RESUMO
The S13 subunit (also called Pad1, Rpn11, and MPR1) is a component of the 19S complex, a regulatory complex essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome. To address the functional role of S13, we combined double-stranded RNA interference (RNAi) against the Drosophila proteasome subunit DmS13 with expression of wild-type and mutant forms of the homologous human gene, HS13. These studies show that DmS13 is essential for 26S function. Loss of the S13 subunit in metazoan cells leads to increased levels of ubiquitin conjugates, cell cycle defects, DNA overreplication, and apoptosis. In vivo assays using short-lived proteasome substrates confirmed that the 26S ubiquitin-dependent degradation pathway is compromised in S13-depleted cells. In complementation experiments using Drosophila cell lines expressing HS13, wild-type HS13 was found to fully rescue the knockdown phenotype after DmS13 RNAi treatment, while an HS13 containing mutations (H113A-H115A) in the proposed isopeptidase active site was unable to rescue. A mutation within the conserved MPN/JAMM domain (C120A) abolished the ability of HS13 to rescue the Drosophila cells from apoptosis or DNA overreplication. However, the C120A mutant was found to partially restore normal levels of ubiquitin conjugates. The S13 subunit may possess multiple functions, including a deubiquitinylating activity and distinct activities essential for cell cycle progression that require the conserved C120 residue.
Assuntos
Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Interferência de RNA , Proteínas Ribossômicas/genética , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Sítios de Ligação , Western Blotting , Ciclo Celular , Linhagem Celular , Drosophila , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Deleção de Genes , Teste de Complementação Genética , Proteínas de Fluorescência Verde , Humanos , Immunoblotting , Proteínas Luminescentes/metabolismo , Metaloendopeptidases/química , Microscopia de Fluorescência , Mutação , Fenótipo , RNA de Cadeia Dupla/metabolismo , Ubiquitina/metabolismoRESUMO
BACKGROUND: Identification of multidrug resistance (MDR) factors is crucial for designing chemotherapeutic strategies. Aberrant expression and dysfunction of proteasome subunits have been involved in malignant transformation and in cell resistance to various cytotoxic drugs. MATERIALS AND METHODS: We analyzed the expression levels of the proteasome subunit Rpn11 in a panel of cancer cell lines, and studied the effect of Rpn11 overexpression on the resistance of mammalian cells to cytotoxic drugs in clonogenic cytotoxicity assay. RESULTS: Rpn11 levels are highly variable in cancer cells; mammalian cells stably overexpressing Rpn11 display moderate resistance to vinblastine, cisplatin and doxorubicin, and also exhibit a slower proliferation rate when compared to the control cells. CONCLUSION: Rpn11-overexpression in mammalian cells affects cell proliferation and the response to cytotoxic drugs, both of which may promote tumor cell escape from chemotherapeutic agents, and may serve as a marker for MDR-cells.
Assuntos
Adenosina Trifosfatases/fisiologia , Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos/fisiologia , Endopeptidases/fisiologia , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/genética , Animais , Células COS/efeitos dos fármacos , Células COS/fisiologia , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Endopeptidases/biossíntese , Endopeptidases/genética , Feminino , Hemaglutininas/biossíntese , Hemaglutininas/genética , Humanos , Melfalan/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Células Tumorais Cultivadas , Vimblastina/farmacologiaRESUMO
Multipotent mesenchymal stromal cells (MSCs) are a type of adult stem cells that can be easily isolated from various tissues and expanded in vitro. Many reports on their pluripotency and possible clinical applications have raised hopes and interest in MSCs. In an attempt to unify the terminology and the criteria to label a cell as MSC, in 2006 the International Society for Cellular Therapy (ISCT) proposed a standard set of rules to define the identity of these cells. However, MSCs are still extracted from different tissues, by diverse isolation protocols, are cultured and expanded in different media and conditions. All these variables may have profound effects on the selection of cell types and the composition of heterogeneous subpopulations, on the selective expansion of specific cell populations with totally different potentials and ergo, on the long-term fate of the cells upon in vitro culture. Therefore, specific molecular and cellular markers that identify MSCs subsets as well as standardization of expansion protocols for these cells are urgently needed. Here, we briefly discuss new useful markers and recent data supporting the rapidly emerging concept that many different types of progenitor cells are found in close association with blood vessels. This knowledge may promote the necessary technical improvements required to reduce variability and promote higher efficacy and safety when isolating and expanding these cells for therapeutic use. In the light of the discussed data, particularly the identification of new markers, and advances in the understanding of fundamental MSC biology, we also suggest a revision of the 2006 ISCT criteria.