bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2021‒10‒10
seventeen papers selected by
Lucas B. Zeiger
Beatson Institute for Cancer Research
  1. Understanding PI3K inhibitor mechanism of action.
  2. The concerted action of oncogenic driver mutations directs global translation in intestinal epithelial cells.
  3. The structural basis of PTEN regulation by multi-site phosphorylation.
  4. Oncogenic KRAS blockade therapy: renewed enthusiasm and persistent challenges.
  5. Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair.
  6. A late endosome signaling hub that couples PI3Kα and WNT/β-catenin signaling in breast cancer.
  7. K-RAS4A: Lead or Supporting Role in Cancer Biology?
  8. Mapping of mTOR drug targets: Featured platforms for anti-cancer drug discovery.
  9. A structural model of a Ras-Raf signalosome.
  10. Quantifying ERK activity in response to inhibition of the BRAFV600E-MEK-ERK cascade using mathematical modelling.
  11. Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry.
  12. CRISPR screens identify cholesterol biosynthesis as a therapeutic target on stemness and drug resistance of colon cancer.
  13. Targeting ARID1A mutations in cancer.
  14. Detection of Kras Gene in Colorectal Cancer Patients through Liquid Biopsy: A Cost-effective Method.
  15. Cholesterol-Lowering Drugs on Akt Signaling for Prevention of Tumorigenesis.
  16. Single-nucleotide variant of PIK3CA H1047R gene assay by CRISPR/Cas12a combined with rolling circle amplification.
  17. Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.
  1. Nat Rev Drug Discov. 2021 Oct 05.
    Understanding PI3K inhibitor mechanism of action.
    Sarah Crunkhorn.
      
    DOI:  https://doi.org/10.1038/d41573-021-00173-w
  2. Mol Cell Oncol. 2021 ;8(4): 1879614
    The concerted action of oncogenic driver mutations directs global translation in intestinal epithelial cells.
    Wouter Smit, Jarom Heijmans.
      Oncogenic transformation of colorectal cancer cells is driven by a set of mutations that cause aberrant signaling of growth factor-receptor pathways. Using organoids, we demonstrate that the most frequent driver mutations in APC, KRAS, SMAD4, and TP53 are enhancers of the global mRNA translational capacity, which is linked to intestinal cell growth in an mTOR-dependent manner.
    Keywords:  Colorectal cancer; driver mutations; global translation; mTOR signaling; protein synthesis
    DOI:  https://doi.org/10.1080/23723556.2021.1879614
  3. Nat Struct Mol Biol. 2021 Oct;28(10): 858-868
    The structural basis of PTEN regulation by multi-site phosphorylation.
    Daniel R Dempsey, Thibault Viennet, Reina Iwase, Eunyoung Park, Stephanie Henriquez, Zan Chen, Jeliazko R Jeliazkov, Brad A Palanski, Kim L Phan, Paul Coote, Jeffrey J Gray, Michael J Eck, Sandra B Gabelli, Haribabu Arthanari, Philip A Cole.
      Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN's catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN's cellular functions.
    DOI:  https://doi.org/10.1038/s41594-021-00668-5
  4. Mol Cancer. 2021 Oct 04. 20(1): 128
    Oncogenic KRAS blockade therapy: renewed enthusiasm and persistent challenges.
    Daolin Tang, Guido Kroemer, Rui Kang.
      Across a broad range of human cancers, gain-of-function mutations in RAS genes (HRAS, NRAS, and KRAS) lead to constitutive activity of oncoproteins responsible for tumorigenesis and cancer progression. The targeting of RAS with drugs is challenging because RAS lacks classic and tractable drug binding sites. Over the past 30 years, this perception has led to the pursuit of indirect routes for targeting RAS expression, processing, upstream regulators, or downstream effectors. After the discovery that the KRAS-G12C variant contains a druggable pocket below the switch-II loop region, it has become possible to design irreversible covalent inhibitors for the variant with improved potency, selectivity and bioavailability. Two such inhibitors, sotorasib (AMG 510) and adagrasib (MRTX849), were recently evaluated in phase I-III trials for the treatment of non-small cell lung cancer with KRAS-G12C mutations, heralding a new era of precision oncology. In this review, we outline the mutations and functions of KRAS in human tumors and then analyze indirect and direct approaches to shut down the oncogenic KRAS network. Specifically, we discuss the mechanistic principles, clinical features, and strategies for overcoming primary or secondary resistance to KRAS-G12C blockade.
