We also analyzed Pgp activity determining the accumulation of daunomycin, a fluorescent sub strate of Pgp, this research in cells treated or untreated with iHDACs, using as a positive control L1210R cells. The presence of an active Pgp in these cell lines was demonstrated by the increased accumulation of DNM observed after addition of the Pgp inhibitor verapamil. Our results in Figure 1C show that no functional Pgp was present in IMIM PC 1, IMIM PC 2 and RWP 1 cell lines, either in the presence or in the absence of TSA. I P D whether the MDR1 mRNA present in pancreatic carcin oma cell lines included the longer or the shorter 50 UTR form, and whether the translational control was a general phenomenon in pancreatic cancer cells. In order to do that, we extended this study to other pancre atic cell lines.

Results in Figure 2B show that neither the pancreatic carcinoma cell lines, IMIM PC 1, IMIM PC 2 and RWP 1 nor the new added cell lines Hs 766 T, BxPC 3 and PANC 1 express the longer 50UTR MDR1 mRNA, suggesting a generalized translational control of Pgp expression in human pancreatic carcinoma cell lines, since the new added cell lines were also negative for Pgp expression in Western blot analysis. We have used K 562 ADR and HTC 15 as positive controls, since both of them express a functional P glycoprotein. Furthermore, Figure 3 shows the effects of TSA on MDR1 mRNA levels, as determined by real time RT PCR in IMIM PC 1, IMIM PC 2, RWP 1, PANC 1, Hs 766 T and BxPC 3 cells. All the cell lines except BxPC 3 show an increase in MDR1 mRNA levels after TSA treatment.

TSA regulates differentially the ABCB1 promoters The longer and the shorter 50 UTR forms of MDR1 mRNA have been related to the existence of two ABCB1 alternative promoters. To determine Drug_discovery the effects of TSA on both ABCB1 gene promoters, we analyzed the levels of MDR1 mRNA after 24 h treatment with TSA in a panel of cell lines of different origin that have been divided in two groups. Group A includes cell lines Translational control of Pgp mRNA in pancreatic carcinoma cell lines After demonstrating that the increase in MDR1 mRNA levels due to iDHACs treatment did not correspond with an increase in Pgp protein expression or activity, and since these results resembled our previous findings in colon carcinoma cell lines, we decided to determine that we have analyzed previously and use the down stream promoter which transcribes the short 50UTR MDR1mRNA. Group B includes cell lines which use the upstream pro moter that transcribes the long 50UTR MDR1 mRNA. Results in Figure 4 clearly demonstrate differential effects of TSA on DSP and USP promoters and suggest that TSA is able to downregulate the USP promoter and upregulate the DSP promoter in the ABCB1 gene.