Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p...Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H2O2 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H2O2, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP. hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.展开更多
Mutations of the p53 tumor suppressor gene are the most frequent genetic alterations detected in human lung cancer. To assess the pathogenic significance of p53 gene alterations in Chinese non small cell lung cancer(...Mutations of the p53 tumor suppressor gene are the most frequent genetic alterations detected in human lung cancer. To assess the pathogenic significance of p53 gene alterations in Chinese non small cell lung cancer(NSCLC),74 paired samples of primary lung cancer and normal lung tissue far away from the cancer were analyzed for mutations of the p53 gene(exons 5 8) using exon specific PCR, single strand conformation polymorphism (PCR SSCP). p53 mutations were observed in 55 4%(41/74) of the samples. No linkages were detected between the incidence of p53 mutations and histological type, lymph node metastasis,age or sex. Significant association between p53 mutations and degree of differentiation in adenocarcinomas, not in squamous cell carcinomas, was observed. The frequency of p53 mutations in smokers(65 3%) was higher than in nonsmokers(33 3%) and reached statistical significance.We also found p53 mutations in 6/7 samples which had tissue invasion and distant metastasis.These results suggest that smoking could be an important factor in lung carcinogenesis,p53 mutation is a worse prognosis indicator in adenocarcinomas and related to high aggressive behavior of human lung cancer.展开更多
Objective： The aim of the study was to establish models of Hep-2 laryngeal cancer cell line of different oxygen supplying, trying to investigate the impact of normoxia, hypoxia, reoxygenation after hypoxia on apoptos...Objective： The aim of the study was to establish models of Hep-2 laryngeal cancer cell line of different oxygen supplying, trying to investigate the impact of normoxia, hypoxia, reoxygenation after hypoxia on apoptosis and expression of proteins HIF-1α and p53 to Hep-2 human laryngeal cancer cell line induced by ^60Co γ-ray. Methods： Human laryngeal cancer Hep-2 cells were divided into 3 groups： group A （normoxia）, group B （hypoxia）, and group C （reoxygenation after hypoxia）. All of the cells were exposed to 5 Gy dosage of γ-ray. Flow cytometry （FCM） was used tomeasure the protein levels of HIF-1α and p53 and to detect cell apoptosis. The protein levels of HIF-la and p53 were also determined by immunohistochemistry and Western blotting. The expression of HIF-la mRNA was determined by RT-PCR. Results： The protein levels of HIF-1α and p53 were evidently increased in group B compared to group A. The protein levels of HIF-1α and p53 in group C were lower compared to group B; the rate of apoptosis in group C was higher than that in group B. Conclusion： Hypoxia decreased the effect of apoptosis induced by ^60Co γ-ray in Hep-2 human laryngeal cancer cell line. The apoptosis pathway maybe related to some other genes or proteins but not p53 in the conditions of hypoxia and reoxygenation after hypoxia.展开更多
The presence of anti-p53 antibody in serum is a biomarker for cancer. However, its high sensitivity detection is still an issue in cancer diagnosis. To tackle this challenge, we used fd phage, a human-safe bacteria-sp...The presence of anti-p53 antibody in serum is a biomarker for cancer. However, its high sensitivity detection is still an issue in cancer diagnosis. To tackle this challenge, we used fd phage, a human-safe bacteria-specific virus nanofiber that can be mass-produced by infecting host bacteria in an error-free manner, and genetically engineered it to display a peptide capable of recognizing and capturing anti-p53 antibody on its side wall. We employed the resultant phage nanofibers as a capture probe to develop a modified version of the enzyme- linked immunosorbent assay （ELISA） method, termed phage-ELISA. We compared it to the traditional ELISA method for the detection of anti-p53 antibody, p53-ELISA, which uses recombinant wild-type p53 protein to capture anti-p53 antibody. We applied phage-ELISA to detect anti-p53 antibody in an experimental group of 316 patients with various types of malignant tumors. We found that a detection rate of 17.7% （56 positive cases） was achieved by phage-ELISA, which was comparable to the detection rate of 20.6% for p53-ELISA （65 positive cases）. However, when both phage and p53 were combined to form antibody-capturing probes for phage/p53-ELISA, a detection rate of 30.4% （96 positive cases） was achieved. Our work showed that owing to the combined capture of the anti-p53 antibody by both phage nanofibers and p53, the phage/p53-ELISA achieved the highest diagnostic accuracy and detection efficiency for the anti-p53 antibody in patients with various types of cancers. Our work suggests that a combination of nanofibers and antigens, both of which capture antibody, could lead to increased detection sensitivity, which is useful for applications in the life sciences, clinical medicine, and environmental sciences.展开更多
文摘Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H2O2 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H2O2, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP. hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.
