However, an increase in Pgp expression mediated by these inhibitors would hamper their combination with other cytotoxic agents that are substrates of Pgp. We have previously selleck compound inves tigated in the human colon carcinoma cell lines SW620, HT 29 and HT 29/M6 the effect of TSA and Suberoylani lide Hydroxamic Acid on Pgp expression, demon strating a translational control of Pgp expression. The MDR1 mRNA produced in these cell lines is 285 bp shorter that the MDR1 mRNA produced in the human MCF 7/Adr and K562/Adr cell lines, both of them expressing Pgp pro tein. The different size of the MDR1 mRNA is due to the use of alternative promoters. Interestingly, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand.

More spe cifically, several RUNDC3B exons are located in the com plementary strand of the ABCB1 gene that corresponds with the intronic region between exon ?1 and exon 1 of the MDR1 mRNA, raising the possibility of transcriptional interference between both genes. The study presented herein has been designed to gain insight in ABCB1 regulation determining whether the translational control of Pgp functions also in pancreatic cancer cell lines, the puta tive regulation of ABCB1 alternative promoters by iHDACs, and whether the expression of the ABCB1 nested gene RUNDC3B interferes with the expression of the MDR1 mRNA isoforms. Methods Cell lines and culture IMIM PC 1, IMIM PC 2, RWP 1, PANC 1, Hs 766 T and BxPC 3 pancreatic carcinoma cell lines, as well as HT 29, HT 29/M6, SW 620, HTC 15, DLD 1, Colo 320 HSR and LS 174 T human colon carcinoma cell lines, were kindly donated by the IMIM cell line re pository.

MCF 7/Adr human breast carcinoma cell line was kindly donated by the Vincent T. Lombardi Cancer Center. Cells were grown at 37 C in 5% CO2, with DMEM or RPMI supplemented with 10% foetal calf serum, 2 mM L glutamine, 1 mM sodium pyruvate when required, 50 U/ml penicillin and 50 ug/ ml streptomycin. The murine leukaemia cell lines L1210, L1210R, K 562 and K562/Adr were grown as previously described. Western blot Treated or untreated cells were washed twice with PBS. After scraping the cells with PBS, they were centrifuged at 1000 x g for 5 minutes. L1210R and K562Adr cells which grow in suspension were washed twice with PBS.

Pgp expression was determined by Western immunoblot using the monoclonal antibody C 219, as previously described, followed by enhanced chemoluminiscence to develop protein bands. Protein Drug_discovery concentration in the cell lysates was determined by the Bradford method. Drug accumulation studies Steady state intracellular accumulation of the fluorescent substrate daunomycin was determined as previ ously described, in the absence or in the presence of verapamil, an inhibitor of Pgp. Real time RT PCR To determine the level of MDR1 mRNA and RUNCD3B mRNA, total RNA from non treated or TSA treated cells was isolated using the TRI reagent.