Apoptosis and Lipolysis of White Adipocytes Induced by Neuropeptide Y- Y5 Receptor Antisense Oligodeoxynucleotides

Objective: To investigate the influence of central administration ofneuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs) on body weight, fat pads of SDrats, and the effects of white adipocytes lipolysis and apoptosis. Methods: Y5 receptor antisense,sense, mismatched ODNs or vehicle was intracerebroventricularly (i. c. v.) injected. Averageadipocyte area was calculated. DNA ladders were measured to evaluate adipocyte apoptosis, and RT-PCRwas used to analyse the expression of Bcl-2 and Bax gene. Results: Central administration of Y5antisense ODNs significantly decreased body weight, and average adipocyte area. DNA fragmentationwas present after electrophoresis at epididymal adipose tissue. The expression of Bcl-2 gene wasdownregulated, while the expression of Bax upregulated. Conclusion: Lipolysis and adipocyteapoptosis may be important mechanisms far 75 antisense therapy.

INHIBITION EFFECT OF VASCULAR ENDOTHELIAL GROWTH FACTOR ANTISENSE OLIGODEOXYNUCLEOTIDES ON C6 GLIOMA

Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by S-P immunohistochemical technique. Tumor cell apoptosis was observed by TUNEL method. Results: Compared with control, VEGF protein expression was inhibited by antisense oligodeoxynucleotides in vitro. And the inhibitory effects increased with the increasing concentration. VEGF positive rate was 82.10% in control group, while in 2.5, 5, 10 mmol/L AODN groups, they were 70.00%, 57.85%, 53.20% respectively. No inhibition effect was found in the cell lines treated with missense and sense oligodeoxynucleotides. In vivo, antisense oligodeoxy-nucleotides therapy also inhibited VEGF protein expression and induced the increase of apoptotic tumor cells. However, it has no effect on tumor cell proliferation. Conclusion: It is hopeful that VEGF antisense oligodeoxynucleotides may be a new gene therapy method to glioma through its antiangiogenesis effect by inhibition of VEGF.

Effect of C-myc Antisense Oligodeoxynucleotides on Hypoxia-induced Proliferation of Pulmonary Vascular Pericytes

To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labeled cDNA, 3H thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c myc antisense ODNs on expression of c myc gene and proliferating cell nuclear antigen (PCNA), and 3H thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c myc and PCNA ( P <0.01), and elevate 3H thymidine incorporation of PC ( P <0.01), but antisense ODNs could significantly inhibit the expression of c myc and PCNA ( P <0.05), and 3H thymidine incorporation of PC ( P <0.01). It was suggested that hypoxia could promote the proliferation of PC by up regulating the expression of c myc gene, but c myc antisense ODNs could inhibit hypoxia induced proliferation of PC by downregulating the expression of c myc gene.

The effect of antisense oligodeoxynucleotides targeting Aurora A kinase on cell proliferation and chemosensitivity to paclitaxel in human lung cancer cell line A549

Objective： Aurora A kinase representing a family of evolutionadly conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpression of Aurora A contributes to the carcinogenesis, chromosomal instability （CIN）, and de-differentiation of lung cancers. To address its possibility as a therapeutic target for lung cancer, we employed the antisense oligodeoxynucleotide （ASODN） technique to inhibit Aurora A expression and investigate its effects on tumor growth and cell cycle of A549, as well as the chemosensitivity to paclitaxel. Methods： Aurora AASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000. Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot respectively. Cell cycle distribution was observed by flow cytometer. MTT assay was used to evaluate cell inhibition ratio before and after transfection. Results： The proliferation of the A549 cells was inhibited by Aurora A ASODN dose and time dependently. It was also observed that the IC50 of A549 cells after 48 hours＇ treatment of ASODN was about 300 nmol/L and under such circumstances, the Aurora A mRNA and protein expression significantly decreased （P 〈 0.05）, along with the induction of accumulation of cells in S phase and the G2-M transition. Furthermore, cell inhibition ratio of the combination of Aurora AASODN and paclitaxel was higher significantly than paclitaxel （P 〈 0.05） or Aurora AASODN alone （P 〈 0.05）. Conclusion： Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensitizing activity to paclitaxel in human lung cancer cell line A549.

