RESUMO
We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a approximately 100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K(m) for folic acid; (b) 6-fold increased transport K(m) for GW1843; and (c) a slightly increased transport V(max) for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 --> Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Resistencia a Medicamentos Antineoplásicos/genética , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Indóis/farmacologia , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Mutação , Quinazolinas/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Transporte Biológico , Northern Blotting , Southern Blotting , Western Blotting , Divisão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cloretos/farmacologia , Análise Mutacional de DNA , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Éxons , Ácido Fólico/química , Ácido Fólico/farmacocinética , Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Humanos , Indóis/química , Concentração Inibidora 50 , Isoindóis , Cinética , Leucovorina/farmacologia , Leucemia/genética , Leucemia/metabolismo , Metotrexato/química , Metotrexato/farmacologia , Mutagênese Sítio-Dirigida , Polimorfismo Conformacional de Fita Simples , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Quinazolinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína Carregadora de Folato Reduzido , Timidilato Sintase/antagonistas & inibidores , Fatores de Tempo , Transfecção , Trimetrexato/farmacologia , Células Tumorais CultivadasRESUMO
Human CEM-7A cells established by gradual deprivation of leucovorin from the growth medium, display 100-fold overexpression of methotrexate transport activity. We found that this was associated with 10-fold reduced folate carrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6. This deletion consisted of the 3'-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3'-UTR. These resulted in a truncated reduced folate carrier protein, which lacks the C-terminal 160 amino acids, but instead contains 58 new C-terminal amino acids obtained from reading through the 3'-UTR. Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face. Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadly migrating approximately 65-90 kDa native reduced folate carrier and a approximately 40-45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS-[3H]MTX and NHS-[3H]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement. The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ácido Fólico/metabolismo , Leucemia/genética , Leucemia/metabolismo , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Regiões 3' não Traduzidas , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/química , Divisão Celular/efeitos dos fármacos , Primers do DNA/genética , DNA Complementar/genética , DNA de Neoplasias/genética , Humanos , Leucemia L1210/genética , Leucemia L1210/metabolismo , Metotrexato/metabolismo , Metotrexato/farmacologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteína Carregadora de Folato Reduzido , Transfecção , Células Tumorais CultivadasRESUMO
CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays >200-fold resistance to methotrexate (MTX) due to defective transport via the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, derived by a gradual deprivation of folic acid from 2.3 microM to 2 nM (LF) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild type Km value for MTX transport but a 31-fold and 9-fold lower Km values for folic acid and leucovorin, respectively, relative to wild type RFC; 2) a 10-fold RFC1 gene amplification along with a >20-fold increased expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulation of MTX transport by anions (i.e. chloride); and 4) a G --> A mutation at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, resulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the human RFC. Upon transfer of CEM/MTX-LF cells to folate-replete medium (2.3 microM folic acid), the more efficient folic acid uptake in CEM/MTX-LF cells resulted in a 7- and 24-fold elevated total folate pool compared with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol/mg of protein, respectively). This markedly elevated intracellular folate pool conferred a novel mechanism of resistance to polyglutamatable (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. trimetrexate and pyrimethamine) by abolishing their polyglutamylation and circumventing target enzyme inhibition.
