Effects of ZNF331 overexpression on human colon cancer cell proliferation and apoptosis / 军事医学

Objective To investigate the effects of ZNF331 overexpression on proliferation and apoptosis of human colon cancer cell HCT116, and the relevant apoptotic mechanism.Methods The lentivirus vector of overexpressed ZNF331,Flag-pLV-Neo-ZNF331,was constructed and packaged.HCT116/p53 +/+(wild type p53)and HCT116/p53 -/-(deficient p53)cells were infected.Clones with ZNF331 overexpression were identified by Western blotting.Cell proliferation assay,colony formation assay and flow cytometry analysis were used to examine the effects of ZNF 331 on cell proliferation and apoptosis.Immunoprecipitation,luciferase reporter gene assay and real-time PCR were performed to detect interactions between ZNF331 and p53, p53 transcriptional activity and the expression of p 53 apoptotic target genes, respectively.Results The lentivirus vector of overexpressed ZNF 331 was successfully generated.Stable clones of ZNF331 overexpression were established.ZNF331 showed no significant effect on cell proliferation of HCT 116/p53 +/+, but inhibited cell proliferation of HCT116/p53 -/-(P<0.01).ZNF331 could interact with p53,dose-dependently inhibit the transcriptional activity of p53 and downregulate the mRNA levels of pro-apoptotic p53 target genes, Puma and p53AIP1 (P<0.05).ZNF331 could suppress p53-induced apoptosis(P <0.01).Conclusion The influence of ZNF331 overexpression on colon cancer cell proliferation is dependent on p 53 status.ZNF331 overexpression can suppress colon cancer cell apoptosis by interacting with p 53 and inhibiting the transcriptional activity of p 53.

Experimental BCSC-1 gene therapy on nasopharyngeal carcinoma mediated by adenovirus / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the therapeutic value of BCSC-1 in tumor gene therapy.</p><p><b>METHODS</b>Recombinant adenovirus Ad5-BCSC-1 was prepared. Cell proliferation was assayed using CellTiter 96 Aqueous one solution cell proliferation assay kit. Ad5-BCSC-1 was injected into tumor with Ad5-egfp or with PBS injection as controls. The injections were repeated one weak later. After another 2 weeks, the mice were sacrificed and the tumors were excised and weighed.</p><p><b>RESULTS</b>The growth of the CNE-2L2 cell infected with Ad5-BCSC-1 in vitro was remarkably slower than that of the controls, the wild type cell and the cell infected with Ad5-egfp. In contrast to the controls, the cells infected with Ad5-BCSC-1 aggregated and formed huge messes in the culture. The average weight of the CNE-2L2 tumors in mice was (2.28 +/- 0.73), (2.07 +/- 0.40), and (0.58 +/- 0.32) g for the tumors injected with PBS, Ad5-egfp, and Ad5-BCSC-1, respectively (Ad5-BCSC-1 vs. PBS or Ad5-egfp, P<0.05).</p><p><b>CONCLUSION</b>Intra-tumor injection of Ad5-BCSC-1 can suppress the growth of CNE-2L2 tumor in nude mice, suggesting that BCSC-1 gene therapy may be effective for tumors with low or no expression of BCSC-1 gene.</p>

Construction and identification of a eukaryotic expression vector for APP695 gene containing green fluorescence protein gene / 中华神经医学杂志

Objective To construct a eukaryofic expression vector for A PP695 gene carrying green fluorescence protein (GFP) gene. Methods RT-PCR was used to amplify the full-length APP695 cDNA. The PCR products and the eukaryotic expression vector pIRES2-EGFP were digested by restriction endonueleases, and the digested APP695 gene was inserted into the digested eukaryotic expression vector. The positive recombinants were identified by PCR analysis, Nhe Ⅰ and Hind Ⅲ digestion and sequence analysis. Results The 2088-bp DNA fragment was amplified by PCR from the plasmid pCB6, and an identical DNA fragment was also amplified from the recombinants. The products of double restriction enzyme digestion were A PP695 gene with a 5.3-kb DNA fragment. Sequence analysis confirmed successful insertion of A PP695 gene into pIRES2-EGFP vector. Conclusion The eukaryotie expression vector pIRES2/APP695-EGFP has been successfully constructed.