Selection of Effective Bcl-2 Antisense Oligodeoxynucleotides with Computer Software and Experimental Assay

Objective： To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods： Bcl-2 gene was used as the target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal secondary structure regions that were predicted free from intramolecular fold or instability of free energy. The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed and determined with morphonological method, electrophoresis and flowcytometry. Results： The results showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to induce apoptosis of leukemia cells significantly. Conclusion： The computational prediction of antisense efficacy is faster than other methods and more efficient, which can potentially speed the development of sequences for both research and clinical applications.

Inhibition of α_1(Ⅰ) collagen gene in vitro transcription by antisense oligodeoxynucleotides

Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on in vitro transcrption α1 (I) collagen gene, isotopes (α-32pGTP) was incorporated into 2 SP6 in vitro transcription systems. Results and Conclu- sion: Oligo 2 (at the transcription start region) could effectively inhibit in vitro transcription of pGEM3-Col13 and the control (random oligodeoxynucleotides) showed no inhibition. However, oligo 1 (at the transcription start region) obviously inhibited the in vitro transcription of pGEM3-Col14, while Oligo 2, which targeted at the down stream region (about 200 bp) of the promoter showed no significant inhibition effect.

Inhibitory Effect of Tissue Transglutaminase (tTG) Antisense Oligodeoxynucleotides on tTG Expression in Cultured Bovine Trabecular Meshwork Cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides （tTG-ASDON） on tTG expression in cultured bovine trabecular meshwork cells （BTMCs） in vitro and explore a new treatment alternative for primary open angle glaucoma （POAG）, the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTCr-ASDON2 were significantly decreased as compared with that of the controls （P〈0.05）. On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

Inhibiting effect of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides on VEGF expression in U937 cells

Objective:To study the effect of VEGF antisense oligodeoxynucleotide(VEGF ASODN)on VEGF expression in acute monocyte leukemic cell line U937 in vitro.Methods:U937 cells were incubated with VEGF ASODN(final concentra-tion as follows:10,20 and 30 μmol/L respectively)or scrambled sequence,compared with negative control.The expression of VEGF mRNA was measured by semi-quantitative RT-PCR,VEGF protein was measured by Western blot.Results:VEGF ASODN obviously inhibited expression of VEGF mRNA in U937 cell,compared with scrambled sequence and negative control(P<0.05).And the inhibition effect was most remarkable after 24 h,which is related with the dose of VEGF ASODN(P<0.05).Scrambled sequence groups had no significant difference compared with negative control groups(P>0.05).VEGF ASODN obvi-ously inhibited expression of VEGF protein,compared with scrambled sequence and negative control(P<0.05).Conclusion:The expressions of VEGF at mRNA and protein levels in leukemic cell line U937 are down-regulated after being treated with VEGF ASODN.

Com bined effect of alphafetoprotein antisense oligodeoxynucleotidesand 5-fluorouracil on human hepato ma cell growth

Objective To investigate the effect of alpha fetoprotein (AFP) antisense phosphorothioate oligodeoxynucleotides in combination with 5 fluorouracil (5 FU) on the growth of BEL 7404 human hepatoma cells in vitro Methods Phosphorothioate oligodeoxynucleotides were synthesized by β cyanoethyl phosphoramidite chemistry AFP gene expression in human hepatoma cells was determined by avidin biotin peroxidase complex (ABC) immunocytochemical method Cell growth in the presence or absence of experimental agents was measured using 3 (4, 5 dimethylthiazol 2 yl) 2, 5 dipheny ltetrazolium (MTT) microculture tetrazolium assay Results AFP antisense oligomers markedly suppressed the growth of BEL 7404 human hepatoma cells in vitro by sequence specific blocking of the AFP gene expression in the cells (P<0 05) 5 FU also inhibited the hepatoma cell growth in a dose dependent manner when used alone (P<0 05) The combined treatment with AFP antisense oligomers and 5 FU showed significantly enhanced hepatoma cell growth inhibition than either AFP antisense or 5 FU treated cells alone (P<0 05) Conclusion Combined use of AFP antisense oligomers and 5 FU could more effectively inhibit the growth of BEL 7404 human hepatoma cells in vitro

