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LMP1-target deoxyribozyme causes S phase arrest and induction radiosensitivity in LMP1-positive cells
Abstracts / Cell Biology International 32 (2008) S1eS67 Radio-sensitivity of gliomas determines radiotherapy efficacy. Evidence demonstrates that methylation of CpG island in the promoter region results in gene silencing. This study was designed to determine the relationship between methylation status of ERCC1 promoter and radio-sensitivity in glioma cell lines. Surviving fraction (SF2) was measured in 4 glioma cell lines (MGR1, MGR2, SF767 and T98G) by using colony forming experiment. Using bisulphate sequencing, we identified hypermethylation in the promoter region of ERCC1 in 4 gliomas cell lines. The result showed that ERCC1 gene promoter CpG islands were methylated in MGR1 and T98G cells, with a SF2 of 0.59 0.09 and 0.70 0.05 respectively. No methylamine of ERCC1 gene promoter CpG islands was found in MGR2 and SF767. SF2 of MGR2 and SF767 were 0.18 0.05 and 0.32 0.08 respectively. There was a statistical difference in the radio-sensitivity between glioma cells with or without methylamine of ERCC1 gene promoter CpG islands (t ¼ -4.437, P<0.05). Our data indicate that methylation status of ERCC1 was associated with radio-sensitivity in gliomas cell lines. It could be used as a new biomarker for predicting the radiosensitivity of human gliomas.
LMP1-TARGET DEOXYRIBOZYME CAUSES S PHASE ARREST AND INDUCTION RADIOSENSITIVITY IN LMP1POSITIVE CELLS Xiao Qian Ma, Li Fang Yang, Li Li Li, Ya Cao Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, PR China This study was designed to determine the molecular mechanism underlying the radiosensitive effect of the LMP1-target deoxyribozyme on nasopharyngeal carcinoma cells (NPC), CNE1-LMP1, which constitutively expresses the LMP1.We observed active DNAzyme induced an S phase growth arrest correlated with reduction in Rb phosphrylation at S473, decrease in the levels of components of the cell cycle moleculars and suppression the interaction of CDK4 and cyclinD1. We showed that the DNAzyme down-regulated the expression of cyclinD1 and E, and sharply decreased in the level of E2F1. All experiments were done on CNE1 cells, which are LMP1-negative. There were no changes at all. These findings demonstrate that LMP1-target DNAzyme induced cell cycle blockade relied on modulated expression and activity of G1 and S phase associated regulatory proteins. Our results suggest that the DNAzyme induced S phase arrest may cause the LMP1-positive cells to be more sensitive, because when combined with radiation treatment, the DNAzyme significantly induced apoptosis in LMP1-positive cells. The DNAzyme may well be a clinical appealing new class of radio-sensitizing agent.
A TUMOR DOWN-REGULATED GENE NOR1 INDUCED BY HYPOXIA PROMOTE APOPTOSIS IN HELA CELLS Jue Ouyang 1, Song Qing Fang* 2, Bo Xiang 1, Ming Hua Wu 1, Shu Ping Peng 1, Li Cao 1, Gui Yuan Li* 1 1 Cancer Research Institute of Central South University Xiang-Yale School of Medicine, Changsha, Hunan, 410078, China 2 Pathology department of the 2nd Xiang-Yale Hospital of Central South University Changsha, Hunan, P.R.China Cobalt chloride could fluctuate the level of reactive oxygen species (ROS) and change the cytosol redox state. In this paper, we found the expression level of nitro oxidored reductase like gene (NOR1), which has the characteristic of reductase, a novel member of nitroreductases in cancer of cervix, nasopharyngeal, liver, stomach, colon and rectum, was significantly lower than those in their corresponding benign tissue shown with combination of tissue array and in situ hybridization (ISH) (p<0.05). However, we failed to indicate the correlation between tumor clinical pathology stage and the expression level of NOR1 (p>0.05). After induced by transition metal cobalt, NOR1 was up-regulated and the level of apoptosis of Hela was increased. Hence the effects of overexpression of NOR1 on Hela were investigated. Results showed that NOR1 was up-regulated after 12 hour with induction and maintained till 24 hour. NOR1 reduced proliferation of the Hela cells and promoted apoptosis. NOR1 also reduced the ability to form colonies in soft agar. The effects of overexpression of NOR1 in Hela were consistent
S33
with those treated by cocl2. Our results suggest that the NOR1 could suppress the tumor growth by promoting apoptosis and suppressing cell proliferation. This work was supported by National nature sciences foundation of china (No. 30400084, No. 30200312), Hunan province nature sciences foundation (No. 04JJ3096).