Inhibition of malignant activities of nasopharyngeal carcinoma cell by ectopic expression of BCSC-1 gene / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study effects of ectopic expression of BCSC-1 gene on the malignant activi-BCSC-1 cDNA was isolated by RT-PCR ties of human nasopharyngeal carcinoma cell CNE-2L2.</p><p><b>METHODS</b>and inserted into pMAL-c2X and pcDNA4/myc-His A. BCSC-1 protein was expressed in prokaryocytes. Rabbit antiserum to BCSC-1 was developed by means of immunization of rabbit with the BCSC-1 protein. Expression of BCSC-1 gene in wild type CNE-2L2 cell (W cell) was examined by real-time RT-PCR and immunofluorescence staining with the antiserum as a probe. pcDNA4/myc-His A-BCSC-1 was transfected into W cell at the presence of LipofectAmine. The cells were selected by G418 and cloned. Ectopic expression of BCSC-1 gene in W cell was examined by Western blot. Cell growth was detected by drawing of growth curves and colony formation tests. Cells were inoculated into nude mice. Size of tumors was assayed once a week. Lungs of the mice were sectioned continuously and metastatic loci in lungs were examined upon a microscope.</p><p><b>RESULTS</b>Rabbit BCSC-1 antiserum was prepared. Expression of BCSC-1 gene in W cell was found to be very low. CNE-2L2 cell with ectopic expression of BCSC-1 gene was developed. Growth in vitro, colony formation, tumorigenesis in nude mice, and lung metastasis of the tumor were profoundly inhibited of the cell with ectopic expression of BCSC-1 gene in comparison with controls, wild type cell and the cell transfected with mock. Conclusion Ectopic expression of BCSC-1 gene exerts profound inhibitive effect on the malignant activities of CNE-2L2 cell.</p>

Ectopic expression of BCSC-1 gene results in enhancement of adhesion and cell cycling blockade of nasopharyngeal carcinoma CNE-2L2 cell / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study mechanisms of reduction of the malignant activities of human naso-pharyngeal carcinoma cell CNE-2L2 induced by ectopic expression of BCSC-1 gene.</p><p><b>METHODS</b>DNA was stained with propidium iodide and assayed upon a flow cytometer. Chromosomes were stained with Hoechest 33258. Adhesion of CNE-2L2 cells was detected by cell aggregation test. Protein expression on CNE-2L2 cells was examined by Western blot.</p><p><b>RESULTS</b>Cell cycle analysis showed that the percentage of CNE-2L2 cells was 55.1%, 43.4%, and 39.4% in G0/G1 phase, 25.2%, 28.7%, and 30.9% in S phase, and 19.7%, 27.9%, and 29.7% in G2/M phase for the cell with ectopic expression of BCSC-1 gene, wild type cell (W cells), and the cell transduced with the mock (M cell). Many mitotic cells were found in W cells and M cells. In contrast, almost no mitotic cell was observed in the cells with ectopic expression of BCSC-1 gene. Ectopic BCSC-1 expression resulted in cell aggregation, enhanced expression of E-cadherin, cx-catenin, and p53.</p><p><b>CONCLUSIONS</b>Ectopic BCSC-1 expression causes enhancement of adhesion of CNE-2L2 cells associated with enhanced expression of E-cadherin and alpha-catenin, arrest of cell in G1 phase, which may be associated with enhanced expression of p53. These alteration may play a role in the reduction of malignant activities of the cells with ectopic expression of BCSC-1 gene.</p>

