Aumento en la transfección genética de la línea celular PC-12 mediada por fármacos que aumentan la fusión celular e inhiben la función lisosomal

Abstract

ABSTRACT: Gene therapy can be defined as the introduction of genetic material into cells with the objective of altering the course of a disease. Efficient and safe genetic vehicles are one of the fundamental requirements for the clinical application of gene therapy. Two classes of genetic vehicles have been used for gene therapy protocols: viral and nonviral vehicles. LipofectamineTM -DNA lipoplexes, one kind of nonviral vehicle, produce a gene transfection generally low (20-25%), which is a limitation for the use of LipofectamineTM in protocols for gene therapy. However, in our laboratory it has been demonstrated that an adequate mixture of compounds, such as inductors of membrane fusion, lysosomotropics and inductors of gene expression and cell division, produce an important increase in the efficiency of gene transfection of C-33 A and HEK 293 cells lines with LipofectamineTM lipoplexes. These compounds can be applied to improve the efficiency of gene transfection of cells which are very difficult to transfect, such as PC-12 cells. PC-12 is a cell line derived from the pheochromocytoma, which is a tumor of the medulla of the suprarenal gland of the rat. PC-12 cells are extensively used in neurobiological and neurochemical studies, which are important in the research of diseases such as the Alzheimer disease. In this work was used the lysosomotropic chloroquine, the polyamine spermidine and the inductors of membrane fusion chlorpromazine and procainamide, in order to improve the efficiency of transfection of PC-12 cell line. Previous studies in HEK 293 cells were used as reference, as well as transfection with LipofectamineTM -pRSβVgal lipoplexes. The cytotoxicity of the drugs and their mixtures on PC-12 cells was determined by trypan blue and Alamar blue techniques. For gene transfection experiments, were used drug concentrations in which cell viability was higher than 80%. Gene transfection with LipofectamineTM -pIRES2-EGFP lipoplexes increased 90% in the presence of chloroquine and decreased until 90% in the presence of chlorpromazine and procainamide. However, the highest gene transfection was attained when chloroquine and chlorpromazine or procainamide were used in combination (150% for chloroquine + chlorpromazine, and 180% for chloroquine + procainamide). The increase in transfection can be attributed to the combined effect of both drugs. On one hand, chlorpromazine and procainamide increased the fusion of membranes and thus improved the penetration of lipoplexes in the cells by endocytosis. On the other hand, chloroquine caused an increase in the phagolysosomal pH that inhibits their enzymes, and caused an alteration in the phagolysosomal membrane, which led to the lipoplexes release in the cytoplasm; the lipoplexes could then reach the nucleus and produce the gene transfection increase. Spermidine, which improves gene expression and cell division, produced an increase in gene transfection of 100%; furthermore, the mixture of spermidine and chloroquine produced an increase of 150%. When mixtures of spermidine plus chlorpromazine or procainamide were used, the effect of the inductors of fusion was dominant and gene transfection was decreased. The mixture of the three kind of drugs did not improve gene transfection. These findings demonstrate that an adequate mixture of compounds with properties of inductors of membrane fusion, lysosomotropic, and inductors of gene expression and cell division, produce an important increase in the efficiency of gene transfection of PC-12 cells. This could lead to a future application in protocols of gene therapy of diseases of the nervous system.

ABSTRACT: Gene therapy can be defined as the introduction of genetic material into cells with the objective of altering the course of a disease. Efficient and safe genetic vehicles are one of the fundamental requirements for the clinical application of gene therapy. Two classes of genetic vehicles have been used for gene therapy protocols: viral and nonviral vehicles. LipofectamineTM -DNA lipoplexes, one kind of nonviral vehicle, produce a gene transfection generally low (20-25%), which is a limitation for the use of LipofectamineTM in protocols for gene therapy. However, in our laboratory it has been demonstrated that an adequate mixture of compounds, such as inductors of membrane fusion, lysosomotropics and inductors of gene expression and cell division, produce an important increase in the efficiency of gene transfection of C-33 A and HEK 293 cells lines with LipofectamineTM lipoplexes. These compounds can be applied to improve the efficiency of gene transfection of cells which are very difficult to transfect, such as PC-12 cells. PC-12 is a cell line derived from the pheochromocytoma, which is a tumor of the medulla of the suprarenal gland of the rat. PC-12 cells are extensively used in neurobiological and neurochemical studies, which are important in the research of diseases such as the Alzheimer disease. In this work was used the lysosomotropic chloroquine, the polyamine spermidine and the inductors of membrane fusion chlorpromazine and procainamide, in order to improve the efficiency of transfection of PC-12 cell line. Previous studies in HEK 293 cells were used as reference, as well as transfection with LipofectamineTM -pRSβVgal lipoplexes. The cytotoxicity of the drugs and their mixtures on PC-12 cells was determined by trypan blue and Alamar blue techniques. For gene transfection experiments, were used drug concentrations in which cell viability was higher than 80%. Gene transfection with LipofectamineTM -pIRES2-EGFP lipoplexes increased 90% in the presence of chloroquine and decreased until 90% in the presence of chlorpromazine and procainamide. However, the highest gene transfection was attained when chloroquine and chlorpromazine or procainamide were used in combination (150% for chloroquine + chlorpromazine, and 180% for chloroquine + procainamide). The increase in transfection can be attributed to the combined effect of both drugs. On one hand, chlorpromazine and procainamide increased the fusion of membranes and thus improved the penetration of lipoplexes in the cells by endocytosis. On the other hand, chloroquine caused an increase in the phagolysosomal pH that inhibits their enzymes, and caused an alteration in the phagolysosomal membrane, which led to the lipoplexes release in the cytoplasm; the lipoplexes could then reach the nucleus and produce the gene transfection increase. Spermidine, which improves gene expression and cell division, produced an increase in gene transfection of 100%; furthermore, the mixture of spermidine and chloroquine produced an increase of 150%. When mixtures of spermidine plus chlorpromazine or procainamide were used, the effect of the inductors of fusion was dominant and gene transfection was decreased. The mixture of the three kind of drugs did not improve gene transfection. These findings demonstrate that an adequate mixture of compounds with properties of inductors of membrane fusion, lysosomotropic, and inductors of gene expression and cell division, produce an important increase in the efficiency of gene transfection of PC-12 cells. This could lead to a future application in protocols of gene therapy of diseases of the nervous system.