ARSENITE UPREGULATES EGR1 IN HaCaT CELL LINE Min Qi 1,2, Wei Qi Zeng 1, Mihee Kim 1, Zhi Min Yuan 1 1 Department of Cancer Cell Biology, Harvard University School of Public Health, Boston, MA 2 Department of Burns and Plastic Surgery, Xiangya Hospital, Central South University, Changsha, P.R. China Arsenite executes its carcinogenic effects by induction of signaling cascades. We analyzed whether arsenite had an impact on the biosynthesis of EGR1. Here we showed by Western blot that arsenite induced a transient synthesis of EGR1 in human keratinocyte HaCaT cell line. This arsenite triggered EGR1 biosynthesis was completely inhibited by the epidermal growth factor receptor-specific tyrosine kinase inhibitor and the mitogen-activated protein kinase inhibitor. These results indicate that activation of EGFR as well as stimulation of the mitogen activated/extracellular signal-regulated protein kinase was essential for arsenite-induced upregulation of EGR1. The fact that low dose of arsenite was sufficient to induce EGR1 biosynthesis suggests that EGR1 may be an integral part of arsenite-triggered signal transduction leading to tumor formation or cell death.
THE ASSOCIATION OF E-CADHERIN/CONNEXIN32 EXPRESSION WITH THE METHYLATION STATUS OF THE E-CADHERIN/CONNEXIN32 GENE IN Hep-G2 HEPATOCELLULAR CARCINOMA CELLS Jian Zhen Ren, Ji Rong Huo*, Wen Fang Hu, Shi Hua Chen, Chang Mei Hu, Nai Xin Kang Department of Gastroenterology, Xiangya 2nd Hospital, Central South University, Changsha, China E-cadherin (E-cad)/Connexin32 (Cx32), a cell adhesion molecule, is regarded as an invasion-suppressor molecule and a prognostic marker in many types of human cancers. Loss of E-cad/Cx32 has been associated with progression and poor survival in hepatocellular carcinoma (HCC). This study was to clarify the role of methylation on E-cad/Cx32 inactivation in HCC cell line (Hep-G2). We examined 5’ CpG island promoter methylation of E-cad/Cx32 in Hep-G2 cell line using methylation-specific PCR (MSP), and further studied the correlation of E-cad/Cx32 gene methylation with E-cad/Cx32 protein expression. We found that hypermethylation of E-cad/Cx32 was involved in Hep-G2 cell line; E-cad/Cx32 protein expression was lost in this cell line. Methylation, which was noted in this cell line, included fully methylated and partially methylated. The fully methylated cell line lacked E-cad/Cx32 protein expression; E-cad/Cx32 expression was reduced in a part of the methylated cell line but preserved in the other part. Treatment of E-cad/Cx32-negative carcinoma cells with the demethylating agent, 5-aza-2’-deoxycytidine, induced re-expression of the gene. These results suggest 5’ CpG island methylation of E-cad/Cx32 gene may play an important part in the inactivation of E-cad/Cx32 in HCC; and the demethylation effect may contribute to enhancing cell adhesion through re-expression of E-cad/Cx32, which may be a potential therapeutic strategy for HCC.
METHYLENETETRAHYDROFOLATE REDUCTASE POLYMORPHISMS AND SUSCEPTIBILITY TO GASTRIC CANCER IN CHINESE POPULATIONS: A META-ANALYSIS Ye Huan Sun, Liang Sun, Chen Yu, Bo Wang, Hong Yuan Cao Department of Epidemiology and Health Statistics, Anhui Medical University, Hefei 230032, China Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene are thought to have significant effects on folate metabolism thus on cancer risk but the reported results are not always consistent. In this meta-analysis we assessed reported studies of associations between polymorphisms of MTHFR susceptibility to gastric cancer in Chinese populations. A
LMP1-TARGET DEOXYRIBOZYME CAUSES S PHASE ARREST AND INDUCTION RADIOSENSITIVITY IN LMP1POSITIVE CELLS Xiao Qian Ma, Li Fang Yang, Li Li Li, Ya Cao Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, PR China This study was designed to determine the molecular mechanism underlying the radiosensitive effect of the LMP1-target deoxyribozyme on nasopharyngeal carcinoma cells (NPC), CNE1-LMP1, which constitutively expresses the LMP1.We observed active DNAzyme induced an S phase growth arrest correlated with reduction in Rb phosphrylation at S473, decrease in the levels of components of the cell cycle moleculars and suppression the interaction of CDK4 and cyclinD1. We showed that the DNAzyme down-regulated the expression of cyclinD1 and E, and sharply decreased in the level of E2F1. All experiments were done on CNE1 cells, which are LMP1-negative. There were no changes at all. These findings demonstrate that LMP1-target DNAzyme induced cell cycle blockade relied on modulated expression and activity of G1 and S phase associated regulatory proteins. Our results suggest that the DNAzyme induced S phase arrest may cause the LMP1-positive cells to be more sensitive, because when combined with radiation treatment, the DNAzyme significantly induced apoptosis in LMP1-positive cells. The DNAzyme may well be a clinical appealing new class of radio-sensitizing agent.
