.05).The rate of ras protein expression was significantly higher in cases with deeper invasion(P<0.05).The frequency of K-ras gene mutation was 22(12.2％).All of them were found in codon 12.The types of mutation included GGT→AGT(1 case),GGT→TGT(1 case),GGT→GCT(2 cases),GGT→GTT(8 cases)and GGT→GAT(10 cases).K-ras mutation was significantly associated with intestinal phenotype(P<0.05).The rates of ras protein expression in cases with mutational type of K-ras gene was higher than those in cases with wild type(P<0.05).The ras protein expression was positively associated with K-ras gene mutation(r=0.61,P<0.05).Conclusion The ras protein expression is correlated with nodal involvement and invasion.K-ras gene mutation and expression of ras protein is related to phenotypic classification,and they might influence the phenotypic transformation in gastric signet ring cell carcinoma.

High-resolution melting analysis for detection of K-ras mutation in colorectal cancer and its clinical significance / 医学研究生学报

Objective The K-ras gene plays a major role in the development , progression, and drug selection for the target treatment of colorectal cancer .The aim of this study was to evaluate the feasibility of detecting K-ras mutation in colorectal cancer by high-resolution melting ( HRM) analysis and to investigate the relationship between K-ras mutation and the clinicopathological parame-ters of colorectal cancer. Methods We collected the tissue samples of colorectal cancer from 179 patients, detected the mutations in codons 12 and 13 of the K-ras gene, and analyzed the relationship between K-ras mutation and the clinicopathological parameters of the patients. Results In the 179 cases of colorectal cancer, K-ras mutation was found in 77 (43.02%), significantly higher in those aged ≥60 years than in those aged <60 years (55.17% vs 33.70%, P<0.05).Multivariate logistic regression analysis showed a significant influence of age on K-ras mutation (OR=1.506, 95%CI:1.028-2.011, P<0.05). Conclusion HRM analysis is a rapid, sensitive, and inexpensive diagnostic tool for the detection of K-ras mutation, and K-ras mutation is associated with the age of colorectal cancer patients .

Identification of cut off value and lower limit of detection for K-ras mutation by PNA-PCR clamping method / 中华胰腺病杂志

Objective To determine the lower limit of detection (LLOD) and cut off values of K-ras mutation detection by peptide nucleic acid (PNA) clamping-PCR.Methods The genomic DNA of pancreatic cancer cell lines (PANC1 and SW1990) with codonl2,13 mutation and the genomic DNA of placenta with K-ras wild type were mixed and diluted serially into samples with different mutation rate (0,0.1％,0.2％,0.4％,0.8％,1.6％,3.1％,6.25％,12.5％,25％,50％),PANC1 cells with 1％ mutation rate and SW1990 cells with 30％ mutation rate and 4 samples with the quantity of DNA was 50,20,5,1 ng and 50,10,5,1 ng was prepared.Codon 12,13 mutation of K-ras was determined by PNA-PCR,and the mutation Ct values,overall Ct values were collected,and the △Ct values (mutation Ct values-overall Ct values) were calculated,and the tests were repeated for 10 times.ROC curve was used to analyze the △Ct values and determine the best cut off values for K-ras mutation,and the positive diagnostic rate,LLOD was evaluated.Results The mutation Ct,△Ct values of codon 12 mutation of PANC1 and codon 13 mutation of SW1990 of all the different mutation rates were statistically significantly different (P ＜ 0.05) when compared with negative control group,but the overall Ct values were not statistically significantly different from that of negative control group.For detection of K-ras codon 12 mutation by ROC curve,the relevant area of ROC curve (AUC) was 0.926,the optimum cut off value of △CT was 11,the sensitivity and specificity were 84％ and 100％,respectively,and the LLOD was 0.4 ng.For detection of K-ras codon 13 mutation by ROC curve,the relevant AUC was 0.906,the optimum cut off value of △CT was 9.5,the sensitivity and specificity were 71％ and 100％,respectively,and the LLOD was 1.5 ng.The mutation detection results of fixed rate further confirmed the LLOD.Conclusions This study successfully defines LLOD and cut off value of PNA clamping-PCR/K-ras method in detection of K-ras 12 and 13 codon mutations.This method meets the requirement of clinical application.

