Ⅰ Experimental Research Of The Effect Of ALDH1a2 Gene To The Biological Behavior Of Human Bladder Cancer Cell Lines Ⅱ Clinical Application Of The Single Needle Running Suture Method In Urethrovesical Anastomosis During Laparoscopic Radical Prostatecto

Bladder cancer has been an alarming public health problem, in accordance with thestatistics, and there are about 200000 newly reported cases of the illness in the worldannually. The incidence rate and mortality rate of bladder cancer are the highest of allurinary system malignant tumors in China, showing an increasing tendency year by year. Atpresent, the research on bladder cancer has made enormous achievement. Hypermethylationof DNA in promoter CpG islands of tumor suppressor genes is known to play a crucial rolein carcinogenesis. The DNMT inhibitor 5-aza-dC is widely used to study the reexpressionof genes silenced by promoter methylation. Except for DNA methylation, recent studieshave demonstrated the importance of histone modification as another epigenetic mechanismin the organization of chromosomal domains and gene regulation. It has also been shownthat histone modification is crucial to the process of DNA methylation in some organisms.There are many dates suggest a functional linkage between DNA methylation and histonemodifications in gene repression. Epithelial cells require retinoid acid (RA) synthesize itlocally and control its concentration by balancing its synthesis and catabolism. RAbiosynthesis begins with the reversible oxidation of retinol (vitamin A) to retinal by theenzyme retinol dehydrogenase, followed by the oxidation of retinal to RA by the aldehydedehydrogenase family of enzymes. ALDH1a2 is a member of the human aldehydedehydrogenase family which catalyzes the oxidation of retinal to RA. Oxidation to RAoccurs locally at the tissue sites where RA affects biological processes. ALDH1a2 itself hasbeen studied mainly in the context of normal prenatal development. There were findings suggest the methylation-silenced expression of ALDH1a2 may be responsible for lower RAlevels and altered RA signaling, contributing to the development of bladder cancer. As aresult, the mechanism of cell apoptosis and re-expression of ALDH1a2 gene in five humanbladder cancer cell lines treated with 5-aza-dC and TSA, alone or in combination wereinvestigated in this experiment.ObjectiveTo investigate the mechanism of cell apoptosis and expression of ALDH1a2 gene infive human bladder cancer cell lines treated with 5-aza-dC and TSA, alone or incombination.Methods1. The methylation status of ALDH1a2 gene promoter in five human bladder cancercell lines and normal human bladder epithelium were determined by MSP.2. The expression of the ALDH1a2 gene mRNA in five human bladder cancer celllines was established by real-time RT-PCR.3. The expression of the ALDH1a2 gene protein in five human bladder cancer celllines was determined by Western blot.4. After treatment with 5-aza-dC and TSA, the apoptosis of five human bladdercancer cell lines and morphological changes were assessed with FITC-Annexin Vand propidium iodide (PI) double staining.Results1. The MSP analysis revealed ALDH1a2 was hypermethylated but showed acharacteristic DNA methylation status in each promoter regions in the five celllines. The BIU-87, T24 cells displayed hypermethylation of the ALDH1a2promoter. The Tumor Stage of 253J and 5637 was lower than that of BIU-87, T24,so was their methylation. Very weak methylation was observed in RT-4. DNAmethylation was not identified in ALDH1a2 gene promoter region in normalbladder tissue samples. DNA demethylation was specifically noted after 5-Aza-dCtreatment but not after TSA treatment, and 5-aza-dC or combined with TSAresulted in demethylation of ALDH1a2 in which the silenced gene was associatedwith DNA hyper-methylation. 2. ALDH1a2 was silenced and TSA was not able to activate gene expression in fivehuman bladder cancer cell lines. ALDH1a2 transcript was faintly in RT-4, 253J,5637 (relative values were 0.168, 0.146, 0.153 respectively) readily detectable inBIU-87 and T24 (relative values were 0.342, 0.402 respectively), after 5-aza-dCtreatment. ALDH1a2 transcript was marked in each cell lines (relative values were0.293, 0.324, 0.421, 0.658, 0.753 respectively) Combined with 5-aza-dC and TSAtreatment which showed a synergistic effect to expression of ALDH1a2 transcript.3. 5-aza-dC treatment of cells reactivated the ALDH1a2 protein, whereas TSA hadlittle effect. The amount of ALDH1a2 protein in combination-treated cells wasabout twice of that in cells treated with 5-aza-dC alone (P＜0.05).4. The trend between the early apoptotic and later apoptotic was different, and theformer was steeper. It was safe to say that early apoptotic is the main mode ofapoptosis and death of human bladder cancer cell lines induced by 5-aza-dC andTSA, and the effect of 5-aza-dC induce cell apoptosis was superior to that of TSA(P＜0.05). The results showed that the percentage of early apoptotic cells was1.40% in Control group, and 2.80% in TSA group, however, 20.20% in 5-aza-dCgroup and 33.80% in 5-aza-dC+TSA group respectively. The groups of TSA,5-aza-dC and 5-aza-dC+TSA were significantly different from control group(P＜0.05). Actually, there was no statistic significant difference in five cell lines.ConclusionsAberrant methylation of ALDH1a2 gene may be the main reason