Inhibitory effects of antisense phosphorothioate oligodeoxynucleotides on pancreatic cancer cell Bxpc-3 telomerase activity and cell growth in vitro

AIM： To investigate the effect of telomerase hTERT gene antisense oligonucleotide （hTERT-ASO） on proliferation and telomerase activity of pancreatic cancer cell line Bxpc-3. METHODS： MTT assay was used to detect the effect of different doses of hTERT-ASO on proliferation of Bxpc-3 cell for different times. To study the anti-tumor activity, the cells were divided into there groups： Control group （pancreatic cancer cell Bxpc-3）; antisense oligonucleotide （hTERT-ASO） group; and nosense oligonucleotide group decorated with phosphorothioate. Telomerase activity was detected using TRAP-PCR-ELISA. Cell DNA distribution was examined using flow cytometry assay. Cell apoptosis was observed by transmission electron microscope in each group. RESULTS： After treatment with 6 mmollL hTERT- ASO, cell proliferation was inhibited in dose- and time- dependent manner. The telomerase activity decreased after treatment with hTERT-ASO for 72 h. Flow cytometry showed the cell number of G0/G1 phase increased from 2.7% to 14.7%, the cell number of S phase decreased from 72.7% to 51.0%, and a sub-G1 stage cell apoptosis peak appeared in front of G1 stage. CONCLUSION： Telomerase antisense oligodeoxy- nucleotide can inhibit the proliferation of pancreatic cancer cell line Bxpc-3 and decrease the telomerase activity and increase cell apoptosis rate in vitro.

Inhibition of Telomerase with hTERT Antisense Increases Susceptibility of Leukemic Cells to CDDP-induced Apoptosis

Objective: To investigated the e?ect of inhibition of telomerase with hTERT antisense on leukemic cells (HL-60 and K562) to CDDP-induced apoptosis. Methods: Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and puri?ed. Telomerase activity was detected by Telomerase PCR ELASA kit and cell apoptosis was observed by morphological method and determined by ?owcytometry. Results: AS PS-ODN could signi?cantly inhibit telomerase activity by down regulat- ing the hTERT expression, and increase the susceptibility of leukemic cells to CDDP-induced apoptosis. Conclusion: Inhibition of telomerase with hTERT antisense can increases the susceptibility of leukemic cells to CDDP-induced apoptosis.

EFFECT OF TWO NEW BCL-2 ANTISENSES ON DRUG-SENSITIVITY OF CELLS FROMN LEUKEMIA PATIENTS

Objective: To investigate the effect of two antisense oligonucleotides on cell surviving, bcl-2 expression and apoptosis of leukemia cells. Methods: The experimental assays were performed with cell culture, immunochemistry and flowcytometry. Results: The two antisense oligodeoxynucleotides, combined with Vp16 or Ara-c or DNR, were able to decline the survival rate of myeleukemic cells, downregulate bcl-2 gene expression and induce apoptosis of leukemic cells significantly, as compared with Vp16 or Ara-c or DNR alone. Conclusion: It is possible for the two new bcl-2 antisenses to be developed into clinical trials for leukemia and tumor with bcl-2 gene overexpression. Key words Leukemia - Antisense oligodeoxynucleotides - bcl-2 gene - drug-resistance CLC number R733.7 Foundation item: this work was supported by the Key Foundation of Science & Technology Program of Guangzhou (No. 2001-Z-037-01); and the Nature Science key Foundation of Guangdong Province(No. 021195).Biography: LEI Xiao-yong(1970–), male, associate professor, doctor of medicine, Institute of Pharmacy and Pharmacology, Nanhua University, majors in tumor pharmacology.