Effect of cetuximab chemotherapy on operation resection rate of wild type k-ras gene in liver metastasis of colorectal cancer / 中国基层医药

Objective To explore the clinical effect and safety of cetuximab plus mFOLFOX6 first-line treatment in k-ras wild type patients with liver metastasis from colorectal cancer.Methods 40 k-ras wild-type colorectal cancer patients with liver metastasis diagnosed by histological detection in our hospital were chosen.20 patients in the observation group were treated with cetuximab plus mFOLFOX6.20 patients in the control group were only given mFOLFOX6.To observe the effect and safety of the two groups.Results RR,operation excision rate and R0 removal rate of observation group (60％,45％,35％) were significantly higher than those of the control group (30％,15％,10％),the differences were statistically significant (x2 =3.95,4.36,4.28,P ＜ 0.05);The PFS of the observation group (16.37 ± 7.24) was higher than (11.52 ± 6.85) in the control group,but the difference was not statistically significant (P ＞ 0.05).In the observation group,there were 2 cases with rash,2 cases of leukopenia,1 case with nausea and vomiting,the incidence rate of adverse reaction was 25 ％ (5/20) ;In the control group,there were 2cases with leukopenia,1 case with nausea and vomiting,there was no rash and paronychia,the incidence rate of adverse reaction was 15％ (3/20),there was no significant difference in incidence rate of adverse reaction between the two groups (P ＞ 0.05).Conclusion Cetuximab combined with FOLFOX6 can improve the performance clinical remission rate and lesion resection rate of k-ras wild type patients with liver metastases from colorectal cancer,and has high safety,which is worthy of clinical application.

Roles of K-ras gene in non-small cell lung cancer / 国际肿瘤学杂志

K-ras gene is one of oncogenes in non-small cell lung cancer,and it can promote tumor cells growth after mutations by several signaling pathways.K-ras mutations frequently occur in lung adenocarcinoma patients with smoking history.In the present study,K-ras mutations are associated with resistence to targated therapy and may be a marker of poor prognosis in patients with non-small cell lung cancer.

K-ras gene and colorectal cancer therapeutic effect / 国际肿瘤学杂志

K-ras gene is one of oncogenes,which promotes cells growth and differentiation.Ras protein loses the activity of GTP enzyme because of K-ras mutations,and that may cause abnormal growth,differentiation of cells and promote the occurrence of tumor.Patients with colorectal carcinoma that carry mutations in K-ras gene do not benefit from the administration of anti-epidermal growth factor receptor (EGFR) polyclonal antibodies.Different solid tumors of colorectal carcinoma have different K-ras mutation rates.Different genotypes of K-ras gene (wild-type or K-ras mutant type) affect significantly on treatment options and prognosis.The efficacy of mutant type patients with anti-EGFR antibodies is poor,and the therapeutic effects of 5-FU and FOLFOX are still unclear.

Evaluation of K-ras status concordance between primary colorectal cancer and related metastatic sites / 中国癌症杂志

Background and purpose:Metastatic colorectal cancer (mCRC) patients with K-ras mutation won’t benefit in the anti-epidermal growth factor receptor (EGFR) treatments. Thus K-ras mutation analysis is mandatory before this treatment. There is controversy that K-ras mutation analysis should be performed on primaries or related metastases. The aim of our study was to evaluate the concordance of K-ras status between primary and related metastases tumors, thus investigate the validity and rigorousness of clinical K-ras testing. Methods:Seventy-six patients with confirmed mCRC treated in Fudan University Shanghai Cancer Center were enrolled. After DNA extraction and PCR amplification, tumor specimens with paired primary tumors and related metastatic sites were put into sequencing analysis. And the K-ras mutation status in exon 2 was assessed. Results: K-ras mutation was detected in 31 out of 76 primary tumours (40.8%) and also 40.8%of the metastatic sites. But discordance was found between primary tumor and metastasis in 15 cases (19.7%):8 primary tumors had a K-ras mutation with a wild-type metastasis, meanwhile 7 primary tumors were wild type with a K-ras-mutated metastasis. Conclusion:Our study indicated that quite a few mCRC cases have different K-ras status between primary tumors and related metastatic sites, and it’s not very rigorous to choose the anti-EGFR treatments merely according to the primary tumor-K-ras mutation.Further study and consultation are needed on this problem.