Effects of human alpha-mannosidase Man2c1 transgene on growth and metastasis of transplanted tumor in mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of human alpha-mannosidase Man2c1 transgene on tumor growth and metastasis in mice.</p><p><b>METHODS</b>Hepatoma cell H22 or squamous epithelial carcinoma cell S180 was subcutaneously inoculated into the right armpit of mice (wild type mice and 28#, 35#, and 54# transgenic mice). Tumor size was measured every week. Mice were sacrificed on day 9 or 10 and then the tumors were exercised and weighted. Tumors and lungs were fixed in formaldehyde and sectioned. The sections were stained with hematoxylin/eosin and examined under microscope. The red blood cells in spleen were destroyed by Tris-NH4Cl. Natural killer (NK) cell activity was detected with Yac-1 cell as target.</p><p><b>RESULTS</b>H22 and S180 tumors grew faster in all the three transgenic mice (28#, 35#, and 54#) than in wild type mice. The average size and weight of tumors between the transgenic mice and wild type mice were significantly different (P<0.05). Most tumors in the transgenic mice invaded the surrounding tissues. In contrast, nearly all the tumors in wild type mice were capsulized. Three of 10 28# transgenic mice, 5 of 10 35# transgenic mice, 3 of 10 54# transgenic mice, and 1 of 10 wild type mice showed lung metastasis of H22 tumor. Two of 6 28# transgenic mice, 3 of 6 35# transgenic mice, 1 of 6 54# transgenic mice, and 0 of 6 wild type mice showed lung metastasis of S180 tumor. No difference of NK activity in spleen cells was observed between the transgenic mice and wild type mice.</p><p><b>CONCLUSIONS</b>hMan2c1 transgene promotes growth, invasion, and metastasis of transplanted H22 and S180 tumors in mice. hMan2cl transgene does not affect NK activity in splenocytes.</p>

Reduction of CD44 expression results in growth inhibition of human nasopharyngeal carcinoma cell CNE-2L2 in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of the inhibition of CD44 gene expression on the growth of human nasopharyngeal carcinoma cell CNE-2L2 in vitro.</p><p><b>METHODS</b>CD44 gene expression in cells was suppressed by siRNA which was introduced into cells through retrovirus infection. Integration of siRNA into genomic DNA was examined by genomic PCR. CD44 gene expression in cells was detected by Western blot analysis. Cell growth in vitro was assayed using Cell Titer 96 AQueous One Solution Cell Proliferation Assay kit Promega. Cells were stained with propidium iodium and cell DNA content was detected upon a flow cytometer.</p><p><b>RESULTS</b>siRNA was integrated into genomic DNA of host cells. The 4 cell pools integrated with one of the 4 siCD44s showed a significant inhibition of CD44 gene expression comparing to the controls, the wild type cell and the cell pool integrated with siegfp. The cell pools integrated with siCD44-1 or siCD44-2 showed the most profound inhibition. Growth of these 2 cells in vitro was compared to that of the controls and was found to be significantly inhibited. Cell DNA content analysis indicated 44.4%, 45.5%, 53.9%, and 53.3% in G0/G1 phase; 39.3%, 40.0%, 27.1%, and 28.2% in S phase; and 16.3%, 14.5%, 19.0%, and 18.5% in G2/M phase for the wild type cell, the cell pool integrated with siegfp, the cell pools integrated with siCD44-1, and the cell pools integrated with siCD44-2, respectively.</p><p><b>CONCLUSION</b>Reduction in CD44 expression inhibit the growth of CNE-2L2 cell and affects the development of cells from G0/G1 into S phase, but may somehow promote cells to develop from S into G2/M phase.</p>

8B7 spectrin--a new member of spectrin family / 中国医学科学院学报

<p><b>OBJECTIVE</b>To analyze the nature of the protein encoded by 8B7cDNA (1 835 bp) and to examine the localization of the protein in cells.</p><p><b>METHODS</b>The nature of the protein was analyzed using Blastn, Blastp, and TMpred. Expressions of 8B7 mRNA in tissues and cells were examined by Northern blotting. Recombinant expression vectors for localization test were constructed and transfected into COS-7 cells. Fluorescence emission in cells was examined upon a laser scan confocal microscope.</p><p><b>RESULTS</b>The protein encoded by 8B7cDNA was 363 amino acids long and contained spectrin repeats. It was completely homologous to the C-terminal 363 amino acids of Enaptin, Nasprin-1, Mynel, and Syne-1 proteins. It belonged to Spectrin super-family and was called 8B7 spectrin. Northern blotting revealed a 1.8 kb mRNA expression in human spleen and small intestine tissues. EGFP-8B7 fusion protein was observed to express on the nuclear membrane and in the cytoplasm in a reticular form in a localization assay on COS-7 cells. The position of fluorescence in COS-7 cells transfected with pEGFP-delta SR8B7 was similar to that in the cells transfected with pEGFP-8B7cDNA.</p><p><b>CONCLUSIONS</b>8 B7 spectrin is a novel member of spectrin super-family. It localizes to the nuclear membrane and the cytoplasm in a reticular form in COS-7 cells. The localization is determined by the C-terminal KASH domain in 8B7 spectrin molecule.</p>