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Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (or PfCRT) were shown to be causative of decreased sensitivity to diverse quinoline-based antimalarials. In this report we describe the identification of a post-translational variant of PfCRT using highly characterized antibodies raised against its N- and C-terminal cytoplasmic domains (e.g., 58 and 26 amino acids, respectively). Western blot analyses of P. falciparum protein extracts with anti N-PfCRT antiserum revealed two polypeptides with apparent molecular masses of 52 kDa and 42 kDa, relative to the calculated molecular mass of PfCRT of 48.7 kDa. The 52 kDa polypeptide was detectable with anti C-PfCRT antiserum, only after alkaline phosphatase treatment of P. falciparum extracts. Detailed epitope mapping of anti N- and C-PfCRT antisera revealed epitopes covering two previously identified phosphorylation sites, Ser411 and Thr416, whereby substitution of these residues with Asp amino acid, to mimic phosphorylated residues, dramatically inhibited anti C-PfCRT binding. Consistently, alkaline phosphatase treatment of P. falciparum extract unmasked the binding of anti C-PfCRT to the 52 kDa polypeptide, suggesting that the 52 kDa but not 42 kDa polypeptide is phosphorylated at its C-terminal Ser411 and Thr416. Interestingly, Pfcrt expressed in HEK-293F human kidney cells showed the same reactive polypeptides with anti N- and C-PfCRT antisera, consistent with PfCRT origin of the two polypeptides (e.g., 42 kDa and 52 kDa), but lacking PfCRT phosphorylation at its C-terminal. Immunohistochemical staining of late trophozoite-infected erythrocytes with anti N-or C-PfCRT antisera showed both polypeptides are localized to the parasite's digestive vacuole. Moreover, both polypeptides are detected in chloroquine-susceptible and -resistant strains of P. falciparum. This is the first report describing a post-translationally modified variant of PfCRT. The physiologic role of the 52 kDa phosphorylated PfCRT in P. falciparum remains to be determined. • Native PfCRT migrates as 42 kDa polypeptide is non-phosphorylated at its C-terminal. • Post-translationally modified PfCRT variant is phosphorylated and migrates as 52 kDa polypeptide. • Antibody to PfCRT N-terminal detects both native and post-translationally modified variant polypeptides. • Antibody to PfCRT C-terminal binds only non-phosphorylated native or dephosphorylated variant PfCRT. [ABSTRACT FROM AUTHOR]

Multidrug resistant tumor cells show collaterally sensitive to a range of non-toxic drugs. In this report, we describe the isolation of several P-glycoprotein-knockout cell clones, using CRISPR/Cas9, from Chinese hamster multidrug resistant model cell line and its parental cells (e.g. , CHORC5 and AuxB1, respectively). All three P-glycoprotein-knockout clones of CHORC5 cells show complete loss of resistance to anti-cancer drugs (e.g., colchicine and doxorubicin), while gaining resistance to well characterized collateral sensitivity drugs (e.g., verapamil, progesterone and NSC73306). A correlation between P-glycoprotein and Sorcin expression levels and a possible role for the latter in low grade resistance to colchicine and doxorubicin was observed. Furthermore, we show that P-glycoprotein expression is necessary for the ROS-mediated mechanism of collateral sensitivity. However, expectantly, P-glycoprotein-knockout clones of CHORC5 cells revealed a dramatic increase in the accumulation of Rhodamine 123, Mito tracker red and doxorubicin, but not Hoechst 33342. The latter findings and their significance to P-glycoprotein collateral sensitivity remain to be determined. [Display omitted] • Knockout of P-glycoprotein expression demonstrates its essential role in collateral sensitivity of multidrug resistant cells. • Collateral sensitivity of multidrug resistant cells is due to P-glycoprotein-dependent rise in reactive oxygen species. • Sorcin may mediate resistance to doxorubicin in P-glycoprotein knockout tumor cells. • Loss of P-glycoprotein expression from multidrug resistant cells unmask putative drug in-flux mechanism or transporter. [ABSTRACT FROM AUTHOR]

