RESUMEN

Resistance to chemotherapeutic drugs presents a big caveat for cancer treatment. In this review we will describe the molecular mechanisms involved in chemoresistance, discussing the mechanisms of resistance related to tumour microenvironment, as well as their intracellular mechanisms. Chemoresistance can also appear as a consequence to treatments with new anticancer drugs. In this sense, we will exemplify this type of resistance discussing mechanisms of action of epidermal growth factor receptor (EGFR) inhibitors. We conclude that the main problem of chemoresistance is due to its pleiotropic and multifactorial nature(AU)

Asunto(s)

Animales , Masculino , Femenino , Quimioterapia/métodos , Quimioterapia , Genes MDR , Resistencia a Múltiples Medicamentos/inmunología , Adenocarcinoma/tratamiento farmacológico , Carcinoma de Células Gigantes/tratamiento farmacológico , Fluorouracilo/uso terapéutico , Muerte Celular , Muerte Celular/fisiología , Genes MDR/efectos de la radiación , Células/patología , Apoptosis/inmunología , Genes MDR/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Resistencia a Antineoplásicos , Resistencia a Antineoplásicos/inmunología

RESUMEN

We investigated the mechanisms responsible for paclitaxel resistance in HME-1 cells (human mammary epithelial cells immortalized with hTERT). These cells were exposed to paclitaxel (10 pM for 7 days) and 20 cellular surviving populations (PSP) were obtained. PSP demonstrated high levels of resistance to paclitaxel cytotoxicity as compared with HME-1 cells. Activation of mdr-1 gene expression was observed in 2 PSP. Protein expression analysis using a C-terminal targeted antibody showed that 13 PSP were negative for p21/WAF1 expression after ionizing radiation (6 Gy) or doxorubicin (100 nM) treatment. Sequencing of the 3 exons of the CDKN1A gene revealed that 13 PSP contained a point mutation in exon 2. This mutation consisted in a T insertion at codon 104 leading to a premature STOP codon appearance. Mismatch amplification mutation assay and RFLP-PCR confirmed the presence of the mutation in 16 PSP. Western blot using an N-terminal targeted antibody demonstrated that the C-terminal-truncated p21/WAF1 protein (14 kDa) was indeed expressed in the 13 PSP. Our data suggest that p21/WAF1 inactivation may confer a strong resistance to paclitaxel in noncancerous breast epithelial cells harboring a p21/WAF1 mutant.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Glándulas Mamarias Humanas/efectos de los fármacos , Paclitaxel/farmacología , Mutación Puntual , Secuencia de Aminoácidos , Disparidad de Par Base , Western Blotting , Codón , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de la radiación , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/efectos de la radiación , Células Epiteliales/efectos de los fármacos , Femenino , Regulación de la Expresión Génica , Silenciador del Gen , Genes MDR/efectos de los fármacos , Genes MDR/efectos de la radiación , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/efectos de la radiación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Dosis de Radiación , Radiación Ionizante , Treonina

RESUMEN

PURPOSE: To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines. MATERIALS AND METHODS: The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy. To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR1 and MYCC-specific cosmid DNA probes. In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels. Dose-response assays to follow loss of function of the MDR1 gene from KB-C1 cells were also performed. RESULTS: A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation. Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells. CONCLUSIONS: Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype. Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Resistencia a Antineoplásicos/efectos de la radiación , Amplificación de Genes , Eliminación de Gen , Genes MDR/efectos de la radiación , Genes myc/efectos de la radiación , Proteínas Proto-Oncogénicas c-myc/análisis , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/efectos de la radiación , Fraccionamiento de la Dosis de Radiación , Relación Dosis-Respuesta en la Radiación , Resistencia a Antineoplásicos/genética , Citometría de Flujo , Genes MDR/efectos de los fármacos , Genes myc/efectos de los fármacos , Humanos , Pruebas de Micronúcleos , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiación , Ensayo de Tumor de Célula Madre

