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The media formulations necessary for deriving and sustaining organoids from epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established. Critical components of organoid media are a set of growth factors that include R-spondins and BMP signalling antagonists such as Noggin or Gremlin 1. Currently, the practical limitations for formulating organoid media of reproducible potency and larger-scale media production that have hampered further technological applications of organoid technology include: the cost of growth factors such as R-spondins and Gremlin 1/Noggin and their production as defined specific activities free of contaminants that may affect organoid growth. Here we report the production of highly pure recombinant Gremlin 1 and R-spondin 1 from bacterial expression for use in organoid media. We detail the workflow for Gremlin 1 and R-spondin 1 expression, purification, quantification of cellular activity, quality control and use in media formulated for culturing organoids derived from a number of tissues. The development of precisely formulated, cost-effective media of defined specific activity will engender the development of novel applications for organoid technology.
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Técnicas de Cultura de Células/economia , Meios de Cultura/química , Peptídeos e Proteínas de Sinalização Intercelular/química , Organoides/crescimento & desenvolvimento , Animais , Bactérias/genética , Bactérias/metabolismo , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células/métodos , Meios de Cultura/economia , Técnicas de Transferência de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/economia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismoRESUMO
Purpose: Constitutively active WNT signaling is a hallmark of colorectal cancers and a driver of malignant tumor progression. Therapeutic targeting of WNT signaling is difficult due to high pathway complexity and its role in tissue homeostasis. Here, we identify the transcription factor ADNP as a pharmacologically inducible repressor of WNT signaling in colon cancer.Experimental Design: We used transcriptomic, proteomic, and in situ analyses to identify ADNP expression in colorectal cancer and cell biology approaches to determine its function. We induced ADNP expression in colon cancer xenografts by low-dose ketamine in vivo Clinical associations were determined in a cohort of 221 human colorectal cancer cases.Results: ADNP was overexpressed in colon cancer cells with high WNT activity, where it acted as a WNT repressor. Silencing ADNP expression increased migration, invasion, and proliferation of colon cancer cells and accelerated tumor growth in xenografts in vivo Treatment with subnarcotic doses of ketamine induced ADNP expression, significantly inhibited tumor growth, and prolonged survival of tumor-bearing animals. In human patients with colon cancer, high ADNP expression was linked to good prognosis.Conclusions: Our findings indicate that ADNP is a tumor suppressor and promising prognostic marker, and that ketamine treatment with ADNP induction is a potential therapeutic approach that may add benefit to current treatment protocols for patients with colorectal cancer. Clin Cancer Res; 23(11); 2769-80. ©2016 AACR.
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Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Proteômica , Animais , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Ketamina/uso terapêutico , Camundongos , Terapia de Alvo Molecular , Regiões Promotoras Genéticas , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MAIN CONCLUSION: The extreme Alb3 C terminus is important for Alb3 stability in a light dependent manner, but is dispensable for LHCP insertion or D1 synthesis. YidC/Oxa1/Alb3 dependent insertion of membrane proteins is evolutionary conserved among bacteria, mitochondria and chloroplasts. Chloroplasts are challenged by the need to coordinate membrane integration of nuclear encoded, post-translationally targeted proteins into the thylakoids as well as of proteins translated on plastid ribosomes. The pathway facilitating post-translational targeting of the light-harvesting chlorophyll a/b binding proteins involves the chloroplast signal recognition particle, cpSRP54 and cpSRP43, as well as its membrane receptor FtsY and the translocase Alb3. Interaction of cpSRP43 with Alb3 is mediated by the positively charged, stromal exposed C terminus of Alb3. In this study, we utilized an Alb3 T-DNA insertion mutant in Arabidopsis thaliana lacking the last 75 amino acids to elucidate the function of this domain (alb3∆C). However, the truncated Alb3 protein (Alb3∆C) proved to be unstable under standard growth conditions, resulting in a reduction of Alb3∆C to 20 % of wild-type levels. In contrast, accumulation of Alb3∆C was comparable to wild type under low light growth conditions. Alb3∆C mutants grown under low light conditions were only slightly paler than wild type, accumulated almost wild-type levels of light harvesting proteins and were not affected in D1 synthesis, therefore showing that the extreme Alb3 C terminus is dispensable for both, co- and post-translational, protein insertion into the thylakoid membrane. However, reduction of Alb3∆C levels as observed under standard growth conditions resulted not only in a severely diminished accumulation of all thylakoid complexes but also in a strong defect in D1 synthesis and membrane insertion.