Detection of K-ras mutation by PNA-PCR/K-ras method in diagnosis of colorectal cancer tissues / 第二军医大学学报

Objective To determine the positive judgement standard of K-ras mutation detection method peptide nucleic acid (PNA)-PCR/K-ras (previously established in our laboratory) and to assess its diagnostic value for colorectal cancer tissues. Methods Plasmidswith K-ras codon 12 mutation and plasmids with K-ras wild-type plasmids were mixed and serially diluted into standard samples (mutation/total: 0, 1/3 200, 1/1 600, 1/800, 1/400, 1/200, 1/100) for six independent tests. The mutation CT, total CT and ΔCT (mutation CT-total CT) values were obtained by PNA-PCR/K-ras method. After the cut-off values of the mutation CT and ΔCT for K-ras diagnosis were identified by ROC analysis, the diagnostic criteria for K-ras mutation was defined by combining both the cut-off values of the mutation CT and ΔCT. A comparison was made between K-ras diagnostic rate by PNA-PCR/K-ns method and direct sequencing for 35 colorectal cancer tissues and their corresponding adjacent noncancerous tissues. Results The mutation CT and ΔCT values for 1/800 and the above standard samples were significantly different from those of the negative samples(P<0. 05), with the optimumcut-off values of mutation CT and ΔCT being 41. 7 and 15. 4, respectively. The diagnostic criteria (mutation CT≤41. 7 or ΔCT≤15. 4) for K-ras mutation was set up asAUC-ROC 0. 955 (P=0. 001). According this diagnostic criteria, the K-ras mutation diagnostic rates in each concentration gradient of the standard samples (0, 1/3 200, 1/1 600, 1/800, 1/400, 1/200, and 1/100) were 0%,66.7%,83.3%,100%, 100%, 100%,and 100%, receptivity, with the upper diagnostic limit being 1/800. The diagnostic rates of K-ras mutation by our method and by direct sequence method for colorectal cancer tissues were 45. 7% (32/70) and 18. 6% (13/70), respectively, showing significant difference (P = 0. 000). Conclusion PNA-PCR/K- ras method has higher sensitivity and positive detection rate than direct sequencing method for colorectal cancer tissues.

Significance of quantitatively detecting K-ras codon 12 and 13 mutations in the tissues of pancreatic cancer and related pancreatic diseases / 中华胰腺病杂志

ObjectiveTo investigate the clinical significance of quantitative detection of K-ras codon 12 and 13 mutations in the tissues of pancreatic cancer and related pancreatic diseaaes. Methods One hundred and thirty samples from surgically removed pancreatic tissue with a conclusive pathological diagnosis (105 cases of pancreatic ductal adenocarcinoma,8 cases of pancreatic adenosquamous carcinoma of the pancreas,2 cases of pancreatic mucinous adenocarcinoma,3 cases of pancreatic endocrine carcinoma,6 cases of duodenal and papillary adenocarcinoma and 6 cases of benign pancreatic diseases ) were collected.Quantitative detection of K-ras codon 12 and 13 mutations was performed by the method of peptide nucleic acidmediated PCR clamping with two different fluorescence labeled probes.Mutation number ＞ 100 copies was used as the criteria to calculate the positive mutation rate.ResultsThe median and quartile of K-ras codon 12mutations of pancreatic ductal adenocarcinoma,adenosquamous carcinoma of the pancreas,pancreatic mucinous adenocarcinoma,pancreatic endocrine carcinoma,duodenal and papillary adenocarcinoma and benign pancreatic diseases were 4062 (495,10800),238 (45,8420),15 (9,21),3 (3,16),2283 (73,5037)and 21(8,56),and the positive mutation rates were 84.8％ (89/105),50.0％ (4/8),0,0,66.7％ (4/6)and 16.7％ (1/6).The quantity of K-ras codon 12 mutation in pancreatic ductal adenocarcinoma was not statistically different from those of adenosquamous carcinoma,duodenal and papillary adenocarcinoma,but it was significantly higher than those in pancreatic mucinous adenocarcinoma,pancreatic endocrine carcinoma,and benign pancreatic diseases (P ＜0.05).The area under ROC of K-ras codon 12 mutation in pancreatic ductal adenocarcinoma was 0.727.The sensitivity and specificity of the K-ras codon 12 mutation for the diagnosis of pancreatic ductal adenocarcinoma were 84.8％,64.0％,respectively.The quantity of K-ras codon 12 was associated with survival of patients with pancreatic ductal adenocarcinoma.The quantity of K-ras codon 13 mutations and the positive mutation rates in pancreatic ductal adenocarcinoma was not statistically different from other pancreatic diseases.ConclusionsThe quantity of K-ras codon 12 mutation has good differential diagnostic and prognostic prediction value for pancreatic ductal adenocarcinoma.

