We present here the cloning and characterization of the EhPgp1 multidrug resistance gene promoter isolated from the Entamoeba histolytica drug-resistant mutant clone C2. The EhPgp1 promoter lacks the typical TATA box and the transcriptional initiation sequences described for other E. histolytica promoters. The major transcription initiation site of the EhPgp1 gene was located at the ATG start codon. The EhPgp1 core promoter located within the first 244 base pairs showed a higher chloramphenicol acetyltransferase expression in the transfected trophozoites of clone C2 than in those of the sensitive clone A. Gel shift assays revealed three specific DNA-protein complexes (Ia, IIa, and IIIc) using nuclear extracts from clone C2, whereas three main complexes (If, IIf, and IIg) were limited to clone A. Competition assays suggested the presence of C/EBP-like and OCTlike proteins in complexes Ia and IIa, respectively, probably involved in the expression of the EhPgp1 gene, whereas complex IIIc was competed by GATA-1, C/EBP, OCT, and HOX oligonucleotides. Thus, differential DNAprotein complexes may be formed by transcriptional factors involved in the regulation of the EhPgp1 gene expression.

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We report here the cloning and transcriptional characterization of the EhPgp5 multidrug resistance gene promoter isolated from the drug-resistant clone C2 of Entamoeba histolytica. The EhPgp5 promoter has the TATA-like motif at ؊31 base pairs; transcription initiates three nucleotides upstream from the ATG in trophozoites grown in 225 M emetine (clone C2(225)), whereas in those grown without the drug (clone C2) a product with no open reading frame was detected. The promoter was active in transfected clone C2 trophozoites, its activity increased when trophozoites were cultured in 40 M emetine, while it was turned off in the drug-sensitive clone A. The first ؊235 base pair kept full promoter activity, suggesting that it has important drug responsive elements. Gel shift assays detected the complex Ib in clone C2, which was augmented in clone C2(225). Competition experiments suggested that complex Ib may be constituted by HOX and AP-1 like factors in clone C2, whereas in clone C2(225), complex Ib was only competed by the HOX sequence. Complexes Ie, detected in clones A and C2 but not in C2(225), and Ia, present in all clones, were competed by the TATA box oligonucleotide. Our results suggest that proteins forming complexes Ib and Ie may be participating in the regulation of the EhPgp5 gene expression.The MDR 1 phenotype in Entamoeba histolytica seems to be a consequence of an increased expression of the P-glycoprotein, although the protein has not been detected (1). However, E. histolytica drug-resistant mutants overexpress mdr-like genes (EhPgp) (2, 3) and common characteristics are shared among the MDR phenotype of this parasite, Plasmodium falciparum, Leishmania tarentolae, and mammalian cells (Refs. 4 -6 and reviewed in Ref. 7). As in some mammalian transformed cells, where only certain mdr genes are expressed, three out of the four EhPgp genes are transcribed in the drug-resistant trophozoites (clone C2) (3, 4). While the EhPgp1 and EhPgp6 genes are transcribed in clone C2 grown in the absence of the drug, the amount of EhPgp5 transcript increases according to the emetine concentration (3, 4).The overproduction of the P-glycoprotein has been proposed to be mediated mainly by transcription, gene amplification, or both (8). However, the amplification alone may not be sufficient to activate the expression of a gene that is normally turned off or transcribed at very low levels (9). Increased mdr gene transcription precedes gene amplification in several mouse cell lines (10), and in human breast cancer and neuroblastoma cell lines (11,12), supporting that the regulation of the mdr genes is principally controlled at the transcriptional level.As occurs in the EhPgp5 gene, the expression of the mouse mdr1b gene is induced by the presence of the drug in the medium. The functional analysis of the mdr1b promoter demonstrated that three nuclear protein binding sequences from Ϫ82 to Ϫ59, from Ϫ123 to Ϫ101, and from Ϫ272 to Ϫ249, participate in the mdr1b gene regulation (13). In addition, DNase I footprinting experiments sho...

SummaryHere, we show the relevance of promoter regions ( ----74 to + + + + 24, ----167 to ----75 and ----259 to ----168 bp) in the transcriptional activation of the multidrug resistance gene EhPgp1 in Entamoeba histolytica , using mutated plasmids and transfection assays. We also demonstrate that both CCAAT/enhancer binding protein sites ( b antibodies and formed a specific complex with the C/EBP probe. The antibodies recognized proteins in the cytoplasm, nucleus and EhkO organelles in immunofluorescence and confocal microscopy experiments. Based on our results, we propose that the C/EBP site at ----54 bp stabilizes the transcription pre-initiation complex, whereas the other site at ----198 bp may be involved in the formation of a multiprotein complex, which provokes DNA folding and promotes the EhPgp1 gene transcription.

We have studied the cellular location and the efflux pump function of the Entamoeba histolytica P-glycoproteins (EhPgps) in drug-sensitive and -resistant trophozoites. Polyclonal antibodies against the EhPgp384 polypeptide (375-759 amino acids) revealed a 147-kDa protein by Western blot. The band intensity correlated with the emetine-resistance of the trophozoites. Through the confocal microscope, using the anti-EhPgp384 and fluorescein secondary antibodies, the EhPgps were found in a complex vesicular network, in the plasma membrane and outside of the cells. Transmission electron microscopy assays confirmed that drug-resistant trophozoites presented four to five times more EhPgps than sensitive cells. Fluorescence co-localization experiments using rhodamine-123 (R123) and the anti-EhPgp384 antibodies suggested the interaction between EhPgps and the drug. R123 efflux kinetics evidenced that the emetine-resistant trophozoites displayed a drug efflux kinetic four times higher than the drug-sensitive trophozoites, which was reduced by verapamil in both cases. EhPgps may participate in avoiding drug accumulation in the trophozoites by two putative mechanisms: (1) the direct extrusion of the drug from the plasma membrane, and (2) an indirect transport mechanism in which the drug is trapped by EhPgps and concentrated within vesicles that drive the drug to the plasma membrane.