RESUMEN

Antigene radiotherapy is based upon damaging selected genes by a high dose of radiation from radionuclides delivered to this gene by a sequence-specific DNA-binding molecule. Here we describe our recent trials of antigene radiotherapy using the human mdr1 gene over-expressed in KB-V1 cells as a model. As a delivery molecule, we used a triplex-forming oligonucleotide (TFO) with a binding site in intron 14 of mdr1. This TFO was labeled with an Auger-electron-emitting radionuclide 125I. Decay of 125I releases a shower of low energy electrons that produce DNA strand breaks mostly within 10 bp from the decay site. Targeting in situ was assessed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. Double-strand breaks in the target sequence were detected in purified nuclei and digitonin-permeabilized cells, but not in the intact cells when TFO were delivered with liposomes. On the basis of these observations we hypothesized that there are cytoplasmic factors that bind such TFO and deliver them into the nucleus, but do not release them inside the nucleus, thus preventing TFO from binding their genomic targets. To test this hypothesis we (i) delivered TFO along with an excess of unlabeled oligonucleotide with an arbitrary sequence ("ballast") and (ii) conjugated TFO with a nuclear localization sequence peptide (NLS). We have found that TFO/NLS conjugates cleaved the target in a concentration-dependent manner regardless of the presence of the "ballast" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the "ballast." These results may provide a new insight into the mechanism of intracellular transport of oligonucleotides.

Asunto(s)

ADN/efectos de los fármacos , ADN/efectos de la radiación , Radioisótopos de Yodo/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Oligodesoxirribonucleótidos/farmacología , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/efectos de la radiación , Genes MDR/efectos de los fármacos , Genes MDR/efectos de la radiación , Humanos

RESUMEN

PURPOSE: Antigene radiotherapy (AR) is based on targeting localized radiodamage to specific sites in the genome by using sequence-specific triplex-forming oligonucleotides (TFO) to carry Auger-electron-emitters (A-Ettr) such as Iodine-125 (125I) to the target gene sequence. The radiodecay of an A-Ettr produces a cascade of low-energy electrons and creates a highly positively-charged daughter atom; delivered by a TFO, it should produce double-strand breaks (dsb) localized to the specific DNA target sequence. The result should be a "knock-out" of the targeted gene. METHODS AND MATERIALS: As a model, we used the MDR1 gene amplified nearly 100 times in the human KB-V1 carcinoma cell line. Chemically modified TFO complementary to the polypurine/polypyrimidine region of the MDR1 gene were synthesized and radiolabeled with 125I-dCTP by the primer extension method. Purified plasmid and genomic DNA and extracted nuclei were treated with 125I-TFO and analyzed for sequence-specific cleavage by electrophoresis in agarose gel and Southern hybridization. RESULTS: We created 125I-TFO that could effectively recognize, bind, and cleave the target sequence in plasmid and genomic DNA. We showed that these 125I-TFO in nanomolar concentrations were able to cleave the target MDR1 gene sequence in a natural environment, i.e., within the eucaryotic nucleus. CONCLUSION: 125I-TFO can effectively introduce sequence-specific dsb to a target within the MDR1 gene, both in purified DNA and inside intact nuclei. Chemically modified TFO conjugated with nuclear localization signal appear to be a promising delivery vehicle for future in vivo trials of AR.

Asunto(s)

Daño del ADN/genética , ADN de Neoplasias/efectos de la radiación , ADN/genética , Genes MDR/genética , Radioisótopos de Yodo/metabolismo , Oligonucleótidos/metabolismo , Radiofármacos/metabolismo , ADN/metabolismo , Cartilla de ADN/genética , Cartilla de ADN/uso terapéutico , ADN de Neoplasias/genética , Electroforesis en Gel de Agar , Genes MDR/efectos de la radiación , Humanos , Radioisótopos de Yodo/uso terapéutico , Hibridación de Ácido Nucleico/métodos , Oligonucleótidos/uso terapéutico , Plásmidos/metabolismo , Radiobiología , Radiofármacos/uso terapéutico , Radioterapia/métodos , Células Tumorales Cultivadas

RESUMEN

Failure of chemotherapy is frequently observed in patients previously treated with radiotherapy. To establish a cellular model for examining this resistance phenotype a series of mammalian tumor cell lines were exposed in vitro to fractionated X-irradiation and were then shown to express resistance to multiple antitumor drugs, including vincristine, etoposide and cisplatin. In these experiments the radiation was delivered as 10 fractions of 5 Gy (dose resulting in 1 log cell kill) given intermittently over several months. We now report that a comparable multidrug-resistance profile is expressed by human SK-OV-3 human ovarian tumor cells exposed in vitro to low dose (2 Gy) twice-daily fractions of X-rays given for 5 days on two consecutive weeks, essentially mimicking clinical practice, involving an overexpression of two MDR-associated proteins, P-glycoprotein and the multidrug resistance protein 1 (MRP1), with the latter being readily detectable by immunocytochemistry.