K-ras gene mutation in clinical diagnosis and risk factors of pancreatic cancer: A review / 第二军医大学学报

The early symptoms and laboratory findings of pancreatic cancer are nonspecific and the outcome of victims are poor. Currently surgical resection is the only means for cure. When pancreatic cancer is clinically suspected, it is usually non-resectable due to metastasis. So prevention and early diagnosis are especially important for pancreatic cancer. The risk factors of pancreatic cancer include male sex, old age, long-term smoking, coffee consumption, alcohol abuse, diabetes, chronic pancreatitis, hereditary factors, obesity, non-familial hypercholesterolemia, high caloric intake, and environmental factors. Many studies have indicated that pancreatic cancer is closely related to K-ras mutations, and patients with high susceptibility have a higher probability of K-ras mutations. This paper reviews the value of K-ras mutation in diagnosis of pancreatic cancer, K-ras mutation in pre-cancerous lesions, and the relation between high-risk factors and K-ras mutations.

Quantitative detection of mutation of K-ras gene at codon 12 / 中华胰腺病杂志

Objective To quantitatively analyze the K-ras gene mutation at codon 12 in pancreatic cancer tissues and the relationship between K-ras gene mutation and clinicopathological parameters. Methods Quantitative detection of K-ras gene at codon 12 in 93 pairs of pancreatic cancer and adjacent tissues were performed by using PNA-mediated PCR clamping with two different fluorescence labeled probes. The quantity of mutation was expressed by percentage of mutation. The percentage of K-ras gene mutation = the copy of K-ras mutation/(copy of wild type K-ras + copy of K-ras mutation) × 100%. Results The percentage of mutation of K-ras gene at codon 12 in pancreatic cancer and adjacent tissues were 83.9% and 65.6%, and the difference was statistically significant(P ＜ 0. 05); and the quantity of mutation were (13.385 ± 1. 745) % and (2. 246 ±0. 728) %, and the difference was also statistically significant(P ＜ 0. 05). The quantity of mutation of K-ras gene at codon 12 was not associated with clinicopathological parameters. Conclusions The percentage of K-ras gene mutation, as well as the quantity of K-ras gene mutation was different in pancreatic carcinoma and adjacent tissues.

Role of K-ras gene mutations in carcinogenesis, diagnosis and treatment of patients with lung adenocarcinoma / 第二军医大学学报

K-ras gene mutalion iscommonly seen in lung adenocarcinoma. Cigarette smoking is thought to be an important reason for K-ras gene mutation. Studies have repotted that K-ras mutations can influence the subtyping and prognosis of lung adenocarcinoma. Recently, the techniques to detect K-ras mutation have been continuously improved, greatly promoting the detection sensitivity of K-ras mutations, even in tissues with small amount of tumor component. Emerging data showed that K-ras mutation can induce primary resistance of lung adenocarcinoma to tyrosine kinase inhibitors (TKIs) and decrease the efficacy of chemotherapy. Therefore, K-ras mutation has attracted considerable attention as a target for diagnosis and anticancer therapy. In this review, we summarize recent studies on K-ras mutations in lung adenocarcinoma, and discuss the development, diagnosis, and treatment of lung adenocarcinoma.

