Inhibition of Leukemic Cell Telomerase Activity by Antisense Phosphorothioate Oligodeoxynucleotides

Objective To evaluate the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynucleotide (ASODN) ontelomerase activity in K562 cells.Methods Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) in K562 cellstreated with ASODN and hTERT mRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR).Results The hTERT mRNA level was decreased, and teloraerase activity was significantly inhibited when the K562 cells were treated withASODN for 48 h.Conclusion It is suggested that hTERT ASODN might specifically inhibit telomerase activity of K562 cells at translation level, and it isfurther proved that hTERT gene has significant correlation with telomerase activity.

Origin and fate of the nucleolar channel system of normal human endometrium^1

Human normal endometrium was examined in ultrathin sections. Nucleolar channel system (NCS) appeared in the endometrial epithelial cells during the early and mid secretory phase of menstrual cycle. The NCS was a hollow ball like structure of different sizes and was composed of 2 to 5 rows of tubules embedded in an amorphous matrix. On its surface there were numerous electron dense particles resembling ribosonies. It was usually located within or associated with the nucleolus. Sometimes, it was close to the nuclear envelope or protruding out from the nucleus. On occasion, NCS with simplified structure was found in the perinuclear cytoplasm. Concepts concerning the genesis, involution and function(s) of the NCS were discussed.

Gene Analysis of Arsenic Trioxide—induced Apoptosis of Lymphoma Cells

Objective The effect of arsenic trioxide on apoptosis gene expression of Raji cell was explored when Raji cells were incubated with 0.5μmol/L of arsenic trioxide for 6h。Methods Cell culture,extraction and isolation of mRNA,preparation of probes labeled with fluorescence,hybridization technique of DNA chip(each chip containing 200 apoptosis genes,Chinese Shanghai Biostar,In.)were used.Results Arsenic trioxide induced significant changes in 10%(20/200 genes)of the apoptosis genes:18 genes were downregulated,only two upregulated.In particular,inhibitors of apoptosis protein,such as X-linked inhibitor of apoptosis protein,were significantly downregulated.P53 and the other apoptosis genes were also downregulatec.Of the upregulated genes,high expression of heat-shock protein could promote apoptosis of Raji cells.Conclusion The inhibitors of apoptosis protein play an important role in the process of arsenic trioxide-induced apoptosis of Raji cells.

Inhibition of Telomerase Activity of Lymphoblastic Leukemic Cells by hTERT Antisense

To investigate the effect of antisense, human telomerase reverse transcriptase (hTERT) mRNA oligodeoxynucleotide on telomerase activity of lymphoblastic leukemic cells. Methods: Telomerase activity was measured by the telomerase PCR ELISA assay kit (TRAP), hTERT protein by immunochemistry and flowcytometry, hTERT mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) assay and gel-image system. Results: Incubation of lymphoblastic leukemic cells (Jurkat, Raji and CEM cell lines) with 10 μmol/L AS PS-ODN could significantly decline the mRNA and hTERT after 72 h, and the telomerase activity was significantly down-regulated or inhibited. Conclusion: The hTERT AS PS-ODN was an excellent inhibitor for telomerase activity of lymphoblastic leukemic cells.

Effects of All－trans Retinoic Acid on hTERT Gene Expression and Telomerase Activity of HL－60 Cells

Objective: To investigate the effects of all-trans retinoic acid (ATRA) on human telomerase reverse transcriptase (hTERT) protein expression and telomerase activity in HL-60 cells. Methods: The expression of hTERT protein was assayed by immunofluorescence using fluoresce isothiocyanate label and telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay with HL-60 cells untreated or treated with ATRA. Cell cycle was analyzed by flow cytometry. Results: After treatment with 1μmol/L ATRA for 24, 48, 72 h, mean fluorescence intensity of hTERT protein in HL-60 cells was 61.87±4.36, 37.47±2.85， 33.45±2.37，respectively. There was a significant decrease in hTERT protein expression compared to the cells untreated, and the effect had statistically significant difference (P<0.05).Telomerase activity was decreased significantly in HL-60 cells treated with 1μmol/L ATRA for 48, 72h as compared to the cells untreated (P<0.05). Conclusion: ATRA could inhibit telomerase activity and hTERT gene expression in HL-60 cells.

