RESUMO
The processes of cell migration and invasion are integral to embryonic development and the functioning of adult organisms. Deregulation of these processes contributes to numerous diseases. Ras GTPases and in particular members of the Rho subfamily of GTPases play critical roles in cell migration and invasion. Here, we provide a collection of protocols to assay these functions. We describe two cell migration assays. The monolayer wound healing assay is very easy to implement, whereas the microliter-scale migration assay allows examination of cell behavior on defined extracellular matrices. We also describe two methods that allow the quantification of tumor cell invasion, a versatile transwell Matrigel invasion assay and an organotypic assay that examines the invasion of glioma cells through a rat brain slice.
Assuntos
Movimento Celular/fisiologia , Invasividade Neoplásica , Animais , Encéfalo/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Glioma/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Oligonucleotídeos , Técnicas de Cultura de Órgãos , RNA Interferente Pequeno , Ratos , Cicatrização/genéticaRESUMO
Members of the Rho family of small GTPases have been shown to be involved in tumorigenesis and metastasis. Currently, most of the available information on the function of Rho proteins in malignant transformation is based on the use of dominant-negative mutants of these GTPases. The specificity of these dominant-negative mutants is limited however. In this study, we used small interfering RNA directed against either Rac1 or Rac3 to reduce their expression specifically. In line with observations using dominant-negative Rac1 in other cell types, we show that RNA interference-mediated depletion of Rac1 strongly inhibits lamellipodia formation, cell migration and invasion in SNB19 glioblastoma cells. Surprisingly however, Rac1 depletion has a much smaller inhibitory effect on SNB19 cell proliferation and survival. Interestingly, whereas depletion of Rac3 strongly inhibits SNB19 cell invasion, it does not affect lamellipodia formation and has only minor effects on cell migration and proliferation. Similar results were obtained in BT549 breast carcinoma cells. Thus, functional analysis of Rac1 and Rac3 using RNA interference reveals a critical role for these GTPases in the invasive behavior of glioma and breast carcinoma cells.
Assuntos
Neoplasias Encefálicas/patologia , Neoplasias da Mama/patologia , Glioblastoma/patologia , Invasividade Neoplásica/fisiopatologia , Interferência de RNA , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Pseudópodes/fisiologia , RNA Interferente Pequeno , Células Tumorais Cultivadas , Proteínas rac de Ligação ao GTP , Proteínas rac1 de Ligação ao GTPRESUMO
The beta-chemokines, MIP-1alpha/CCL3, MIP-1beta/CCL4, and RANTES/CCL5, play a critical role in the selective accumulation and activation of macrophages in inflamed tissues. Herein, we demonstrate that the binding of each of these beta-chemokines to their cognate receptors, CCR1 and CCR5, in either macrophages or in CCR1- or CCR5-transfected CHO cells, induced actin reorganization and the formation of lamellipodia that are characteristic of the activation of the Rho family GTPase, Rac. A dominant negative mutant of Rac, but not dominant negative mutants of RhoA or Cdc42, blocked MIP-1alpha-induced lamellipodia formation. Moreover, this MIP-1alpha-induced Rac activation and consequent lamellipodia formation is Gi- and phosphoinositide-3 kinase (PI3K)-mediated. Thus, Rac activation is critical for both CCR1- and CCR5-triggered signaling cascades mediating beta-chemokine-induced reorganization of the actin cytoskeleton, a process essential for effective recruitment and activation of macrophages in inflammation.