The value of the K-ras mutations in FNA samples of pancreas on the diagnosis of pancreatic cancer / 中华胰腺病杂志

Objective To investigate the diagnostic value of the K-ras mutations in FNA samples for early detection of pancreatic cancer. Methods FNA samples of 27 patients with pancreatic cancers, 9 patients with other malignant tumors and 14 patients with non malignant pancreatic mass (NMPM) were collected. DNA was extracted, and K-ras gene was amplified through PNA-mediated PGR clamping, the products were sequenced to determine the mutation type. Results The positive rate of K-ras mutations in pancreatic cancers,other malignant tumors and NMPM were 88.9%, 44.4%, 35.7%. There was significant difference in K-ras gene mutations in FNA samples between pancreatic cancer and other malignant tumors ( P = 0. 013 ) and NMPM ( P = 0. 001 ). The sensitivity, specificity, positive predictive value, negative predictive value,accuracy of K-ras mutations in FNA samples of pancreatic cancers were 88.9%, 55.6%, 85.7%, 62.5%,80.6% when compared with other malignant tumors, and the difference between the two groups was significant (P =0. 013) ;Those were 88.9%, 64.3%, 82.8%, 75.0%, 80. 5% when compared with NMPM, and the difference between the two groups was significant ( P = 0. 001 ). When cytology of FNA samples and K-ras mutations was combined, the positive rate of pancreatic cancer was up to 96.3%. Conclusions The detection of K-ras mutations in EUS-FNA samples helped improve the positive diagnostic rate of pancreatic cancer.

Peptide nucleic acid-mediated one-step PCR assay in detection of K-ras mutation / 第二军医大学学报

Objective: To establish a peptide nucleic acid-mediated one-step PCR assay for detecting K-ras mutation, and to evaluate its diagnostic value. Methods: We developed a one-step polymerase chain reaction (PCR) approach with melting curve analysis using wild-type specific peptide nucleic acid (PNA) and fluorescent dye SYBR Green I to determine the genotypes in codon 12 and 13 of K-ras oncogene. The result of our method was compared with that of restriction fragment length polymorphism (RFLP) analysis. Results: Our method simultaneously examined condon 12 and 13 of K-ras oncogene, and was easy to perform. The sensitivity of our method was 0.001% when in a 105-fold excess of wild-type K-ras DNA. The detection limit was 102 copies. Our method could be used for large sample test, with the time of a single test being 1 hour. The limit of traditional method was 103 copies and the sensitivity was 0.01%. The testing time was about 2 days for samples which needed only 1 hour using our method. Conclusion: Our method has obvious advantage over RFLP analysis; it is suitable for detecting K-ras mutation in large samples and can be used as an effective method for early diagnosis of pancreatic cancer.

Screening for pancreatic caner by peptide nucleic acid-mediated one-step K-ras mutation detection assay / 中华胰腺病杂志

Objective To investigate the diagnostic value of determination of the genotypes in codon 12 and 13 of K-ras oncogene in blood samples of patients with pancreatic carcinoma(PC).Methods Blood samples were obtained from 54 patients with pathologically confirmed PC,and 33 healthy controls.The DNAs were obtained in these samples.and then genotype of K-ras mutation was detected by using the PNA-clamping real-time quantitative PCR.Then the correlation between the K-ras genotypes of blood DNA and the clinical characteristics was analyzed.Results K-ras mutations were found in 74.1%(40/54)of patients with PC.There was no such mutation in control samples.The mutations of K-ras was associated with age,lymph node and vessel invasion.poorly differentiated tumor,CA19-9,while it was not associated with sex,tumor location,size of tumor,clinical staging and pathological type.Conclusions The one-step method was highly sensitive for detecting K-ras mutation in blood samples.Detection of circular blood cells harboring K-ras mutation suggested the tumor was highly invasive with poor prognosis.

