RESUMO
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.
Assuntos
Anticorpos/imunologia , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Reações Cruzadas/imunologia , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Roedores/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Sequência Conservada , Epitopos/química , Epitopos/imunologia , Humanos , Soros Imunes/biossíntese , Soros Imunes/imunologia , Soros Imunes/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Ratos , Ratos Wistar , Alinhamento de SequênciaRESUMO
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.
Assuntos
Bilirrubina/farmacocinética , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Trofoblastos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bilirrubina/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Linhagem Celular , Polaridade Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Difusão , Feminino , Humanos , Proteína 3 Homóloga a MutS , Propionatos/farmacologia , Quinolinas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfobromoftaleína/farmacologia , Ácido Taurocólico/farmacologia , Trítio , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacosRESUMO
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.