PMC

Effect of estrogen and 4-hydroxytamoxifen (4-OHT) on expression of MUC1 isoform specific mRNA in T47D and MCF7 cells analyzed by RT-PCR. Human T47D (clone 10) and MCF7 cells were cultured in DMEM medium with or without estrogen and 4-OHT supplements as described in "Materials and Methods". Total RNA was extracted and MUC1 isoform specific RT-PCR was performed. Lane M-DNA marker; lane 1 – cells grown in the medium without estrogen; lanes 2, 3 and 4 – cells grown with 0.1 nM, 1 nM and 10 nM estrogen, respectively; lanes 5, 6 and 7 – cells grown with 10 nM estrogen and with 10 nM, 100 nM and 1 μM 4-OHT supplements, respectively.

Expression of the MUC1 isoform specific mRNA in human mammary epithelial cells detected by RT-PCR. Total RNA from human MCF-7, T47D and MDA-231 cells and RNA from mouse DA3 cells stably transfected with MUC1/SEC (lane – SEC), MUC1/TM (lane – TM) and MUC1/Y (lane – Y) cDNA (positive controls) were extracted and RT-PCR amplification of the MUC1 isoform specific fragments were performed. Lanes 1, 4, 7, 10, 13, 16 and SEC – PCR performed with MUC1/SEC specific primers; lanes 2, 5, 8, 11, 14, 17 and TM – PCR performed with MUC1/TM specific primers; lane 3, 6, 9, 12, 15, 18 and Y – PCR performed with MUC1/Y specific primers.

Expression of the MUC1 isoform specific mRNA in mouse DA3 cells transfected with plasmids pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 and ppolyIII. DA3 cells were transfected with plasmid DNA. Cells transfected with ppolyIII were used as negative controls. DA3 cells stably transfected with MUC1/SEC, MUC1/TM and MUC1/Y cDNA were used as positive control. Total RNA was extracted 48 hrs after transfection and cDNA was synthesized. PCR amplification of MUC1 isoform specific fragments were performed using isoform specific primers. PCR products were separated by electrophoresis on 1.2% agarose gel and stained with ethidium bromide. A – Schematic structure of the pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 plasmids and the table of the MUC1/SEC, MUC1/TM and MUC1/Y mRNA expression in transfected DA3 cells. B – RT-PCR of the MUC1 isoform specific RNA extracted from DA3 cells transiently transfected with indicated plasmids. Lane M-DNA marker; lanes 1, 4, 7, 10 and 13 (negative control) – PCR performed with MUC1/SEC specific primers; lanes 2, 5, 8, 11 and 14 (negative control) – PCR performed with MUC1/TM specific primers; lanes 3, 6, 9, 12 and 15 (negative control) – PCR performed with MUC1/Y specific primers. Positive control: DA3 cells stably transfected with MUC1 isoform specific cDNA. Lane SEC – cells expressed MUC1/SEC; lane TM – cells expressed MUC1/TM and lane Y – cells expressed MUC1/Y RNAs.

ERα binding to EREs detected in the MUC1 promoter. The EMSA of the complexes (C1, C2 and C3) developed by ERα from T47D nuclear lysate and ³²P-labeled oligonucleotides containing the MUC1 promoter EREs (for details see "Materials and Methods"). A: Binding of ERα to ³²P-labeled oligonucleotides ERE1, ERE2, ERE3, ERE4 and ERE5, containing 1/2 consensus core sequence of the classical ERE, and to palindrome like ERE (putative ERE-6 and Vit ERE). Lanes 1, 3, 5, 7, 10, 12, 19 and 26 – complexes developed in absence of anti-ERα Ab; lanes 2, 4, 6, 8, 11, 13, 20 and 27 – partial or complete inhibition of complex formation by anti-ERα Ab; lanes 14, 21 and 28 – binding of ERα to ³²P-ERE2, Vit-ERE and ERE6, respectively, in the presence of "cold" Oct1 oligonucleotide; lanes 15–18 – binding of ERα to ³²P-ERE2 in presence of decreasing amount of the "cold" ERE2; lanes 22–25 – binding of ERα to ³²P-Vit ERE in presence of decreasing amount of the "cold" Vit ERE (* indicates an intermediate complex developed when binding reaction was kept on ice); lanes 29–32 – binding of ERα to ³²P-ERE6 in presence of decreasing amount of the "cold" ERE6. B: Effects of anti-ERα Ab and normal rabbit serum (NRS) on ERα binding to MUC1 specific and mutated EREs. Lanes 1, 6 and 11 – binding of ERα in absence of anti-ERα Ab; lanes 2, 7 and 12 – partial or complete inhibition of binding complexes by anti-ERα Ab; lanes 3, 8 and 13 – binding of ERα in presence of NRS; lanes 4, 9 and 14 – binding of ERα to mutated ERE in absence of anti-ERα Ab; lanes 5, 10 and 15 – binding of ERα to mutated ERE in presence of anti-ERα Ab.