of genetranscriptional inactivation in human bladder cancer cell lines. Cell apoptosis andre-expression of ALDH1a2 gene were detected after either treatment with 5-aza-dC aloneor in combination with TSA. ObjectiveTo study the promoter methylation status and expression of ALDH1a2 gene in fivehuman bladder cancer cell lines and normal bladder epithelium.Methods1. Five human bladder cancer cell lines (RT-4,253J, 5637, BIU-87, T24) were culturedin vitro.2. The methylation status of ALDH1a2 gene promoter of Human bladder cancer celllines and normal bladder epithelium was determined by MSP.3. The expression of the ALDH1a2 gene mRNA was detected by RT-PCR.4. The expression of the ALDH1a2 protein was detected by Western blot.Results1. The MSP analysis revealed ALDH1a2 gene was hypermethylated and showed acharacteristic DNA methylation status in the five cell lines. The BIU-87, T24 cellsdisplayed hypermethylation of the ALDH1a2 promoter, and the tumor stage of 253J and5637 was lower than that of BIU-87, T24, so was their methylation. Very weakmethylation was observed in RT-4. Normal DNA methylation was identified in normalbladder epithelium.2. ALDH1a2 gene mRNA was detected in normal bladder epithelium, but undetectedin five human bladder cancer cell lines.3. ALDH1a2 protein was expressed in normal bladder epithelium, but not in fivehuman bladder cancer cell lines.ConclusionsALDH1a2 gene was expressed and DNA methylation status was in normal status innormal bladder epithelium. The Aberrant methylation of ALDH1a2 gene may be the main cause for gene transcriptional inactivation in human bladder cancer cell lines. ObjectiveTo study the effect of treatment of 5-aza-dC and TSA, alone or in combination toALDH1a2 gene promoter methylation status and expression and cell apoptosis in fivehuman bladder cancer cell lines.Methods1. Human bladder cancer cell lines RT-4,253J, 5637, BIU-87, and T24 were culturedand treated with 5-aza-dC or (and) TSA.2. The methylation statuses of ALDH1a2 gene promoter in Human bladder cancercell lines RT-4,253J, 5637, BIU-87, T24 were determined by MSP.3. After the treatment with 5-Aza-dC and (or) TSA. The expression of the ALDH1a2gene mRNA in Human bladder cancer cell lines RT-4,253J, 5637, BIU-87, T24were detected by RT-PCR.4. The expressions of the ALDH1a2 protein in Human bladder cancer cell linesRT-4,253J, 5637, BIU-87, T24 were detected by Western blot.5. Cultured human bladder cancer cell lines RT-4,253J, 5637, BIU-87, and T24 wereassessed for antigen surface expression by adding the specific antibodies to thesample tube (AnnexinⅤ/PI conjugated). Fluorescence was analyzed by flowcytometry employing a Coulter Flow Cytometer (FACS). All reaction conditionsand flow parameters were standardized. Machinery lose cells were located at thefirst quadrant (annexinⅤ-/PI +); Later apoptotic and necrosis cells at the secondquadrant (annexinⅤ+/PI +); living cells at the third quadrant (annexinⅤ+/PI-); early apoptotic cells at the 4th quadrant (annexinⅤ-/PI-).Results 1. The MSP analysis revealed ALDH1a2 was hypermethylated but showed acharacteristic DNA methylation statue in each promoter regions in the five cell lines.The BIU-87, T24 cells displayed hypermethylation of the ALDH1a2 promoter; TheTumor Stage of 253J and 5637 was lower than that of BIU-87, T24, so was theirmethylation; Very weak methylation was observed in RT-4. DNA demethylationwas specifically noted after 5-Aza-dC treatment but not after TSA treatment (p＜0.05), and 5-aza-dC or combined with TSA resulted in demethylation of ALDH1a2(p＜0.05).2. ALDH1a2 was silenced in five human bladder cancer cell lines and TSA was notable to activate gene expression. ALDH1a2 transcript was faintly in RT-4, 253J,5637 readily detectable in BIU-87 and T24, after 5-aza-dC treatment. ALDH1a2transcript was marked in each cell lines Combined with 5-aza-dC and TSAtreatment which showed a synergistic effect to expression of ALDH1a2 transcript.3. 5-aza-dC treatment of cells reactivated the ALDH1a2 protein, whereas TSA hadlittle effect. The amount of ALDH1a2 protein in combination-treated cells wasabout twice of that in cells treated with 5-aza-dC alone.4. The trend between the early apoptotic and later apoptotic was different, and theformer was steeper. It was safe to say that early apoptotic was the main mode ofapoptosis and death of human bladder cancer cell lines induced by 5-aza-dC andTSA. The effect of 5-aza-dC induce cell apoptosis was superior to that of TSA. Theresults showed that the percentage of early apoptotic ceils was 1.40% in Controlgroup, and 2.80% in TSA group, however, 20.20% in 5-aza-dC group and 33.80%in 5-aza-dC+TSA group respectively. The groups of TSA, 5-aza-dC and5-aza-dC+TSA were significantly different from control group (p＜0.05). Actually,there was no statistic significant difference in five cell lines.ConclusionsAberrant methylation of ALDH1a2 gene is main cause for gene transcriptionalinactivation in human bladder cancer cell lines. Re-expression of ALDH1a2 gene wasdetected after either treatment with 5-aza-dC alone or in combination with TSA. Earlyapoptotic was the main mode of apoptosis and death of human bladder cancer cell lines induced by 5-aza-dC and TSA, the effect of 5-aza-dC induce cell apoptosis was superior tothat of TSA, the treatment of 5-aza-dC and TSA combined induce cell apoptosissignificantly.