Assuntos
Proteínas de Transporte/genética , Antagonistas do Ácido Fólico/farmacologia , Ácido Fólico/metabolismo , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Marcadores de Afinidade/metabolismo , Transporte Biológico , Northern Blotting , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Glutamatos/farmacologia , Humanos , Cinética , Leucemia/metabolismo , Metotrexato/metabolismo , Pirimetamina/farmacologia , Pirimidinas/farmacologia , Proteína Carregadora de Folato Reduzido , Relação Estrutura-Atividade , Tetra-Hidrofolatos/farmacologia , Trimetrexato/farmacologia , Células Tumorais CultivadasAssuntos
Proteínas de Transporte/genética , Epiderme/metabolismo , Genes/genética , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Animais , Epiderme/química , Expressão Gênica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA/análise , RNA/genética , RNA/isolamento & purificação , Proteína Carregadora de Folato ReduzidoRESUMO
Methotrexate is widely used in the treatment of severe psoriasis. However, little is currently known about the mechanisms underlying its therapeutic activity in the skin. Methotrexate has been shown to be carried into cells through the reduced folate carrier (RFC-1). The recent cloning and characterization of the human gene encoding this transmembranal carrier enabled us to investigate RFC-1 gene expression in human skin. Biopsies were obtained from the skin of healthy and psoriatic volunteers. RNA extracted from these biopsies was analyzed by the reverse transcriptase-polymerase chain reaction technique. While RFC-1 gene expression was barely detectable in the uninvolved skin of psoriatic patients and in the skin of healthy volunteers, high levels of RFC-1 transcripts were found in biopsies obtained from psoriatic plaques. To further investigate this pattern of gene expression, we studied skin biopsies by in situ hybridization with a labeled antisense riboprobe specific for the RFC-1 gene. The RFC-1 gene was found to be weakly expressed in the epidermis, in biopsies obtained from the skin of healthy subjects as well as in those from the uninvolved skin of psoriatic patients. In contrast, in biopsies obtained from psoriatic plaques, high levels of RFC-1 gene transcripts were found mostly in the spinous layer of the epidermis. These results suggest the existence of a specific methotrexate carrier in the human epidermis, and may bear relevance to the cutaneous manifestations of methotrexate toxicity.
Assuntos
Proteínas de Transporte/genética , Ácido Fólico/metabolismo , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Psoríase/metabolismo , Pele/metabolismo , Adulto , Idoso , Linhagem Celular , Feminino , Humanos , Masculino , Metotrexato/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/análise , Proteína Carregadora de Folato Reduzido , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Prolonged cell culture of human leukemia cells at folate concentrations in the (sub)physiological range (1-5 nM) rather than at 'standard' supraphysiological concentrations of 2-10 microM folic acid elicited a number of regulatory aspects of the reduced folate carrier (RFC), the membrane transport protein for natural reduced folate cofactors and folate-based chemotherapeutic drugs such as methotrexate (MTX). One subline of human CCRF-CEM leukemia cells grown under folate-restricted conditions (CEM-7A) exhibited a 95-fold increased Vmax for uptake of [3H]-MTX. The increased uptake of MTX in CEM-7A cells is based on at least two factors: (a) a constitutive 10-fold overexpression of the RFC1 gene and RFC1 message; and (b) a 7-9-fold up-regulation of RFC transport activity under low intracellular reduced folate concentrations. This second component appeared to be regulatable by changes in the cellular folate, purine and methylation status as judged from a 7-9 fold down-regulation of RFC transport activity after short term (1-2 hr) incubation of CEM-7A cells with reduced folate cofactors (25 nM LV), purines (100 microM adenosine) or S-adenosylmethionine (100 microM), respectively. Gradual folate restriction in the cell culture medium of CEM/MTX cells, a subline of CCRF-CEM resistant to MTX due to defective transport via the RFC, revealed the up-regulated expression of an altered RFC protein that is characterized by a 35-fold decreased Km for folic acid and a 10-fold decreased Km for the reduced folate cofactor LV compared to the RFC expressed in CCRF-CEM and CEM-7A cells. As a result of the markedly increased efficiency of folic acid uptake in CEM/MTX cells, intracellular folate pools were 7-fold higher than in CCRF-CEM cells when both cell lines were incubated in the presence of 2 microM folic acid. The high intracellular folate pools in CEM/MTX cells appeared to impair the polyglutamylation of antifolates and confer resistance to ZD1694, an antifolate drug that depends on polyglutamylation for its biological activity. Collectively, these studies provide a better insight into the basic regulation of RFC-mediated membrane transport of clinically active antifolates. In addition, these studies may also provide an opportunity to exploit the transport system as a target for biochemical modulation by which it may contribute to an improved efficacy of folate-based chemotherapy in a clinical setting.
