Emetine resistant mutants of Entamoeba histolytica overexpress mRNAs for multidrug resistance

Department of Tropical Public Health, Harvard School of Public Health, Boston, MA.
Archivos de investigación médica 02/1990; 21 Suppl 1:183-9.
Source: PubMed


Although drug therapy is critical for control of amoebiasis, little is known about mechanisms of drug resistance by E. histolytica parasites. Here we tested the hypothesis that multidrug resistant (mdr) amoeba mutants, similar to mdr tumor cells, are drug resistant based upon overexpression of a P-glycoprotein pump that effluxes drugs from the cells. Using primers to conserved regions of the human P-glycoprotein and the polymerase chain reaction (PCR), we identified multiple 344 base par segments of amoeba DNA similar to the mammalian P glycoprotein. The amino acid sequences of amoeba mdr-like PCR products were from 53 to 97 identical with each other, 55 to identical to human mdr1 sequences, and 41-44% identical with P. falciparum mdr-like sequences. On northern blots, the mdr-like PCR products identified amoeba mRNAs 4.5-5 kilobases long, similar to the 5 kilobase mRNAs reported for the mammalian mdr gene. These mRNAs were increased at least seven times in emetine resistant mutant clone C2 amoebae versus wild-type clone A parasites. Further, the expression of the mdr-like mRNAs was increased three to four times when clone C2 mutants were grown under drug pressure versus the same parasites grown without emetine. In contrast, the number of genomic copies of the mdr-like DNA segments was not increased in the mutant clone C2 versus the wild-type clone A amoebae, and no rearrangements of the mdr-like DNA segments by the mutant were identified on Southern blots. In conclusion there appears to be a family of mdr-like genes in E. histolytica, which may be involved in drug resistance by the parasite.

15 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian multidrug-resistant cell lines, selected for resistance to a single cytotoxic agent, display cross-resistance to a broad spectrum of structurally and functionally unrelated compounds. These cell lines overproduce a membrane protein, the P-glycoprotein, which is encoded by a member(s) of a multigene family, termed mdr or pgp. The amino acid sequence of the P-glycoprotein predicts an energy-dependent transport protein with homology to a large superfamily of proteins which transport a wide variety of substances. This report describes the isolation and characterization of two Drosophila homologs of the mammalian mdr gene. These homologs, located in chromosomal sections 49EF and 65A, encode proteins that share over 40% amino acid identity to the human and murine mdr P-glycoproteins. Fly strains bearing disruptions in the homolog in section 49EF have been constructed and implicate this gene in conferring colchicine resistance to the organism. This work sets the foundation for the molecular and genetic analysis of mdr homologs in Drosophila melanogaster.
    Molecular and Cellular Biology 09/1991; 11(8):3940-8. DOI:10.1128/MCB.11.8.3940 · 4.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.
    Molecular and Cellular Biology 07/1992; 12(6):2855-65. DOI:10.1128/MCB.12.6.2855 · 4.78 Impact Factor
  • Source

    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/1994; 305(2):229-39. DOI:10.1016/0027-5107(94)90243-7 · 3.68 Impact Factor
Show more


15 Reads
Available from