Purpose: Overexpression of the multidrug resistance proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) is associated with treatment failure in acute myeloid leukemia (AML) and other malignancies. The Pgp modulator cyclosporin A has shown clinical efficacy in AML, whereas its analogue PSC-833 has not. Cyclosporin A is known to also modulate MRP-1, and we hypothesized that broad-spectrum multidrug resistance modulation might contribute to its clinical efficacy.Experimental Design: We studied the effects of cyclosporin A and PSC-833 on in vitro drug retention and cytotoxicity in resistant cell lines overexpressing Pgp, MRP-1, and BCRP and on nuclear-cytoplasmic drug distribution and cytotoxicity in cells overexpressing LRP. Cellular drug content was assessed by flow cytometry and nuclearcytoplasmic drug distribution by confocal microscopy.Results: Cyclosporin A enhanced retention of the substrate drug mitoxantrone in cells overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR), and BCRP (8226/MR20, HEK-293 482R) and increased cytotoxicity 6-, 4-, 4-, and 3-fold, respectively. Moreover, cyclosporin A enhanced nuclear distribution of doxorubicin in 8226/MR20 cells, which also express LRP, and increased doxorubicin cytotoxicity 12-fold without an effect on cellular doxorubicin content, consistent with expression of wild-type BCRP, which does not efflux doxorubicin. Cyclosporin A also enhanced nuclear doxorubicin distribution in a second cell line with LRP overexpression, HT1080/DR4. PSC-833 enhanced mitoxantrone retention and cytotoxicity in cells overexpressing Pgp, but had no effect in cells overexpressing MRP-1, BCRP, or LRP.Conclusions: Cyclosporin A modulates Pgp, MRP-1, BCRP, and LRP, and this broad-spectrum activity may contribute to its clinical efficacy.

Double minute chromosomes (dmin) are small chromatin bodies consisting of genes amplified in an extrachromosomal location. dmins are uncommon in hematologic malignancies; they are seen primarily in acute myeloid leukemia, with amplification of the MYC oncogene or, less frequently, the MLL transcription factor. Nine patients with hematologic malignancies with dmin were seen at the Roswell Park Cancer Institute between 1985 and 2000; eight had acute myeloid leukemia and one a myelodysplastic syndrome. Fluorescence in situ hybridization (FISH) demonstrated MYC amplification on dmin in four patients, but MLL amplification was not seen. Spectral karyotyping showed that the dmin derived from chromosome 11 in one patient and from chromosome 19 in two others without MYC or MLL amplification; derivation from these chromosomes was confirmed by FISH with chromosome paint probes. The dmin of chromosome 11 origin hybridized to a bacterial artificial chromosome (BAC) RP11-112M22 that maps to 11q24.3 and is predicted to contain ETS1 and other markers, including D11S11351 and D11S4091. The dmin of chromosome 19 origin in one patient hybridized to BACs RP11-46I12 and RP11-110J19; in the other patient, these clones did not hybridize with the dmin, but were found to be amplified on a marker chromosome that was derived from chromosome 19 in that patient's cells. These BACs have been mapped to 19q12-19q13.1 and 19q11-19q13.1, respectively, and are predicted to contain the markers D19S409 and D19S919 and the gene for ubiquinol-cytochrome C reductase, Rieske iron-sulfur polypeptide1 (UQCRFS1). dmin originating from chromosome 19 have not been reported previously in hematologic malignancies.

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Multidrug resistance (MDR) mediated by the ATP-binding cassette proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1), breast cancer resistance protein (BCRP) and the vault protein lung resistance protein (LRP) is implicated in treatment failure in acute myeloid leukemia (AML). Pgp, MRP-1 and BCRP mediate energy-dependent cellular drug efflux, while LRP blocks cytoplasmic-nuclear drug transport. MDR modulators are non-cytotoxic drugs that block the activity of MDR proteins. In clinical trials, the Pgp modulator cyclosporine A (CsA) improved treatment outcome in AML, while its non-immunosuppressive, non-nephrotoxic analogue PSC-833 has not, despite being a potent Pgp modulator in preclinical models. CsA is known to also modulate MRP-1, and we hypothesized that a broad spectrum of MDR modulation might contribute to its clinical efficacy. We studied the effects of CsA and PSC-833 on in vitro drug uptake, retention and cytotoxicity in drug-selected and transfected resistant cell lines overexpressing Pgp, MRP-1 or BCRP, and on nuclear-cytoplasmic drug distribution and cytotoxicity in cell lines overexpressing LRP. Cellular drug content was measured by flow cytometry, and nuclear-cytoplasmic drug distribution was assessed by confocal microscopy. CsA enhanced uptake and retention of the substrate drug mitoxantrone in cells overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR) and BCRP (8226/MR20) and increased cytotoxicity 7-, 4- and 4-fold, respectively. Moreover, CsA increased the nuclear content of doxorubicin in 8226/MR20 cells, which co-express LRP with BCRP, and increased doxorubicin cytotoxicity 12-fold. The effect of CsA on nuclear drug content and cytotoxicity in 8226/MR20 cells occurred without an effect on cellular doxorubicin content, consistent with the fact that 8226/MR20 cells co-express wild type BCRP (BCRPR482), which does not efflux doxorubicin. Moreover the BCRP modulator fumitremorgin C had no effect on 8226/MR20 content, nuclear uptake nor cytotoxicity of doxorubicin. CsA also enhanced nuclear doxorubicin content in a second cell line with LRP-mediated resistance, HT1080/DR4. PSC-833 enhanced mitoxantrone retention and cytotoxicity in cells overexpressing Pgp, but, in contrast to CsA, had no effect on mitoxantrone uptake, retention nor cytotoxicity in cells expressing MRP-1 nor BCRP, and had no effect on doxorubicin nuclear content nor cytotoxicity in cells expressing LRP. Thus CsA is a broad-spectrum MDR modulator, with activity against Pgp, MRP-1, BCRP and LRP, while PSC-833 only modulates Pgp. The broad-spectrum activity of CsA may contribute to its clinical efficacy. These findings support identification and testing of other broad-spectrum MDR modulators.