ABSTRACT
Based on the high level of identity among human, mouse, and rat MRP1 protein sequence, we produced a specific polyclonal antibody (MRP1-A23) against a synthetic polypeptide covering the C-terminus of the human protein. Western blot analysis showed a reactivity against human MRP1 similar to that obtained with the monoclonal QCRL1 antibody. Differently from other available antibodies against human MPR1, MRP1-A23 also detected both rat and mouse MRP1. No cross-reactivity was observed with either human or mouse MRP2 while MRP1-A23 weakly cross-reacted with rat MRP2 in the protein region ranging from 1512 to 1533 amino acids. These data indicate that MRP1-A23 allows specific MRP1 detection in both human and rodent tissues and may provide an important tool in the study of MRP1 expression and function in both experimental and clinical materials.
Subject(s)
Antibodies/immunology , Antibodies/isolation & purification , Antibody Specificity , Cross Reactions/immunology , Membrane Transport Proteins , Multidrug Resistance-Associated Proteins/immunology , Rodentia/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Blotting, Western , Conserved Sequence , Epitopes/chemistry , Epitopes/immunology , Humans , Immune Sera/biosynthesis , Immune Sera/immunology , Immune Sera/isolation & purification , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Molecular Weight , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/chemistry , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunology , Rats , Rats, Wistar , Sequence AlignmentABSTRACT
To evaluate mechanisms that mediate passage of unconjugated bilirubin (UCB) across placenta, the transport of [3H]UCB was studied in the human trophoblastic, BeWo cell line. When plotted against the unbound UCB concentration [Bf], uptake exhibited saturative kinetics with a similar apparent Km ( approximately 30 nM) for BeWo cells grown either in polarized (Transwell) or non-polarized fashion (dish). UCB release from cells, but not uptake, was inhibited by sulfobromophthalein but not by taurocholate, and almost abolished by MK571, a specific inhibitor of the activity of multidrug resistance-associated proteins (MRPs). MRP1 and MRP5 were both present in BeWo cells and the expression of MRP1, but not MRP5, was markedly higher in polarized cells. These data indicate that UCB is taken up from the fetal circulation by a still undefined, saturative process not shared by other organic anions and is then excreted to maternal circulation by proteins of the MRP family.
Subject(s)
Bilirubin/pharmacokinetics , Multidrug Resistance-Associated Proteins , Trophoblasts/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Bilirubin/metabolism , Biological Transport/drug effects , Biological Transport/physiology , Cell Line , Cell Polarity , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Diffusion , Female , Humans , MutS Homolog 3 Protein , Propionates/pharmacology , Quinolines/pharmacology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sulfobromophthalein/pharmacology , Taurocholic Acid/pharmacology , Tritium , Trophoblasts/cytology , Trophoblasts/drug effectsABSTRACT
The detection of the multridrug resistance-associated proteins is becoming increasingly important in assessing tumor sensitivity to treatment. In this work we describe a new, rapid, sensitive, and robust method for the detection of MRP1 expression based on direct RT-in situ-PCR technology and fluorochrome-modified (dCTP(Cy3)) nucleotides. MRP1 expression was found in both placenta (BeWo) and liver (Hep G2)-derived tumor cell line as well as in small cell lung carcinoma. In liver-derived cells, MRP1 expression was detected by RT-in situ-PCR but not by in situ hybridization, suggesting a higher sensitivity of in situ amplification for the low level of expression in Hep G2 cells. RT solution PCR confirmed the presence of MRP1 in BeWo and Hep G2 cells, although the level of the gene expression was lower in liver cells. This method represents a viable alternative to conventional immunohistochemistry, and may be useful in the evaluation of MRP1 expression in different tissue or cell lines.