Effects of antisense oligonucleotides targeting VEGF on radio sensitivity of uterine cervix cancer Hela cells

Objective： To determine the impact of antisense oligonucleotides targeting vascular endothelial growth factor （VEGF） on radiosensitivity of uterine cervix cancer Hela cells. Methods： VEGF antisense oligodeoxynucleotides （ASODN） was transfected into Hela cells by liposome-mediated method. Cells transfected with the oligodeoxynuclecotide and saline were used as control groups. Cells were irradiated by 6 MV X ray at the dose of 0 Gy, 2 Gy, 4 Gy and 6 Gy respectively. The expression of VEGF mRNA was determined by RT-PCR. Apoptosis were evaluated using FCM. Cloning efficiency was determined by colony formation assay. Results： The expression of VEGF mRNAwas inhibited by ASODN （P 〈 0.01） in Hela cells. The inhibited activation which was influenced by radiation resulted in increasing apoptosis （P 〈 0.01） and inhibiting plating efficiency （P 〈 0.01）. Conclusion： The expression of VEGF induced by X irradiation in Hela cells can be blocked by VEGF ASODN. Treatment with VEGF might increase apoptosis in HeLa cells and enhance radiosensitivity.

Inhibitory effects of antisense oligodeoxynu-cleotide on expression of vascular endothelia growth factor by human hepatocarcinoma cells

BACKGROUND: It is widely recognized that the growth of solid tumor depends on angiogenesis. Vascular endothelia growth factor (VEGF) is an endothelial cell-specific mito- gen that promotes angiogenesis in solid tumor. Inhibition of angiogenesis is considered a promising approach for cancer therapy, and treatments including administration of antisense drugs and RNA interference for the VEGF gene are geared to the suppression of tumor angiogenesis. METHODS: As a new approach for gene therapy of hepato- cellular carcinoma (HCC), four groups of antisense oli- godeoxynucleotide (ASODN) (A-Cap, A-AUG, A-UGA and A-Exon-3) were used to block the expression of VEGF, then VEGF mRNA and protein were detected by RT-PCR and Western blot. RESULTS: After treatment with ASODN, the relative VEGF mRNA levels of A-Cap, A-AUG, A-UGA, and A- Exon-3 were decreased significantly to (32±9)%, (63 ± 1)%, (86 ±3)%, and (70 ±5)%, respectively(F=64.18, P< 0.001). The relative VEGF protein levels of A-Cap, A- AUG, A-UGA and A-Exon-3 were decreased significantly to (41 ±5)%, (59 ±3)%, (88 ±7)%, and (79 ±9)% respec- tively (F =60.64, P<0.001). CONCLUSIONS: Among the four ASODNs, the ASODN for Cap structure showed the strongest inhibitory effect and that for A-UGA, the least (P <0.05 ). The inhibitory effect of ASODN on the expression of VEGF proteins was similar to that of VEGF mRNA expression.

Inhibition of telomerase with human telomerase reverse transcriptase antisense increases the sensitivity of tumor necrosis factor-α-induced apoptosis in prostate cancer cells

Aim： To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase （hTERT） antisense on tumor necrosis factor-α （TNF-α-induced apoptosis in prostate cancer cells （PC3）. Methods： Antisense phosphorothioate oligodeoxynucleotide （AS PS-ODN） was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol （TRAP） and polymerase chain reaction enzyme-linked immunoassay （PCR-ELISA）. hTERT mRNA was measured by reverse transcription PCR （RT-PCR） assay and gel-image system, hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-（4, 5-dimethylthiazol-2-yl）-2,5-Diphenyltetrazolium （MTT） assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. Results： The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide （S PS-ODN） and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-α treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-α treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. Conclusion： hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-α- induced apoptosis of PC3 cells.