A TUMOR DOWN-REGULATED GENE NOR1 INDUCED BY HYPOXIA PROMOTE APOPTOSIS IN HELA CELLS Jue Ouyang 1, Song Qing Fang* 2, Bo Xiang 1, Ming Hua Wu 1, Shu Ping Peng 1, Li Cao 1, Gui Yuan Li* 1 1 Cancer Research Institute of Central South University Xiang-Yale School of Medicine, Changsha, Hunan, 410078, China 2 Pathology department of the 2nd Xiang-Yale Hospital of Central South University Changsha, Hunan, P.R.China Cobalt chloride could fluctuate the level of reactive oxygen species (ROS) and change the cytosol redox state. In this paper, we found the expression level of nitro oxidored reductase like gene (NOR1), which has the characteristic of reductase, a novel member of nitroreductases in cancer of cervix, nasopharyngeal, liver, stomach, colon and rectum, was significantly lower than those in their corresponding benign tissue shown with combination of tissue array and in situ hybridization (ISH) (p<0.05). However, we failed to indicate the correlation between tumor clinical pathology stage and the expression level of NOR1 (p>0.05). After induced by transition metal cobalt, NOR1 was up-regulated and the level of apoptosis of Hela was increased. Hence the effects of overexpression of NOR1 on Hela were investigated. Results showed that NOR1 was up-regulated after 12 hour with induction and maintained till 24 hour. NOR1 reduced proliferation of the Hela cells and promoted apoptosis. NOR1 also reduced the ability to form colonies in soft agar. The effects of overexpression of NOR1 in Hela were consistent
S33
with those treated by cocl2. Our results suggest that the NOR1 could suppress the tumor growth by promoting apoptosis and suppressing cell proliferation. This work was supported by National nature sciences foundation of china (No. 30400084, No. 30200312), Hunan province nature sciences foundation (No. 04JJ3096).
ARSENITE UPREGULATES EGR1 IN HaCaT CELL LINE Min Qi 1,2, Wei Qi Zeng 1, Mihee Kim 1, Zhi Min Yuan 1 1 Department of Cancer Cell Biology, Harvard University School of Public Health, Boston, MA 2 Department of Burns and Plastic Surgery, Xiangya Hospital, Central South University, Changsha, P.R. China Arsenite executes its carcinogenic effects by induction of signaling cascades. We analyzed whether arsenite had an impact on the biosynthesis of EGR1. Here we showed by Western blot that arsenite induced a transient synthesis of EGR1 in human keratinocyte HaCaT cell line. This arsenite triggered EGR1 biosynthesis was completely inhibited by the epidermal growth factor receptor-specific tyrosine kinase inhibitor and the mitogen-activated protein kinase inhibitor. These results indicate that activation of EGFR as well as stimulation of the mitogen activated/extracellular signal-regulated protein kinase was essential for arsenite-induced upregulation of EGR1. The fact that low dose of arsenite was sufficient to induce EGR1 biosynthesis suggests that EGR1 may be an integral part of arsenite-triggered signal transduction leading to tumor formation or cell death.
THE ASSOCIATION OF E-CADHERIN/CONNEXIN32 EXPRESSION WITH THE METHYLATION STATUS OF THE E-CADHERIN/CONNEXIN32 GENE IN Hep-G2 HEPATOCELLULAR CARCINOMA CELLS Jian Zhen Ren, Ji Rong Huo*, Wen Fang Hu, Shi Hua Chen, Chang Mei Hu, Nai Xin Kang Department of Gastroenterology, Xiangya 2nd Hospital, Central South University, Changsha, China E-cadherin (E-cad)/Connexin32 (Cx32), a cell adhesion molecule, is regarded as an invasion-suppressor molecule and a prognostic marker in many types of human cancers. Loss of E-cad/Cx32 has been associated with progression and poor survival in hepatocellular carcinoma (HCC). This study was to clarify the role of methylation on E-cad/Cx32 inactivation in HCC cell line (Hep-G2). We examined 5’ CpG island promoter methylation of E-cad/Cx32 in Hep-G2 cell line using methylation-specific PCR (MSP), and further studied the correlation of E-cad/Cx32 gene methylation with E-cad/Cx32 protein expression. We found that hypermethylation of E-cad/Cx32 was involved in Hep-G2 cell line; E-cad/Cx32 protein expression was lost in this cell line. Methylation, which was noted in this cell line, included fully methylated and partially methylated. The fully methylated cell line lacked E-cad/Cx32 protein expression; E-cad/Cx32 expression was reduced in a part of the methylated cell line but preserved in the other part. Treatment of E-cad/Cx32-negative carcinoma cells with the demethylating agent, 5-aza-2’-deoxycytidine, induced re-expression of the gene. These results suggest 5’ CpG island methylation of E-cad/Cx32 gene may play an important part in the inactivation of E-cad/Cx32 in HCC; and the demethylation effect may contribute to enhancing cell adhesion through re-expression of E-cad/Cx32, which may be a potential therapeutic strategy for HCC.
METHYLENETETRAHYDROFOLATE REDUCTASE POLYMORPHISMS AND SUSCEPTIBILITY TO GASTRIC CANCER IN CHINESE POPULATIONS: A META-ANALYSIS Ye Huan Sun, Liang Sun, Chen Yu, Bo Wang, Hong Yuan Cao Department of Epidemiology and Health Statistics, Anhui Medical University, Hefei 230032, China Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene are thought to have significant effects on folate metabolism thus on cancer risk but the reported results are not always consistent. In this meta-analysis we assessed reported studies of associations between polymorphisms of MTHFR susceptibility to gastric cancer in Chinese populations. A