P-glycoprotein, member of the B-subfamily of the ATP-binding cassette (ABC) superfamily (e.g., ABCB1), has been demonstrated to confer resistance to clinically relevant anticancer drugs. Paradoxically, ABCB1-expressing multidrug resistant (MDR) cells are hypersensitivity or collateral sensitivity to non-toxic drugs. In this report, we demonstrate the capacity of trifluoperazine (TFP), a calmodulin inhibitor, to confer a collateral sensitivity onto ABCB1-overexpressing MDR cells. We show TFP-induced collateral sensitivity to be linked to ABCB1 expression and ATPase activity, as such phenotype is abolished in ABCB1-knockout MDR cells (CHORC5ΔABCB1 clones A1-A3) or with inhibitors of ABCB1 ATPase. TFP-induced collateral sensitivity is mediated by apoptotic cell death, due to enhanced oxidative stress. The findings in this study show for first time the use TFP as a collateral sensitivity drug, at clinically relevant concentrations. Moreover, given the use of trifluoperazine in the treatment for symptoms of schizophrenia and the role of ABCB1 transporter in tissue blood barriers and other physiologic functions, the finding in this study may have implications beyond cancer chemotherapy. [Display omitted] • Trifluoperazine is selectively lethal to ABCB1-expressing multidrug resistant cells. • Trifluoperazine-induced Collateral sensitivity is mediated by oxidative cell death. • ABCB1 knockout or inhibition of ATPase activity reverses the collateral sensitivity to trifluoperazine. • Potential clinical use of trifluoperazine to prevent the rise of drug resistant tumor cells. [ABSTRACT FROM AUTHOR]

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a phospho-protein with three identified phosphorylation sites (Ser33, Ser411 and Thr416) at its cytosolic N- and C-termini. In this study, we report on the characterization of PfCRT anti-serum and show the presence of three epitope-specific immunoglobulin (IgG) pools (i.e. , IgG-P1, P2, and P3), each recognizing a different epitope in PfCRT cytoplasmic C-terminal. IgG-P2 bound the heptapeptide sequence (408NEDSEGE414), including Ser411. The effect of Ser411 phosphorylation on the binding specificity of IgG-P2 was confirmed using heptapeptides and full-length PfCRT with substitutions of Ser411 with aspartic acid (phospho-serine mimic) and alanine residues. Moreover, using purified IgG-P2, we show the presence of PfCRT homodimer that has un-phosphorylated Ser411 and migrates with an apparent molecular mass of 90 kDa on SDS-PAGE. In addition, parasite lysates showed PfCRT to be more phosphorylated at Ser411 in CQ-sensitive (3D7) than CQ–resistant (Dd2-H) strains of P. falciparum. Taken together, the findings of this study suggest a role for Ser411 phosphorylation in PfCRT structure-function. • High resolution mapping of anti-PfCRT antisera to its C-terminal sequence. • Isolation of three reactive IgG pools in PfCRT antisera against different epitopes, with IgG pool 2 specific to Ser411 phosphorylation status. • Demonstrating the presence of PfCRT homodimer, ∼90 kDa, encoding un-phosphorylated Ser411. • PfCRT phosphorylation status at Ser411 may predict P. falciparum resistance to chloroquin. [ABSTRACT FROM AUTHOR]

The multidrug resistant associated protein 1 (or MRP1, ABCC1) encodes two cytoplasmic linker domains (L0 and L1) composed of highly charged sequences with multiple protein kinase A/C phosphorylation sites. In this report we made use of the scanning peptide approach to identify MRP1 linker L1 (L1 MRP1 ) interacting proteins. Scanning heptapeptides covering L1 MRP1 126 amino acids (Ile 846 - Leu 972 ) were synthesized and used in pull-down assays to isolate proteins from cell extracts (human multidrug resistant SCLC cell line; H69/AR). The results show four high affinity binding sequences in L1 MRP1 domain [ 866 FLRTYAST 867 ; 906 SAGKQLQRQLSSS 912 ; 925 ISRHHNSTA 927 and 954 AQTGQVKLSVYW 959 ] that bound ∼55 kDa and 110 kDa polypeptides. The latter polypeptides were identified by mass spectrometry as α- and β-tubulin monomers and dimers. Western blotting with monoclonal antibodies to α- and β-tubulin proteins confirmed the mass-spectrometry results. Moreover, using recombinant full-length GST-Linker 1 fusion polypeptide (GST-L1 MRP1 ), we confirmed the peptide scanning approach demonstrating specific binding of tubulin to GST-L1 MRP1 . Intriguingly, substitutions of serine residues in L1 MRP1 by aspartic acid, but not alanine, abolished its binding to tubulin, suggesting that phosphorylation of Ser 871, 915, 930, and 961 within L1 MRP1 may modulate its binding to tubulin. Taken together, the results of this study suggest possible interaction between MRP1 and tubulin that is modulated by phosphorylation of specific sequences in the L1 MRP1 domain. [ABSTRACT FROM AUTHOR]