Asunto(s)

Subfamilia B de Transportador de Casetes de Unión a ATP/efectos de la radiación , Genes MDR/efectos de la radiación , Neoplasias Ováricas/radioterapia , Relación Dosis-Respuesta en la Radiación , Resistencia a Múltiples Medicamentos/efectos de la radiación , Resistencia a Antineoplásicos/efectos de la radiación , Femenino , Humanos , Modelos Biológicos , Neoplasias Ováricas/tratamiento farmacológico , Insuficiencia del Tratamiento , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiación

RESUMEN

Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix (OPF) and the FIX&PERM Cell Permeabilization Kit (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346: 1085-7) as well as Petersen et al. (Clin Chem 1997;43: 2039-46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Antígenos/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteína de Retinoblastoma/análisis , Proteína p53 Supresora de Tumor/análisis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/inmunología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de la radiación , Antígenos/metabolismo , Antígenos/efectos de la radiación , Línea Celular , Citoplasma/inmunología , Citoplasma/metabolismo , Citoplasma/efectos de la radiación , Epítopos/análisis , Epítopos/metabolismo , Epítopos/efectos de la radiación , Citometría de Flujo/métodos , Genes MDR/inmunología , Genes MDR/efectos de la radiación , Humanos , Linfocitos/inmunología , Linfocitos/metabolismo , Linfocitos/efectos de la radiación , Linfocitos/ultraestructura , Microondas , Proteínas Nucleares/análisis , Proteínas Nucleares/inmunología , Proteínas Nucleares/efectos de la radiación , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/efectos de la radiación , Juego de Reactivos para Diagnóstico , Reproducibilidad de los Resultados , Proteína de Retinoblastoma/inmunología , Proteína de Retinoblastoma/efectos de la radiación , Proteína p53 Supresora de Tumor/inmunología , Proteína p53 Supresora de Tumor/efectos de la radiación

RESUMEN

The human multidrug resistance 1 (MDR1) gene encoding P-glycoprotein is often overexpressed in various human tumors after chemotherapy. During treatment with various chemotherapeutic agents, the MDR1 gene is activated at the transcriptional level and/or amplified, resulting in overexpression. Our previous studies demonstrated that an inverted CCAAT box (Y-box) might be a critical cis-regulatory element regulating UV or drug-induced MDR1 gene expression. We have now established various cell lines from human head and neck cancer KB cells which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene driven by various MDR1 promoter deletion constructs. Transient transfection of antisense YB-1 expression constructs resulted in a decrease of both YB-1 protein levels and DNA binding activity to the inverted CCAAT box, as determined by Western blot and gel mobility shift assays. The limited expression and binding activity due to expression of antisense YB-1 constructs were also observed when cells were treated with UV. CAT activity of constructs containing the Y-box was enhanced after treatment with UV irradiation as well as genotoxic agents such as cisplatin and etoposide. Moreover, this activation was reduced by 50-80% by transfection of antisense YB-1 expression constructs. In contrast, transfection of antisense YB-1 expression constructs had no effect on CAT activity driven by MDR1 promoter constructs not containing the Y-box. These data indicate that YB-1 is directly involved in MDR1 gene activation in response to genotoxic stress.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/metabolismo , Genes MDR/efectos de la radiación , Neoplasias de Cabeza y Cuello/genética , Proteínas de Neoplasias/biosíntesis , Factores de Transcripción , ADN sin Sentido , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Factores de Transcripción NFI , Proteínas Nucleares , Unión Proteica , Activación Transcripcional , Células Tumorales Cultivadas , Rayos Ultravioleta , Proteína 1 de Unión a la Caja Y