文摘Mutations of the p53 tumor suppressor gene are the most frequent genetic alterations detected in human lung cancer. To assess the pathogenic significance of p53 gene alterations in Chinese non small cell lung cancer(NSCLC),74 paired samples of primary lung cancer and normal lung tissue far away from the cancer were analyzed for mutations of the p53 gene(exons 5 8) using exon specific PCR, single strand conformation polymorphism (PCR SSCP). p53 mutations were observed in 55 4%(41/74) of the samples. No linkages were detected between the incidence of p53 mutations and histological type, lymph node metastasis,age or sex. Significant association between p53 mutations and degree of differentiation in adenocarcinomas, not in squamous cell carcinomas, was observed. The frequency of p53 mutations in smokers(65 3%) was higher than in nonsmokers(33 3%) and reached statistical significance.We also found p53 mutations in 6/7 samples which had tissue invasion and distant metastasis.These results suggest that smoking could be an important factor in lung carcinogenesis,p53 mutation is a worse prognosis indicator in adenocarcinomas and related to high aggressive behavior of human lung cancer.
文摘Objective： The aim of the study was to establish models of Hep-2 laryngeal cancer cell line of different oxygen supplying, trying to investigate the impact of normoxia, hypoxia, reoxygenation after hypoxia on apoptosis and expression of proteins HIF-1α and p53 to Hep-2 human laryngeal cancer cell line induced by ^60Co γ-ray. Methods： Human laryngeal cancer Hep-2 cells were divided into 3 groups： group A （normoxia）, group B （hypoxia）, and group C （reoxygenation after hypoxia）. All of the cells were exposed to 5 Gy dosage of γ-ray. Flow cytometry （FCM） was used tomeasure the protein levels of HIF-1α and p53 and to detect cell apoptosis. The protein levels of HIF-la and p53 were also determined by immunohistochemistry and Western blotting. The expression of HIF-la mRNA was determined by RT-PCR. Results： The protein levels of HIF-1α and p53 were evidently increased in group B compared to group A. The protein levels of HIF-1α and p53 in group C were lower compared to group B; the rate of apoptosis in group C was higher than that in group B. Conclusion： Hypoxia decreased the effect of apoptosis induced by ^60Co γ-ray in Hep-2 human laryngeal cancer cell line. The apoptosis pathway maybe related to some other genes or proteins but not p53 in the conditions of hypoxia and reoxygenation after hypoxia.
基金Acknowledgements This study was supported by grants from the National Natural Science Foundation of China （No. 81028010）, Ministry of Science and Technology （No. 2014DFA31740） and the Department of Science and Technology of Jilin Province, China （Nos. 20130206009YY and 20130727034YY）. Y. Z., Z. G. J., P. H. Q. and C. B. M. also would like to thank the financial support from National Sdence Foundation （Nos. CMMI-1234957 and CBET-1512664）, National Institutes of Health （Nos. EB015190 and CA200504）, Department of Defense Peer Reviewed Medical Research Program （No. W81XWH- 12-1-0384）, Oklahoma Center for the Advancement of Science and Technology （No. HR14-160） and Oklahoma Center for Adult Stem Cell Research （No. 434003）.
文摘The presence of anti-p53 antibody in serum is a biomarker for cancer. However, its high sensitivity detection is still an issue in cancer diagnosis. To tackle this challenge, we used fd phage, a human-safe bacteria-specific virus nanofiber that can be mass-produced by infecting host bacteria in an error-free manner, and genetically engineered it to display a peptide capable of recognizing and capturing anti-p53 antibody on its side wall. We employed the resultant phage nanofibers as a capture probe to develop a modified version of the enzyme- linked immunosorbent assay （ELISA） method, termed phage-ELISA. We compared it to the traditional ELISA method for the detection of anti-p53 antibody, p53-ELISA, which uses recombinant wild-type p53 protein to capture anti-p53 antibody. We applied phage-ELISA to detect anti-p53 antibody in an experimental group of 316 patients with various types of malignant tumors. We found that a detection rate of 17.7% （56 positive cases） was achieved by phage-ELISA, which was comparable to the detection rate of 20.6% for p53-ELISA （65 positive cases）. However, when both phage and p53 were combined to form antibody-capturing probes for phage/p53-ELISA, a detection rate of 30.4% （96 positive cases） was achieved. Our work showed that owing to the combined capture of the anti-p53 antibody by both phage nanofibers and p53, the phage/p53-ELISA achieved the highest diagnostic accuracy and detection efficiency for the anti-p53 antibody in patients with various types of cancers. Our work suggests that a combination of nanofibers and antigens, both of which capture antibody, could lead to increased detection sensitivity, which is useful for applications in the life sciences, clinical medicine, and environmental sciences.