Assuntos
Proteínas de Transporte/metabolismo , Antagonistas do Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Metotrexato/metabolismo , Metotrexato/farmacologia , Adenosina/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Divisão Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Humanos , Cinética , Leucovorina/farmacologia , Proteína Carregadora de Folato Reduzido , S-Adenosilmetionina/farmacologia , Tetra-Hidrofolatos/metabolismo , Timidilato Sintase/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
We are studying the mechanism underlying chemosensitization of anticancer-drug cytotoxicity in wild-type and multidrug-resistant (MDR) mammalian cells. We show here that the chemosensitizers, reserpine and verapamil, display a dramatic potentiation of taxol, anthracycline and Vinca alkaloids cytotoxicity in P-glycoprotein-(P-gp)-deficient hamster and human nasopharyngeal carcinoma cells. We have therefore utilized this phenomenon to probe for the putative P-gp-independent component of cytotoxicity chemosensitization. These chemosensitizers yielded a marked increase in the accumulation of taxol in parental hamster and human carcinoma cells that are devoid of P-gp. These chemosensitizers and non-ionic detergents brought about a pronounced increase in the accumulation of structurally and mechanistically diverse lipophilic chromophores in parental and MDR hamster cells. Furthermore, non-toxic concentrations of these non-ionic detergents yielded a marked potentiation of taxol cytotoxicity in parental cells. These findings were consistent with a chemosensitizer-mediated, P-gp-independent increase in membrane permeability. Thus, several aspects of chemosensitizers' interaction with lipid bilayers and biomembranes were studied. In this respect, like various mild detergents, chemosensitizers induced a dose-dependent leakage of carboxyfluorescein encapsulated in liposomes. Like specialized membrane fluidizers, various chemosensitizers induced a dose-dependent membrane fluidization (and sometimes rigidification) in both liposomes and various wild-type and MDR animal and human cells, as revealed by diphenylhexatriene fluorescence polarization. Furthermore, a favorable correlation was observed between the ability of chemosensitizers to permeabilize lipid bilayers and their capacity to potentiate anticancer-drug cytotoxicity. Thus, we propose that chemosensitizer-mediated changes in the physical properties of biomembranes, including altered fluidity and increased permeability, may be important factors in achieving potentiation of anticancer-drug cytotoxicity in wild-type and MDR mammalian cells. This study offers a basis for the chemosensitizer-mediated potentiation of drug toxicity to healthy tissues, thus emphasizing the importance of a prior evaluation of the potential untoward toxicity when simultaneously using MDR chemosensitizers and cytotoxic agents in the clinic.
Assuntos
Antineoplásicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Fluidez de Membrana/efeitos dos fármacos , Reserpina/farmacologia , Verapamil/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Células CHO , Cricetinae , Sinergismo Farmacológico , Humanos , Células Tumorais CultivadasRESUMO
We describe an ornithine decarboxylase (ODC) activity-based assay for the quantitation of multidrug resistance (MDR) and its reversal by MDR modulators in cultured mammalian cells. ODC catalyzes the first and rate-limiting step in polyamine biosynthesis. The activity of this enzyme rises rapidly after growth initiation, such as after addition of serum-containing medium to quiescent mammalian cells. This increase in enzyme activity is prevented when growth is arrested, such as after treatment with cytotoxic drugs. In this assay cultures of drug-sensitive animal and human carcinoma cells as well as their MDR sublines were exposed to various concentrations of different cytotoxic agents for 6-48 h. A dose-dependent decrease in ODC activity was obtained with a variety of chemotherapeutic agents including anthracyclines, vinca alkaloids, epipodophyllotoxins, actinomycin D, antifolates, and cisplatinum. Anticancer drug resistance levels were calculated as the 50% inhibitory concentration of ODC activity obtained with drug-resistant cells divided by that obtained with sensitive cells. These cytotoxicity determinations correlated favorably with those obtained by the well-established colony formation assay. The ODC assay also proved useful in the assessment of MDR reversal with modulators of the MDR phenotype. Therefore, these studies show that the ODC assay could be useful for the reliable determination of drug resistance levels in cultured mammalian cells and for the assessment of drug resistance reversal by various modulators of the MDR phenotype.