Study About Cisplatin-resistance Of Bladder Cancer Cells And Their DNA Methylation Pattern

Objective: To investigate the DNA methylation profile of cisplatin-resistance inbladder cancer cell, and explore the alteration of genes and proteins which are involved incisplatin-resistance.Methods:(1) The build-up of cisplatin-resistant bladder cancer cells (T24/DDP): Thecisplatin-resistant bladder cancer cells were built up in T24bladder cancer cells (T24/DDP)with continued increasing of concentrations (0.2-2.0mg/L). After the built-up of T24/DDPcell line, a series of biological parameters were measured including cisplatin sensitivity,cellular morphology, growth curve and cell cycle.(2) DNA methylation profile and selection of drug-resistant genes in T24/DDP cellline: The DNA methylation profile was conducted with DNA methylation chips in T24andT24/DDP cells, and genes with hypermethylation or hypomethylation were screened bystatistical methods. The mRNA expression of selected genes with altered methylation wasanalyzed with real time-PCR. Then, the methylation of these genes was further validatedwith methylation-specific PCR (MSP).(3) The biological impacts of5-aza-dC on T24/DDP cells: The demethylation wasconducted in T24/DDP cells by5-aza-dC treatment. Then, a series of cellular biologicalparameters were measured.(4) The molecular mechanisms of demethylation with5-aza-dC in T24/DDP cells:The expression of genes and proteins were analyzed with real time-PCR and western blotin T24/DDP cells treated by5-aza-dC.Results: (1) The cisplatin-resistant bladder cancer cells (T24/DDP) were successfully built upin T24cell line. T24cells were almost100%died of cisplatin treatment at theconcentration of20ug/ml, whereas it was only30%in T24/DDP cells. The doubling timeof T24and T24/DDP cells was72.5and90.2hours respectively. There was no obviousdifference in morphology between T24and T24/DDP cells. However, a decrease in numberof mitochondria was observed in T24/DDP cells. Moreover, T24/DDP cells showedsignificant increase in percentage of G0/G1and decrease in G2/M, compared to T24cells.(2) There are1120sites with altered methylation in genome of T24/DDP cellscompared to T24cells. Then,40genes with hypermethylation and18genes withhypomethylation were identified with statistical methods. The mRNA expression of30genes among the identified58genes was significantly different between T24and T24/DDPcells with real time-PCR. On the basis of references, we validated the20genes with MSPand found that15genes showed consistent results with methylation chips, including14genes with hypermethylation and1with hypomethylation.(3) We found that7out of14validated genes with hypermethylation weredemethylated by5-aza-dC, including ABCC6， CCNA1， HPP1， ITGA4， RASSF1A,RUNX3， and TMS1, while the methylation status of the rest8genes was not changed.Moreover, we found that5-aza-dC treatment resulted in increased sensitivity to cisplatin,and increase in percentage of cell apoptosis and S phage in T24/DDP cells.(4) The mRNA and protein expression of the7genes were significantly enhancedafter treatment of5-aza-dC, including ABCC6， CCNA1， HPP1， ITGA4， RASSF1A,RUNX3，and TMS1.Conclusions:(1) T24/DDP cells show well resistance to cisplatin, suggesting that the method ofcontinued increasing in concentration of cisplatin is reliable for inducing cisplatin-resistantcells.(2) There are substantial changes in DNA methylation status in genome DNA ofT24/DDP cells. (3) Treatment of5-aza-dC (5μM) can demethylate genes with hypermethylationpartially in T24/DDP cells.(4) It is postulated that7genes may play important roles in mediatingcisplatin-resistance in T24cells, including ABCC6， CCNA1， HPP1， ITGA4，RASSF1A, RUNX3，and TMS1.