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1.
Cancer Res ; 71(3): 725-35, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21266362

RESUMO

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell-like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G(2)/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease.


Assuntos
Carcinoma de Células Renais/patologia , Técnicas de Cultura de Células/métodos , Neoplasias Renais/patologia , Animais , Ciclo Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Sefarose , Especificidade da Espécie
2.
Cancer Res ; 71(3): 716-24, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21266363

RESUMO

The culture of tumor cell lines in three-dimensional scaffolds is considered to more closely replicate the in vivo tumor microenvironment than the standard method of two-dimensional cell culture. We hypothesized that our method of encapsulating and maintaining viable and functional pancreatic islets in agarose-agarose macrobeads (diameter 6-8 mm) might provide a novel method for the culture of tumor cell lines. In this report we describe and characterize tumor colonies that form within macrobeads seeded with mouse renal adenocarcinoma cells. Approximately 1% of seeded tumor cells survive in the macrobead and over several months form discrete elliptical colonies appearing as tumor cell niches with increasing metabolic activity in parallel to colony size. The tumor colonies demonstrate ongoing cell turnover as shown by BrdU incorporation and activated caspase-3 and TUNEL staining. Genes upregulated in the tumor colonies of the macrobead are likely adaptations to this novel environment, as well as an amplification of G(1)/S cell-cycle checkpoints. The data presented, including SCA-1 and Oct4 positivity and the upregulation of stem cell-like genes such as those associated with the Wnt pathway, support the notion that the macrobead selects for a subpopulation of cells with cancer stem cell or cancer progenitor properties.


Assuntos
Carcinoma de Células Renais/patologia , Técnicas de Cultura de Células/métodos , Neoplasias Renais/patologia , Células-Tronco Neoplásicas/patologia , Animais , Apoptose/fisiologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Processos de Crescimento Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Neoplásicas/metabolismo , Sefarose , Células Tumorais Cultivadas
3.
Proc Natl Acad Sci U S A ; 103(28): 10666-71, 2006 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-16815977

RESUMO

Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder that is characterized by retrograde axonal degeneration that primarily affects long spinal neurons. The disease is clinically heterogeneous, and there are >20 genetic loci identified. Here, we show a physical interaction between spastin and atlastin, two autosomal dominant HSP gene products. Spastin encodes a microtubule (MT)-severing AAA ATPase (ATPase associated with various activities), and atlastin encodes a Golgi-localized integral membrane protein GTPase. Atlastin does not regulate the enzymatic activity of spastin. We also identified a clinical mutation in atlastin outside of the GTPase domain that prevents interaction with spastin in cells. Therefore, we hypothesize that failure of appropriate interaction between these two HSP gene products may be pathogenetically relevant. These data indicate that at least a subset of HSP genes may define a cellular biological pathway that is important in axonal maintenance.


Assuntos
Adenosina Trifosfatases/metabolismo , Axônios/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Transdução de Sinais/fisiologia , Paraplegia Espástica Hereditária/enzimologia , Paraplegia Espástica Hereditária/genética , Adenosina Trifosfatases/genética , Animais , Células COS , Chlorocebus aethiops , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP , Genes Dominantes , Heterogeneidade Genética , Células HeLa , Humanos , Proteínas de Membrana , Mutação , Transdução de Sinais/genética , Espastina
4.
Cell Transplant ; 14(7): 427-39, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16285251

RESUMO

For clinical xenogenic islet transplantation to be successful, several requirements must be met. Among them is a sizeable and reliable source of fully functional and microbiologically safe islets. The inherent variability among porcine pancreases, with respect to islet yield, prompted us to develop a Biopsy Score technique to determine the suitability of each pancreas for islet isolation processing. The Biopsy Score consists of an assessment of five variables: warm ischemia time, pancreas color, fat content, islet size, and islet demarcation, each of which is assigned a value of -1 or +1, depending on whether or not the established criteria is met. For determination of islet size and demarcation, fresh biopsies of porcine pancreases are stained with dithizone (DTZ) solution and examined under a dissecting microscope. Based on the scoring of such biopsies in pancreases from 26-56-month-old sows, we report here that the presence of large (>100 microm diameter), well-demarcated islets in the pancreas biopsy is a reliable predictor of isolation success. Encapsulation of the isolated porcine islets within the inner layer of a 1.5% agarose and an outer layer of 5.0% agarose macrobead, containing 500 equivalent islet number (EIN), provides for extended in vitro functional viability (>6 months of insulin production in response to glucose), as well as for comprehensive microbiological testing and at least partial isolation of the xenogeneic islets from the host immune system. All microbiological testing to date has been negative, except for the presence of porcine endogenous retrovirus (PERV). Taken together, we believe that the Biopsy Score enhancement of our islet isolation technique and our agarose-agarose macroencapsulation methodology bring us significantly closer to realizing clinical porcine islet xenotransplantation for the treatment of insulin-dependent diabetic patients.


Assuntos
Transplante das Ilhotas Pancreáticas/normas , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/microbiologia , Pâncreas/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Cápsulas , Ilhotas Pancreáticas/química , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Camundongos , Pâncreas/química , Pâncreas/patologia , Segurança , Suínos , Sobrevivência de Tecidos
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