In Vitro Anti-tumor Immune Response Induced by Dendritic Cells Transfected with Recombinant Adenovirus Carrying Mutant K-ras Genes / 华中科技大学学报（医学）（英德文版）

The specific anti-tumor immune response induced by mouse bone marrow dendritic cells (DCs) transfected with recombinant adenovirus carrying mutant k-ras genes was investigated. DCs were generated from mouse bone marrow in the presence of rmGM-CSF (3.3 ng/mL) and rmIL-4 (1.3 ng/mL) and detected by FACS, and then transfected with the recombinant adenovirus encoding mutant k-ras gene. The efficacy of transfection and T cell stimulating activity of DCs were detected. CTL activity of the mice vaccinated with DCs was observed. The results showed that DCs had dendritic veiled morphology. BmDCs highly expressed B7-1 (80 %), B7-2 (77 %), MHC Ⅱ (70 %), CD11c (65 %), CD40 (70 %) and CD54 (96 %) with FACS, and no significant difference in the expression was observed before and after the transfection (P>0.05). The DCs transfected by mutant k-ras gene could significantly stimulate lymphocytes proliferation as compared with those transfected by Ad-c or non-modified DCs (P<0.05). DC vaccine transfected by mutant k-ras gene could induce CTL activity against Lewis lung cancer, but not against B16. The specific cytotoxicity against Lewis lung cancer in Ad-k-ras/12-transduced DC group was significantly higher than those in the control, vector and non-transfected DCs groups (P<0.05). It was concluded that special antitumor response could be induced by DCs transfected with recombinant adenovirus carrying mutant k-ras genes.

Detection of gene mutations with PCR and anti-colon cancer monoclonal antibody in tissue and stool of patients with colorectal cancer / 中国临床药理学与治疗学

AIM: To detect sequence and mutation of K-ras oncogene in tissue and stool DNA of patients with colorectal cancer in order to provide a method of noninvasive and simple colorectal cancer diagnosis. METHODS: DNA was separated and purified from colorectal cancer tissue or stool of patient with colorectal cancer, then the K-ras gene was amplified by PCR and PCR products were cloned, the K-ras gene was sequenced, and the mutation was identified. The expression of color/colorectal cancer antigen was inspected by immunohistochemical technique. Stool sample of patient with colorectal cancer was detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: K-ras gene sequence of the stool was completely same as that of the tissue of the patient;K-ras mutation was detected in one case. There was relativity between the mutation of K-ras gene and the pathology type of colorectal cancer and the expression level of colorectal cancer antigen in stool sample. CONCLUSION: It is feasible that colorectal tumors can be detected by a noninvasive method based on the molecular pathogenesis of the disease. Detecting K-ras gene mutations of stool DNA can provide bases for the screening, early detection, and prognosis to patients with colorectal cancer.

THE STUDY OF INFLUENCE OF LONG-TERM SMOKING ON THE EXPRESSION AND MUTATION OF p53 AND K-ras GENE IN RAT LUNGS / 解剖学报

Objective To observe the influence of long-term smoking on the expression of p53 and K-ras in rat lung tissues, and to study the relationship of smoking to the mutation of p53 and K-ras gene. Methods The model of Wistar rat smoking was built up. Seventy-two healthy male Wistar rats were divided into the experimental group and control group at random. The rats of the experimental group were compelled to smoke, and the rats of the control group were given the same condition as the experimental group was, without smoking. The rats of the experimental group were smoked for 6 months. At the end of each month, 6 rats were chosen from the two groups respectively, their lung tissues were sampled and immunohistochemistry was applied to observe the expression of p53 and K-ras in lung tissues. Finally, the mutation which might happen in the exon 5, 6, 7-8 of p53 and the exon 1 of K-ras was examined by PCR-SSCP. Results The p53 protein was expressed in cell nucleus and K-ras in cytoplasm. The positive ratio of protein expression was increased with the extension of smoking time. The mutation of p53 was increased as the smoking time extended. But the effect of smoking time was not that significant on the mutation of K-ras.Conclusion Smoking can strengthen the expression of p53 and K-ras protein and can also result in gene mutation. As the time of smoking extended, those phenomenons were tending to rise. That provided the theoretical evidence which can be used to judge the lesion of lung tissues caused by smoking and help the early diagnosis of smoking-related lung carcinomas. It is of great theoretical and application values.