Abstract: The human ABCG2 protein, a member of ABC transporter family, was shown to transport anti-cancer drugs and normal cell metabolites. Earlier studies have demonstrated the expression of ABCG2 in hematopoietic stem cells and erythroid cells; however little is known about the expression and activity of ABCG2 in mature erythrocytes. In this report, we show that ABCG2 in mature human erythrocytes migrates with an apparent molecular mass of 140kDa, under reducing conditions, on Fairbanks SDS gel system. In contrast, tumor cells expressing higher levels of ABCG2 show no detectable homodimers, when resolved under identical reducing conditions. Analysis of the same membrane extracts from tumor cells and human erythrocytes on Laemmli SDS gel system, where samples are boiled in the presence of increasing concentrations of disulfide reducing conditions and then analyzed, migrate with an apparent molecular mass of 70kDa or a monomer. Drug transport studies using Pheophorbide A, a substrate of ABCG2, show the protein to be active in erythrocytes. Furthermore, Fumitremorgin C, a specific inhibitor of ABCG2 increases the accumulation of Pheophorbide A in erythrocytes and drug-resistant cells but not in the parental drug-sensitive cells. Given the ability of ABCG2 to transport protoprophyrin IX or heme, these findings may have implications on the normal function of erythrocytes. [Copyright &y& Elsevier]

The multidrug resistance protein (ABCC1 or MRP1) causes resistance to multiple drugs through reduced drug accumulation. We have previously demonstrated direct interaction between MRP1 and unmodified drugs using photoreactive drug analogues. In this study, we describe the use of [125I]iodoaryl azido-rhodamine123 (IAARh123)—a photoactive drug analogue of rhodamine 123, to study the effects of mild detergents and denaturing agents on MRP1-drug binding in membrane vesicles prepared from HeLa cells transfected with the MRP1 cDNA. Our results show that the zwitterionic detergent CHAPS and a nonionic detergent Brij35 inhibited the photolabeling of MRP1 with IAARh123. Sodium deoxycholate (SDC) and octyl-β-glucoside (OG), structurally similar to CHAPS and Brij35 and disrupting the lipid bilayer, showed a modest increase of MRP1 photolabeling with IAARh123. Proteolytic digestion of IAARh123 photolabeled MRP1 labeled in the presence or absence of various detergents (CHAPS, SDC, or OG) revealed identical photolabeled peptides. Consistent with the drug-binding results, non-toxic concentrations of CHAPS and Brij35 reversed vincristine and etoposide (VP16) toxicity in MRP1 expressing cells. Taken together, the results of this study show that MRP1–drug interaction can occur outside the lipid bilayer environment. However, this interaction is inhibited with certain mild detergents. [Copyright &y& Elsevier]

Highlights: [•] P-glycoprotein mediates the collateral sensitivity of MDR cells to steroid hormones. [•] Stimulation of P-glycoprotein ATPase by steroid hormones is likely causative of collateral sensitivity in MDR cells. [•] Steroid hormones synergize with mETC decoupling drugs to induce collateral sensitivity. [•] Verapamil negatively modulates steroid hormones-induced collateral sensitivity. [ABSTRACT FROM AUTHOR]

The use of anti-cancer chemotherapy often leads to the rise of multidrug-resistant (MDR) tumors. We have previously reported the overexpression of a 40 kDa protein (P-40) in several MDR tumor cell lines. In this report we describe the cloning of a 1.4 kb cDNA with an open reading frame of 344 amino acids that encodes the P-40 protein. Analysis of the P-40 amino acid sequence showed it is identical to the human annexin I (Anx-I) protein. The identity of the isolated P-40 cDNA as Anx-I was confirmed by the specific binding of IPM96 mAb to a 40 kDa protein following the in vitro expression of P-40 full-length cDNA. Northern blot analysis of total RNA from drug-sensitive and -resistant cells revealed an increase in P-40 (or Anx-I) mRNA in drug-resistant cells relative to drug-sensitive cells. Transfection of Anx-I cDNA into drug-sensitive MCF-7 cells was carried out without further drug selection and showed 2- to 5-fold increase in resistance of transfected cells to adriamycin, melphalan, and etoposide. Conversely, transfection of reverse Anx-I cDNA into SKOV-3 cells decreased the expression of Anx-I without affecting the expression of other members of the annexin family and showed a 3- to 8-fold increase in sensitivity to these drugs. Of interest was the correlation between the presence of Anx-I and MDR in MDA-MB-231 cells when compared to MCF-7 cells. MDA-MB-231 cells show 3- to 20-fold increase in resistance to adriamycin, melphalan, and etoposide in the absence of detectable levels of P-glycoprotein (P-gp1), the multidrug resistance protein (MRP1) or the breast cancer resistance protein (BCRP). Taken together, these results provide the first direct evidence for the role of Anx-I in MDR of tumor cells. [Copyright &y& Elsevier]

The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs. Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions. This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide > Rhodamine 6G > vinblastine > doxorubicin > MK571 (a quinoline-based drug) while colchicine had no effect. The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein. In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8 kDa polypeptide that comprises the last two transmembrane domains of LmrA. [Copyright &y& Elsevier]