Low levels of ABCG2 expression in adult AML blast samples

Abstract

Previous reports have suggested that the adenosine triphosphate-binding cassette protein ABCG2 (breast cancer resistance protein [BCRP], mitoxantrone resistance [MXR]) is associated with drug resistance in acute myeloid leukemia (AML). The aims of this study were to determine the level of ABCG2 mRNA expression necessary to produce drug resistance and to define the ABCG2 levels in normal bone marrow (BM), peripheral blood (PB), cord blood (CB), and adult AML blast cell populations. First, using transduced clonal cell lines expressing varying levels of ABCG2, we found that ABCG2 expression conferred resistance to mitoxantrone and topotecan, but not to idarubicin. Next, we developed a real-time reverse transcription-polymerase chain reaction assay for measuring ABCG2 mRNA expression levels in clinical samples. Normal BM and PB contained low levels of ABCG2 mRNA, while higher levels were measured in CB mononuclear cells, CD34 ؉ , and Ac133 ؉ populations, consistent with the known stem cell enrichment in these populations. Next, we studied the ABCG2 mRNA levels in 40 specimens from newly diagnosed adult AML patients. Only 7% of these samples contained ABCG2 mRNA levels within the range of our drugresistant clone, although another 78% were higher than normal blood and bone marrow. Flow cytometry revealed very small subpopulations of ABCG2-expressing cells in the cases we examined. Our data suggest that high levels of ABCG2 mRNA expression in adult AML blast specimens are relatively uncommon and that ABCG2 expression may be limited to a small cell subpopulation in some cases. (Blood. 2002;100:4594-4601)

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2

“…Though verapamil is generally used to identify the SP phenotype in the Hoechst 33342 dye exclusion assay, it is insufficient in specificity to demonstrate the contribution of ABCG2 to the SP phenotype of limbal epithelial cells. As ABCG2-specific inhibitors, fumitremorgin C [33], 5D3 monoclonal antibody [13,22], and TPS-A [23] are reported. Our SP analysis of limbal epithelial cells using 5D3 and TPS-A showed that both 5D3 and TPS-A effectively inhibit Hoechst 33342 dye efflux from these cells (Fig.…”

“…When SP cells were incubated with an anti-ABCG2 monoclonal antibody, 5D3, recognizing an external plasma membrane epitope on living cells [13,22], prior to Hoechst 33342 dye exclusion assay, a marked decrease in SP cell dye efflux was observed ( Fig. 2A).…”

“…After incubation in incubation medium for 90 min at 37°C, (R)+-verapamil (Sigma, St. Louis, MO) or tryprostatin A (TPS-A; provided by Dr. H. Osada (RIKEN Institute)) was added to one portion (final concentration: 50 lM), after which both portions were incubated for a further 30 min at 37°C. Antibody-mediated inhibition assay using anti-ABCG2 monoclonal antibody (5D3; eBioscience, San Diego, CA) or isotype control (mouse IgG 2b ; eBioscience) was performed as previously described [22]. Hoechst 33342 dye (Sigma) was then added to both portions (final concentration: 3 lg/ml), and incubation continued for another 90 min at 37°C.…”

Mutational Studies of G553 in TM5 of ABCG2: A Residue Potentially Involved in Dimerization

“…Chimeric fusion proteins containing two ABCG2 monomers either with or without a flexible linker peptide were shown to be functional (13) supporting the idea of homodimer formation. In addition, coimmunoprecipitation experiments using two different tags on the ABCG2 monomers also suggested homodimer formation (31). A disulfide bridge formed between cysteines 603 of the monomers in the predicted extracellular loop connecting TM5 and TM6 was described as non-essential for trafficking and transport (14).…”

Flow cytometry‐based assessment of mitoxantrone efflux from leukemic blasts varies with response to induction chemotherapy in acute myeloid leukemia

“…There are 49 human genes in the ATP-binding cassette (ABC) transporter superfamily encoding transmembrane proteins, which mediate drug efflux via ATP-dependent pumps (17)(18)(19)(20)(21). Among the ABC transporters, expression of multidrug resistance protein 1 (MDR1 or P-glycoprotein) and breast cancer resistance protein 1 (BCRP1) have been associated with chemotherapy resistance in AML in some studies (22)(23)(24)(25)(26)(27)(28) although not in all (29)(30)(31)(32). One possible reason for the conflicting prognostic relevance suggested for ABC transporter expression in AML is the variety of methods used to assess that expression in previous studies (33).…”