Antisense Oligodeoxynucleotide Inhibits Expression of Re-com binantPorcine Follicle-Stim ulating Horm one Receptor

To assess the role of follicle stimulating hormone receptor(FSHR) gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and 125 I FSH binding were determined in Chinese hamster ovary cells expression recombinant porcine FSHR (pFSHR CHO cells). An 18 mer phosphorothioate endcapped antisense ODN that corresponded to the region surrounding the translation initiation codon of the porcine FSHR cDNA was synthesized. An 18 mer nonsense sequence of identical nucleotide composition, which had little homology to known DNA sequences, was synthesized for use as a control. pFSHR CHO cells were cultured in 24 well plates (10 5 cells/well) in the absence or presence of 1 20 μmol/L antisense or nonsense ODN for 24 h and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for 125 I FSH binding activity. Treatment with 10 μmol/L antisense ODN caused a paradoxical increase in porcine FSHR mRNA from 0.89±0.06 to 1.64±0.08 ng/mg total RNA ( P <0.05). Transfection with lipofectamine and 0.33 μmol/L antisense ODN caused an increase in porcine mRNA from 0.95±0.08 to 1.53±0.07 ng/mg total RAN. This was probably due to upregulation of mRNA synthesis resulting from inhibition of porcine FSHR protein translation. The nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 μmol/L) inhibited membrane binding of 125 I FSH by 13.6± 0.8 % ( P <0.05) in 24 h. Treatment of cells with antisense ODN (10 μmol/L) for 48 h resulted in a 76±1.5 % ( P <0.05) inhibition of 125 I FSH binding. In contrast, transfection with lipofectamine and 0.33 μmol/L antisense ODN at 0 h caused a 76.1±1.3 % ( P <0.05) reduction in binding within 24 h. Binding had returned to 52.3±2.3 % ( P < 0.05) of normal by 48 h. These results indicate that an antisense ODN corresponding to the region of the translation start site of the porcine FSHR cDNA is an effective specific inhibitor of porcine FSHR synthesis and that inhibition of receptor synthesis causes a decrease in functional membrane bound FSHR.

Effect of Antisense Oligodeoxynucleotide Directed to NF-κB-RelA on Bcl-x_L mRNA in Extended Drug Resistance Leukemia Cell Line HL- 60/E6

To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.

Transfection of Antisense Oligodeoxynucleotide Inhibits Heparanase Gene Expression and Invasive Ability of Human Pancreatic Cancer Cell in vitro

Extracellular matrix （ECM） degradation is an essential step that allows tumor cells to penetrate a tissue barrier and become metastatic. Heparanase （HPSE） is an endoglycosidase that specifically degrades heparin sulfate proteoglycans （HSPG）, a chief component of ECM, HPSE is not expressed in normal epithelial cells but can be detected in a variety of human carcinomas including pancreatic cancer. In the present study, human pancreatic cancer cell line Panc-1 was transfected with HPSE antisense oligodeoxynucleotide （AS-ODN） in vitro, then the inhibitory effect of ASODN on HPSE gene expression and invasive ability of Panc-1 cells in vitro was examined. The HPSE mRNA and protein expression of Panc-1 cells transfected with AS-ODN was significantly inhibited. However, there were no marked inhibitory effects in Panc-1 cells treated with nonsense oligodeoxynucleotide （NS-ODN）. Moreover, a modified Boyden chamber assay demonstrated that transfection with HPSE AS-ODN significantly inhibited invasive potential of Panc-1 cells in vitro after AS-ODN transfection. This suggests that HPSE AS-ODN may contribute to the inhibition of HPSE mRNA and protein expression, and results in a decrease of the invasive ability of Panc-1 in vitro.