RESUMEN: La terapia génica puede definirse como la introducción de material genético en células con el objetivo de modificar el curso de una enfermedad. Uno de los requerimientos para la aplicación clínica de la terapia génica es contar con vehículos para la transferencia de genes seguros y eficientes. Los vehículos genéticos deben transferir el gen apropiado a las células específicas en tal forma que se exprese el gen en la cantidad requerida para corregir la enfermedad. Dos clases de vehículos se han usado para protocolos de terapia génica, vehículos virales y no-virales. Lipocomplejos de LipofectaminaTM -DNA, un tipo de vehículo noviral, produce una transfección generalmente baja (20-25%), lo que es una limitación para el uso de LipofectaminaTM en protocolos de terapia génica. En este laboratorio se ha demostrado que una mezcla adecuada de compuestos que inducen fusión de membrana, o lisosomotrópicos, e inductores de expresión genética y de división celular, producen un importante aumento en la eficiencia de la transfección genética de las líneas celulares C-33 A y HEK 293 con lipocomplejos de LipofectaminaTM. Estos compuestos pueden aplicarse a la transfección genética de líneas celulares que son difíciles de transfectar, como la línea PC-12. PC-12 es una línea derivada del feocromocitoma, un tumor que proviene de la glándula suprarrenal de rata. Estas células son ampliamente utilizadas en estudios neurobiológicos y neuroquímicos que son de suma importancia en la investigación de enfermedades como el Alzheimer. En este trabajo se usó el lisosomotrópico cloroquina, la poliamina espermidina y los inductores de fusión de membranas cloropromacina y procainamida, para mejorar la eficiencia de transfección de la línea PC-12. Los estudios previos en la línea celular HEK 293 se usaron como referencia, así como la transfección con lipocomplejos de LipofectaminaTM -pRS?Vgal. La citotoxicidad de los fármacos y de sus mezclas en las células PC-12 fue determinada por las técnicas azul tripan y azul Alamar. Para los experimentos de transfección se utilizaron concentraciones de los fármacos que permitieran una viabilidad mayor al 80%. La transfección genética con lipocomplejos de LipofectaminaTM -pIRES2-EGFP aumentó 90% en presencia de cloroquina y disminuyó en 90% en presencia de cloropromacina y procainamida. Sin embargo, la mayor transfección se obtuvo con mezclas de cloroquina con cloropromacina o procainamida (150% para cloroquina + cloropromacina y 180% para cloroquina + procainamida). Este aumento en la transfección puede atribuirse al efecto combinado de ambos fármacos. Por una parte, la cloropromacina y la procainamida aumentan la fusión de membranas lo que aumenta la penetración de lipocomplejos en las células por endocitosis. Por otra parte, la cloroquina causa un aumento en el pH del fagolisosoma que inhibe sus enzimas; además, causa una alteración en la membrana del fagolisosoma, lo cual en conjunto lleva a una liberación de los lipocomplejos al citoplasma, que pueden alcanzar el núcleo y producir el aumento en la transfección. La espermidina, que aumenta la expresión genética y la división celular, produjo un aumento en la transfección de 100% y la mezcla de espermidina y cloroquina la aumentaron a 150%. Cuando se usaron mezclas de espermidina más cloropromacina o procainamida, el efecto de los inductores de fusión celular predomina y la transfección genética disminuyó. La mezcla de los tres tipos de fármacos empleados no mejoró la transfección de las células PC-12 Estos hallazgos demostraron que la mezcla adecuada de compuestos con propiedades de inducción de fusión de membranas, de lisosomotrópico o de inducir la expresión genética y la división celular, producen un aumento importante en la eficiencia de la transfección genética de la línea celular PC-12, que pudiera conducir en el futuro a la aplicación de protocolos de terapia génica para enfermedades del sistema nervioso.