RESUMO
Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines' sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Hidroxiureia/farmacologia , Leucemia Mieloide Aguda/patologia , Zidovudina/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citarabina/farmacologia , Dano ao DNA/efeitos dos fármacos , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Sinergismo Farmacológico , Histonas/metabolismo , Humanos , Hidroxiureia/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Ubiquitinação , Zidovudina/uso terapêuticoRESUMO
A gradually increasing number of transient neonatal zinc deficiency (TNZD) cases was recently reported, all of which were associated with inactivating ZnT2 mutations. Here we characterized the impact of three novel heterozygous ZnT2 mutations G280R, T312M, and E355Q, which cause TNZD in exclusively breastfed infants of Japanese mothers. We used the bimolecular fluorescence complementation (BiFC) assay to provide direct visual evidence for the in situ dimerization of these ZnT2 mutants, and to explore their subcellular localization. Moreover, using three complementary functional assays, zinc accumulation using BiFC-Zinquin and Zinpyr-1 fluorescence as well as zinc toxicity assay, we determined the impact of these ZnT2 mutations on vesicular zinc accumulation. Although all three mutants formed homodimers with the wild type (WT) ZnT2 and retained substantial vesicular localization, as well as vesicular zinc accumulation, they had no dominant-negative effect over the WT ZnT2. Furthermore, using advanced bioinformatics, structural modeling, and site-directed mutagenesis we found that these mutations localized at key residues, which play an important physiological role in zinc coordination (G280R and E355Q) and zinc permeation (T312M). Collectively, our findings establish that some heterozygous loss of function ZnT2 mutations disrupt zinc binding and zinc permeation, thereby suggesting a haploinsufficiency state for the unaffected WT ZnT2 allele in TNZD pathogenesis. These results highlight the burning need for the development of a suitable genetic screen for the early diagnosis of TNZD to prevent morbidity.
Assuntos
Proteínas de Transporte de Cátions , Permeabilidade da Membrana Celular/genética , Haploinsuficiência , Mutação , Zinco/deficiência , Substituição de Aminoácidos , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Humanos , Recém-Nascido , Células MCF-7 , Mutação de Sentido Incorreto , Ligação Proteica , Zinco/metabolismoRESUMO
Zinc plays a crucial role in numerous key physiological functions. Zinc transporters (ZnTs) mediate zinc efflux and compartmentalization in intracellular organelles; thus, ZnTs play a central role in zinc homeostasis. We have recently shown the in situ dimerization and function of multiple normal and mutant ZnTs using bimolecular fluorescence complementation (BiFC). Prompted by these findings, we here uncovered the heterodimerization, altered subcellular localization, and function of multiple ZnTs in live cells using this sensitive BiFC technique. We show that ZnT1, -2, -3, and -4 form stable heterodimers at distinct intracellular compartments, some of which are completely different from their homodimer localization. Specifically, unlike the plasma membrane (PM) localization of ZnT1 homodimers, ZnT1-ZnT3 heterodimers localized at intracellular vesicles. Furthermore, upon heterodimerization with ZnT1, the zinc transporters ZnT2 and ZnT4 surprisingly localized at the PM, as opposed to their vesicular homodimer localization. We further demonstrate the deleterious effect that the G87R-ZnT2 mutation, associated with transient neonatal zinc deficiency, has on ZnT1, ZnT3, and ZnT4 upon heterodimerization. The functionality of the various ZnTs was assessed by the dual BiFC-Zinquin assay. We also undertook a novel transfection competition assay with ZnT cDNAs to confirm that the driving force for heterodimer formation is the core structure of ZnTs and not the BiFC tags. These findings uncover a novel network of homo- and heterodimers of ZnTs with distinct subcellular localizations and function, hence highlighting their possible role in zinc homeostasis under physiological and pathological conditions.
