RESUMEN
Many cancers and leukemias are associated with strong dominant oncogenic mutations that activate tyrosine kinases and other classes of molecules, including transcription factors and antiapoptotic mechanisms. Some of these events can be targeted with small molecules or antibody-based therapeutics, but many remain intractable. In addition, cancer-related enzyme targets can often mutate, and drug-resistant variants are selected. Therapies directed at the mRNA encoding dominant oncogenes could provide a more global set of technologies for cancer treatment. To test this concept, we have used the model of transformation of hematopoietic cells by the chimeric Bcr-Abl oncogene, a highly activated tyrosine kinase. Our results show that tandem arrays of miRNA mimics, but not single miRNA mimics, directed against the Abl portion of the mRNA and introduced by lentiviral vectors can effectively alter the leukemogenic potency when the degree of suppression of expression of Bcr-Abl is reduced >200-fold from control levels. Only methods capable of such dramatic sustained reduction in the level of expression of highly activated kinase oncogenes are likely to be effective in controlling malignant cell populations.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Terapia Genética , Leucemia Linfoide/terapia , MicroARNs/genética , Animales , Apoptosis , Proteínas de Fusión bcr-abl/genética , Lentivirus/genética , Ratones , Ratones EndogámicosRESUMEN
Murine prostate stem cells express integrin alpha 6, which modulates survival, proliferation, and differentiation signaling through its interaction with the extracellular protein laminin. When plated in vitro in laminin containing Matrigel medium, 1 of 500-1,000 murine prostate cells can grow and form clonogenic spheroid structures that we term prostate spheres. Prostate spheres can be serially passaged individually or in bulk to generate daughter spheres with similar composition, demonstrating that sphere-forming cells are capable of self-renewal. Spheres spontaneously undergo lineage specification for basal and transit-amplifying cell types. P63-expressing cells localized to the outer layers of prostate spheres possess higher self-renewal capacity, whereas cells toward the center display a more differentiated transit-amplifying phenotype, as demonstrated by the expression of the prostate stem cell antigen. When dihydrotestosterone is added to the medium, the androgen receptor is stabilized, is imported to the nucleus, and drives differentiation to a luminal cell-like phenotype. A fraction of sphere cells returned to an in vivo environment can undergo differentiation and morphogenesis to form prostate tubular structures with defined basal and luminal layers accompanied by prostatic secretions. This study demonstrates self-renewal and multilineage differentiation from single adult prostate stem/progenitor cells in a specific in vitro microenvironment.
Asunto(s)
Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Próstata/citología , Células Madre/citología , Animales , Línea Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Próstata/fisiología , Células Madre/fisiologíaRESUMEN
The Polycomb group transcriptional repressor Bmi-1 is often upregulated in prostate cancer, but its functional roles in prostate stem cell maintenance and prostate cancer are unclear. Loss- and gain-of-function analysis in a prostate sphere assay indicates that Bmi-1 expression is required for self-renewal activity and maintenance of p63(+) stem cells. Loss of Bmi-1 blocks the self-renewal activity induced by heightened ß-catenin signaling, suggesting that Bmi-1 is required for full activity of another self-renewal pathway. In vivo, Bmi-1 expression is necessary for normal prostate tubule regeneration. Altered self-renewal and proliferation through Bmi-1 modulation diminishes the susceptibility of prostate cells to transformation. In an in vivo prostate regeneration system, Bmi-1 inhibition protects prostate cells from FGF10-driven hyperplasia and slows the growth of aggressive Pten-deletion-induced prostate cancer. We conclude that Bmi-1 is a crucial regulator of self-renewal in adult prostate cells and plays important roles in prostate cancer initiation and progression.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Proteínas Nucleares/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/etiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Células Madre/citología , Animales , Diferenciación Celular , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Próstata/citología , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Células Madre/metabolismo , beta Catenina/metabolismoRESUMEN
The successful isolation and cultivation of prostate stem cells will allow us to study their unique biological properties and their application in therapeutic approaches. Here we describe step-by-step procedures on the basis of previous work in our laboratory for the harvesting of primary prostate cells from adolescent male mice by a modified enzymatic procedure; the isolation of an enriched population of prostate stem cells through cell sorting; and the cultivation of prostate stem cells in vitro and characterization of these cells and their stem-like activity, including in vivo tubule regeneration. Normally, it will take approximately 8 h to harvest prostate cells, isolate the stem cell-enriched population and set up the in vitro sphere assay. It will take up to 8 weeks to analyze the unique properties of the stem cells, including their regenerative capacity in vivo.
Asunto(s)
Células Madre Adultas/citología , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Próstata/citología , Células Madre Adultas/fisiología , Células Madre Adultas/trasplante , Animales , Diferenciación Celular , Proliferación Celular , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Próstata/fisiología , Regeneración , Células MadreRESUMEN
The ability to isolate prostate stem cells is essential to explore their role in prostate development and disease. In vitro prostate colony- and sphere-forming assays were used to quantitatively measure murine prostate stem/progenitor cell enrichment and self-renewal. Cell surface markers were screened for their ability to positively or negatively enrich for cells with enhanced growth potential in these assays. Immunohistochemical and FACS analyses demonstrate that specific cell surface markers can be used to discriminate prostate stromal (CD34(+)), luminal epithelial (CD24(+)CD49f(-)), basal epithelial (CD24(+)CD49f(+)), hematopoietic (CD45(+), Ter119(+)), and endothelial (CD31(+)) lineages. Sorting for cells with a CD45(-)CD31(-)Ter119(-)Sca-1(+)CD49f(+) antigenic profile results in a 60-fold enrichment for colony- and sphere-forming cells. These cells can self-renew and expand to form spheres for many generations and can differentiate to produce prostatic tubule structures containing both basal and luminal cells in vivo. These cells also localize to the basal cell layer within the region of the gland that is proximal to the urethra, which has been identified as the prostate stem cell niche. Prostate stem cells can be isolated to a purity of up to 1 in 35 by using this antigenic profile. The remarkable similarity in cell surface profile between prostate and mammary gland stem cells suggests these markers may be conserved among epithelial stem cell populations.