Whole Cag A Gene Amplification of Helicobacter pylori and Its Fingerprinting by Restriction Fragment Length Polymorphism

To setup a method of amplification for the whole Cag A gene of H elicobacter pylori and its fingerprinting by restriction fragm entlength polym orphism(RFL P) ,nested PCR was employed in com bination with TD- PCR to am plify the gene and Eco RΙ and Hind were used to generate the RFL P fingerprinting.Target DNA fragm ents from 13of2 0 samples were successfully amplified and the relevant RFL P fingerprintings were obtained.It is concluded thatthe m ethod can be used to amplify the whole Cag A gene and Cag A gene has apparent diversity of RFL P profile.

Paclitaxel-induced activation of murine peritoneal macrophage in vitro

Objective:To study the effects of paclitaxel on macrophage activation.Methods:Mouse macrophages were isolated by peritoneal lavage and cultured in RPMI 1640 medium according to the following groups:paclitaxel(5μmol/L) group,IFN-γ(5U/L) group,paclitaxel (5μmol/L) and IFN-γ (5U/L) combination group, and control group(without paclitaxel and IFN-γ).24 hours later,supernatants were collected for nitric oxide(NO) assessment using the Griess reagent, and tumor necrosis factor-α(TNF-α) assessment using the enzyme linked immunosorbent assay.Antibody-dependent cell-mediated cytotoxicity(ADCC) of the macrophages was assessed using the method of hemoglobin-enzyme release assay (Hb-ERA).Results:Paclitaxel induced the production of higher levels of NO(8.86±1.16μmol/L) and TNF-α(120.2±10.2pg/ml),and enhanced the ADCC of macrophages[(20.61±1.13)%].The differences were significant compared with the control group[no NO and TNF-α detected,ADCC (15.37±1.93)%](P<0.01).Paclitaxel and IFN-γ in combination induced the production of higher levels of NO(22.85±0.91μmol/L) and TNF-α(358.6±27.5pg/ml),and enhanced the ADCC of macrophages[(42.49±3.09)%].The differences were significant compared with paclitaxel or IFN-γ[NO 8.09±1.13μmol/L,TNF-α 124.8±9.6pg/ml,ADCC (23.32±2.63)%] alone(P<0.01).Conclusion:These findings indicate that paclitaxel can promote NO and TNF-α production,enhance ADCC of macrophages,and induce macrophage activation.The active effects are more significant with paclitaxel and IFN-γ combination.

Anti-Tumor Effect of CDA-Ⅱ on a Human Glioma Cell

OBJECTIVE To examine the effect of uroacitide (CDA-Ⅱ ), an extraction product from normal human urine, on proliferation and differentiation of human glioma SWO-38 cells. METHODS The effects of CDA-Ⅱ on cellular survival and colony formation were determined by MTT and colony-formation assays. The in vivo anti-tumor effect of CDA-Ⅱ was examined on transplanted SWO-38 cells in nude mice. In addition, the aterations in cell morphology induced by CDA-Ⅱ were observed by H&E staining. RESULTS Treatment of SWO-38 cells with 1-5 mg/ml of CDA-Ⅱ for 3 days, resulted in 39.49% ± 5.27%-65.05% ± 5.89% growth inhibition with an IC50 of 2.52 mg/ml. Based on the colony-formation assay, 10 days of CDA-Ⅱ treatment at a level of 0.3-2.1 mg/ml caused 23.45% ± 0.62%-96.22% ± 1.01% inhibition with an IC50 of 1.03 mg/ml. Furthermore, the inhibitory response was dose-dependent. CDA-Ⅱ at dosage of 500 mg/kg or 2,000 mg/kg per day for 4 weeks significantly suppressed the growth of human glioma SWO 38 cells in nude mice, with inhibition being 47.77% and 79.94%, respectively (P <0.05, n=10). H&E staining and light microscopy revealed that CDA-Ⅱ induced differentiation of the SWO-38 cells. CONCLUSION CDA-Ⅱ has a significant anti-tumor effect on the human glioma SWO-38 cells, and is a potential and natural anti-glioma therapeutic reagent.