Assuntos
Proteínas de Transporte/metabolismo , Frações Subcelulares/metabolismo , Sequência de Bases , Primers do DNA , Dimerização , Feminino , Humanos , Células MCF-7 , Microscopia de FluorescênciaRESUMO
Zinc transporters (ZnTs) facilitate zinc efflux and zinc compartmentalization, thereby playing a key role in multiple physiological processes and pathological disorders, presumed to be modulated by transporter dimerization. We recently proposed that ZnT2 homodimerization is the underlying basis for the dominant negative effect of a novel heterozygous G87R mutation identified in women producing zinc-deficient milk. To provide direct visual evidence for the in situ dimerization and function of multiple normal and mutant ZnTs, we applied here the bimolecular fluorescence complementation (BiFC) technique, which enables direct visualization of specific protein-protein interactions. BiFC is based upon reconstitution of an intact fluorescent protein including YFP when its two complementary, non-fluorescent N- and C-terminal fragments (termed YN and YC) are brought together by a pair of specifically interacting proteins. Homodimerization of ZnT1, -2, -3, -4, and -7 was revealed by high subcellular fluorescence observed upon co-transfection of non-fluorescent ZnT-YC and ZnT-YN; this homodimer fluorescence localized in the characteristic compartments of each ZnT. The validity of the BiFC assay in ZnT dimerization was further corroborated when high fluorescence was obtained upon co-transfection of ZnT5-YC and ZnT6-YN, which are known to form heterodimers. We further show that BiFC recapitulated the pathogenic role that ZnT mutations play in transient neonatal zinc deficiency. Zinquin, a fluorescent zinc probe applied along with BiFC, revealed the in situ functionality of ZnT dimers. Hence, the current BiFC-Zinquin technique provides the first in situ evidence for the dimerization and function of wild type and mutant ZnTs in live cells.
Assuntos
Proteínas de Transporte de Cátions/química , Multimerização Proteica , Zinco/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Teste de Complementação Genética , Humanos , Proteínas Luminescentes/química , Células MCF-7 , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Quinolonas/química , Compostos de Tosil/química , TransfecçãoRESUMO
Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.
Assuntos
Proteínas de Transporte de Cátions , Doenças do Recém-Nascido , Modelos Moleculares , Mutação de Sentido Incorreto , Dobramento de Proteína , Multimerização Proteica/genética , Zinco/deficiência , Substituição de Aminoácidos , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Citoplasma , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Escherichia coli , Proteínas de Escherichia coli , Feminino , Humanos , Lactente , Recém-Nascido , Doenças do Recém-Nascido/genética , Doenças do Recém-Nascido/metabolismo , Judaísmo , Masculino , Proteínas de Membrana Transportadoras , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.
Assuntos
Arginina/metabolismo , Ácido Fólico/metabolismo , Absorção Intestinal/genética , Síndromes de Malabsorção/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Arginina/química , Arginina/genética , Humanos , Síndromes de Malabsorção/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Transportador de Folato Acoplado a PrótonRESUMO
Hereditary folate malabsorption (HFM) patients harbor inactivating mutations including R113S in the proton-coupled folate transporter (PCFT), an intestinal folate transporter with optimal activity at acidic pH. Here we identified and characterized a novel R113C mutation residing in the highly conserved first intracellular loop of PCFT. Stable transfectants overexpressing a Myc-tagged wild-type (WT) and mutant R113C PCFT displayed similar transporter targeting to the plasma membrane. However, whereas WT PCFT transfectants showed a 22-fold increase in [(3)H]folic acid influx at pH 5.5, R113C or mock transfectants showed no increase. Moreover, WT PCFT transfectants displayed a 50% folic acid growth requirement concentration of 7 nM, whereas mock and R113C transfectants revealed 24- to 27-fold higher values. Consistently, upon fluorescein-methotrexate labeling, WT PCFT transfectants displayed a 50% methotrexate displacement concentration of 50 nM, whereas mock and R113C transfectants exhibited 12- to 14-fold higher values. Based on the crystal structure of the homologous Escherichia coli glycerol-3-phosphate transporter, we propose that the cationic R113 residue of PCFT is embedded in a hydrophobic pocket formed by several transmembrane helices that may be part of a folate translocation pore. These findings establish a novel loss of function mutation in HFM residing in an intracellular loop of PCFT crucial for folate transport.