EFFECTS OF CALM /AF10 ANTISENSES ON PRIMARY LEUKEMIC CELLS WITH CALM /AF10 FUSION TRANSCRIPTS IN VITRO

Objectives: To define the involvement of CALM and AF10 fusion transcripts in primary leukaemias with t(10; 11). Methods: The AF10 and CALM fusion in five t(10; 11) leukemia samples were checked by reverse transcriptase-polymerase chain reaction (RT-PCR), and effects of CALM/AF10 antisense phosphorothioate oligodeoxynucleotides (AS PS-ODNs) on chemotherapy sensitivity and apoptosis of leukemia cells in vitro were observed. Results: Five different-sized AF10-CALM products and four different-sized CALM/AF10 products were detected by RT-PCR. The chemotherapy sensitivity of leukemic cells with t(10; 11) to drugs in vitro was lower than that of leukemic cells without t(10; 11). AS PS-ODNs increased the chemotherapy sensitivity and apoptotic rate. There were 4 cases positive at 5 μmol/L concentration, all cases positive at 10 μmol/L and 20 μmol/L concentration, P<0.01 vs only chemotherapeutic drugs (3 cases positive), and chemotherapeutic drugs + S-PS-ODNs (10 μmol/L) (3 cases positive). After cells were treated with 10 μmol/L AS-PS-ODNs + chemotherapeutic drugs for 48 h, 72 h, 96 h, the apoptotic indexes were 14.22±2.86, 29.39±3.57, and 41.26±4.52, respectively. These were significantly higher than those of only chemotherapeutic drugs-treated cells and chemotherapeutic drugs + S-PS-ODNs-treated cells at corresponding time (P<0.01). There was no difference between only drugs group and S-PS-ODNs group at corresponding time (P>0.05). Conclusion: The CALM and AF10 fusion transcripts are involved in the pathogenesis of haematological malignancies with t(10, 11), and is associated with a poor prognosis. AS-PS-ODNs might be useful in therapy of t(10, 11) leukemia. Key words AF10 - CALM - Fusion transcript - Primary leukemia cell - In vitro sensitivity - Antisense oligodeoxynucleotide CLC number R733.7 Biography: LIU Ge-xiu (1968–), male, associate professor, Institute of Hematology, Medical College, Jinan University, majors in hematology.

Effect of MUC2 Antisense Oligodeoxynucleotide on Cell Proliferation,Adhesion,and Proteolytic Enzyme in Human Gastric Carcinoma in vitro

Objective：To investigate the effect of MUC2 antisense oligodeoxynucleotide （ASODN） on cell proliferation, adhesion and proteolytic enzyme inhuman gastric carcinoma cell line （SGC7901）. Methods： Phosphorothioate MUC2 ASODN was synthesized and packaged by lipofectin, and then transfected to SGC7901 cells. The expression of MUC2 mRNA and protein after transfection was detected by reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemical method respectively, and the effect of MUC2 ASODN on cell proliferation, adhesion and proteolytic enzyme was determined by flow cytometry（FCM）, MTT method, Rose Bengal and immunohistochemical method. Results： Compared with the blank control group, ASODN efficiently downregulated the expression of MUC2 mRNA and protein in SGC7901 cells 48h after transfection（P〈0.01）. Various concentrations of ASODN could significantly inhibit the growth of SGC7901, and the inhibition peaked at the 48th hour after transfection（P〈0.05）. The apoptosis rate of the experimental group was about 4.38%, and the percentage of S-phase cells rose while G0/G1-phase cells fell because most of them were blocked at S-phase. In addition, cells treated with MUC2 ASODN showed lower adhesion ability with matrix and endothelial cells than control cells in vitro（P〈0.01）. By immunohistochemical method, the upregulation of E-cadherin proteins and the downregulation of MMP2 and cathepsinD proteins were also observed（P〈0.05）. Conclusion： MUC2 ASODN could efficiently inhibit SGC7901 cell proliferation, reduce cell adhesion ability and downregulate the expression levels of proteolytic enzyme in vitro.