RESUMEN
TGF-beta 1 is a secreted polypeptide that elicits an antiproliferanve response in many cell types. However, many epithelial cancer cell lines are resistant to TGF-beta 1 growth inhibition. We investigated the in vitro growth suppressive effect of TGF-beta 1 on five ovarian cancer cell lines. Two of these (OVCAR-3 and AZ364) were growth inhibited by TGF-beta 1. The other three cell lines (SKOV-3, AZ224 and AZ547), were resistant to the antiproliferative action of the cytokine. All five cell lines produce TGF-beta 1 mRNA at very different levels and also secrete the TGF-beta 1 polypeptide, but mainly in a biologically latent form as tested by ELISA; this probably explains the fact that the TGF-beta 1 autocrine growth inhibition circuit is not active, even in sensitive cell lines. Even complete activation of the in vitro secreted latent form would be insufficient to induce growth arrest when compared to the levels of exogenous TGF-beta 1 needed to induce growth arrest in sensitive cell lines. The TGF-beta 1 receptor type I mRNA is expressed by all five ovarian cancer cell lines, but two of them (AZ224 and AZ547) lack detectable TGF-beta 1 receptor type II mRNA expression. Since TGF-beta 1 signaling requires both receptor types, the lack of receptor type II in two cell lines may explain their resistance to growth inhibition. Further experiments should be carried out on receptors and downstream components to pinpoint the cause of resistance in the SKOV cell line.
Asunto(s)
Receptores de Activinas Tipo I , Neoplasias Ováricas/patología , Factor de Crecimiento Transformador beta/fisiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/análisis , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/análisis , Receptores de Factores de Crecimiento Transformadores beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales CultivadasRESUMEN
Recombinant adeno-associated virus (rAAV) vectors have emerged as vehicles for gene therapy. In addition, anti-neoplastic properties have been attributed to wild-type AAV. To take advantage of both features and to overcome technical problems associated with rAAV preparation, we developed a production method in which rAAV particles are amplified in an infectious cycle in the presence of wtAAV. This results in a 10(3)-10(4)-fold amplification of rAAV input particles. rAAV-GFP particles generated by this method were used to transduce ovarian cancer cell lines to evaluate their potential in ovarian cancer gene therapy, in comparison to a rAd-GFP vector. The transduction efficiency of NIH-OVCAR3, MDAH 2774 and SKOV3 cells with rAAV-GFP particles was low (< 1%) and did not improve by increasing the number of particles/cell. Repeated administration and continued exposure of NIH-OVCAR3 and MDAH 2774 improved transduction to over 3%. In contrast, these cell lines were more efficiently transduced by rAAV-GFP in the presence of adenovirus (approximately 15%) and by rAd-GFP (> 50%). These results indicate that in contrast to rAd vectors, rAAV particles are not suitable for therapeutic gene transfer in ovarian cancer cells unless efficient help can be provided to mediate ss to ds DNA conversion.
Asunto(s)
Adenoviridae/genética , Carcinoma/metabolismo , Dependovirus/genética , Vectores Genéticos , Neoplasias Ováricas/metabolismo , Transducción Genética/métodos , Carcinoma/terapia , ADN Recombinante/genética , ADN Viral/genética , Femenino , Terapia Genética , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Inmunohistoquímica , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/inmunología , Técnicas de Amplificación de Ácido Nucleico , Neoplasias Ováricas/terapia , Células Tumorales CultivadasRESUMEN
Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.