Assuntos
Proteínas de Transporte/genética , Ácido Fólico/metabolismo , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/metabolismo , Mutação Puntual , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Arginina/química , Sequência de Bases , Sítios de Ligação/genética , Células CHO , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Criança , Consanguinidade , Cricetinae , Cricetulus , Primers do DNA/genética , Receptores de Folato com Âncoras de GPI , Homozigoto , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Metotrexato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120-6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414-8422, 2005). Here we explored the ability of the wild type (WT) R482-as well as the mutant G482-and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Resistencia a Medicamentos Antineoplásicos/genética , Antagonistas do Ácido Fólico/farmacologia , Mutação/genética , Proteínas de Neoplasias/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/química , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Doxorrubicina/química , Doxorrubicina/farmacologia , Fluoruracila/química , Fluoruracila/farmacologia , Antagonistas do Ácido Fólico/química , Compostos Heterocíclicos com 3 Anéis/metabolismo , Humanos , Indóis/química , Indóis/farmacologia , Lipídeos/química , Estrutura Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Paclitaxel/química , Paclitaxel/farmacologia , Pirimetamina/análogos & derivados , Pirimetamina/química , Pirimetamina/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Rodaminas , Transfecção , Trimetrexato/química , Trimetrexato/farmacologiaRESUMO
Folate cofactors are one-carbon donors essential for the biosynthesis of purines and thymidylate. Mammalian cells are devoid of folate biosynthesis and are therefore folate auxotrophs that take up folate vitamins primarily via the reduced folate carrier (RFC). In this study, we showed that the human RFC (hRFC) gene can serve as a novel selectable marker for the overproduction of recombinant proteins. Toward this end, a hemagglutinin (HA) epitope tagged hRFC (hRFC-HA) was introduced into a bicistronic vector (pIRES2-EGFP), upstream of an enhanced green fluorescent protein (EGFP) reporter gene. Chinese hamster ovary cells deficient in RFC activity were isolated and transfected with this construct, followed by gradual deprivation of leucovorin, the sole folate source in the growth medium. Only cells with hRFC-HA overexpression were able to take up leucovorin and thereby survive these selective conditions. Western blot and immunofluorescence analyses confirmed that the hRFC-HA was overexpressed at extremely high levels, properly glycosylated and sorted out to the plasma membrane. This resulted in a approximately 450-fold increase in [3H]methotrexate influx and approximately 100-fold increased sensitivity to methotrexate, relative to untransfected RFC-deficient cells. Flow cytometric analysis consistently revealed that EGFP was overexpressed approximately 100-fold above the autofluorescence level. Overproduction of hRFC-HA and EGFP was stably maintained for at least 2 months in a constant concentration of leucovorin. These results establish a novel RFC-based metabolic selection system for the efficient overexpression of recombinant proteins. Furthermore, the possible implications to subcellular transporter localization and restoration of MTX sensitivity in drug-resistant tumors by RFC-based gene therapy are discussed.
Assuntos
Marcadores Genéticos , Proteínas de Membrana Transportadoras/genética , Animais , Transporte Biológico , Western Blotting , Células CHO , Cricetinae , Proteínas de Fluorescência Verde/genética , Proteínas de Membrana Transportadoras/metabolismo , Metotrexato/metabolismo , Metotrexato/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Carregadora de Folato Reduzido , Frações Subcelulares/metabolismoRESUMO
Felix 01 (F01) is a bacteriophage originally isolated by Felix and Callow which lyses almost all Salmonella strains and has been widely used as a diagnostic test for this genus. Molecular information about this phage is entirely lacking. In the present study, the DNA of the phage was found to be a double-stranded linear molecule of about 80 kb. 11.5 kb has been sequenced and in this region A + T content is 60%. There are relatively few restriction endonuclease cleavage sites in the native genome and clones show this is due to their absence rather than modification. A restriction map of the genome has been constructed. The ends of the molecule cannot be ligated although they contain 5' phosphates. At least 60% of the genome must encode proteins. In the sequenced portion, many open reading frames exist and these are tightly packed together. These have been examined for homology to published proteins but only 1 to 17 shows similarity to known proteins. F01 is therefore the prototype of a new phage family. On the basis of restriction sites, codon usage and the distribution of nonsense codons in the unused reading frames, a strong case can be made for natural selection that reacts to mRNA structure and function.
Assuntos
DNA Viral , Fagos de Salmonella/genética , Sequência de Bases , Códon , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Genes Virais , Genoma Viral , Fases de Leitura Aberta , Mapeamento por Restrição , Salmonella/virologia , Fagos de Salmonella/crescimento & desenvolvimento , Fagos de Salmonella/patogenicidade , Proteínas Virais/biossíntese , Proteínas Virais/química , Proteínas Virais/genética , VirulênciaRESUMO
Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate the interaction of FGFs with their receptors (FGFRs) and are thought to play a critical role in receptor dimerization. Biochemical and crystallographic studies, conducted mainly with FGF-2 or FGF-1 and FGF receptors 1 and 2, suggests that an octasaccharide is the minimal length required for FGF- and FGFR-induced dimerization and subsequent activation. In addition, 6-O-sulfate groups are thought to be essential for binding of HS to FGFR and for receptor dimerization. We show here that oligosaccharides shorter than 8 sugar units support activation of FGFR2 IIIb by FGF-1 and interaction of FGFR4 with FGF-1. In contrast, only relatively long oligosaccharides supported receptor binding and activation in the FGF-1.FGFR1 or FGF-7.FGFR2 IIIb setting. In addition, both 6-O- and 2-O-desulfated heparin activated FGF-1 signaling via FGFR2 IIIb, whereas neither one stimulated FGF-1 signaling via FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required for activating FGFs is dictated by the specific FGF and FGFR combination. These different requirements may reflect the differences in the mode by which a given FGFR interacts with the various FGFs.