    Keywords:  Covalent inhibitor; Drug resistance; Gene mutation; KRAS; Targeted therapy
    DOI:  https://doi.org/10.1186/s12943-021-01422-7
  5. Nucleic Acids Res. 2021 Oct 04. pii: gkab871. [Epub ahead of print]
    Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair.
    Linlin Yang, Changxian Shen, Adriana Estrada-Bernal, Ryan Robb, Moumita Chatterjee, Nikhil Sebastian, Amy Webb, Xiaokui Mo, Wei Chen, Sunil Krishnan, Terence M Williams.
      KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance.
    DOI:  https://doi.org/10.1093/nar/gkab871
  6. Mol Cell Oncol. 2021 ;8(4): 1954470
    A late endosome signaling hub that couples PI3Kα and WNT/β-catenin signaling in breast cancer.
    Samuel J Rodgers, Sabryn A Hamila, Christina A Mitchell, Lisa M Ooms.
      AKT is the central phosphoinositide 3-kinase (PI3K) signaling effector, however, PIK3CA (p110α subunit of PI3Kα)-mutant estrogen receptor-positive (ER+) breast cancers exhibit minimal AKT activation and the downstream signaling is poorly characterized. We discovered that a subset of PIK3CA-mutant ER+ breast cancers exhibit increased inositol polyphosphate 4-phosphatase type II (INPP4B) expression, which promotes late endosome formation and glycogen synthase kinase 3 beta (GSK3β) trafficking, leading to enhanced Wingless-related integration site (WNT)/catenin beta 1 (β-catenin) activation.
    Keywords:  INPP4B; PIK3CA; WNT/β-catenin; cell proliferation; late endosome
    DOI:  https://doi.org/10.1080/23723556.2021.1954470
  7. Front Mol Biosci. 2021 ;8 729830
    K-RAS4A: Lead or Supporting Role in Cancer Biology?
    Veronica Aran.
      The RAS oncogene is one of the most frequently mutated genes in human cancer, with K-RAS having a leading role in tumorigenesis. K-RAS undergoes alternative splicing, and as a result its transcript generates two gene products K-RAS4A and K-RAS4B, which are affected by the same oncogenic mutations, are highly homologous, and are expressed in a variety of human tissues at different levels. In addition, both isoforms localise to the plasma membrane by distinct targeting motifs. While some evidence suggests nonredundant functions for both splice variants, most work to date has focused on K-RAS4B, or even just K-RAS (i.e., without differentiating between the splice variants). This review aims to address the most relevant evidence published regarding K-RAS4A and to discuss if this "minor" isoform could also play a leading role in cancer, concluding that a significant body of evidence supports a leading role rather than a supporting (or secondary) role for K-RAS4A in cancer biology.
    Keywords:  K-Ras4A; K-Ras4B; K-ras; alternative splicing; cancer
    DOI:  https://doi.org/10.3389/fmolb.2021.729830
  8. Pharmacol Ther. 2021 Oct 05. pii: S0163-7258(21)00214-X. [Epub ahead of print] 108012
    Mapping of mTOR drug targets: Featured platforms for anti-cancer drug discovery.
    Raef Shams, Yoshihiro Ito, Hideyuki Miyatake.
      The mammalian/mechanistic target of rapamycin (mTOR) is a regulatory protein kinase involved in cell growth and proliferation. mTOR is usually assembled in two different complexes with different regulatory mechanisms, mTOR complex 1 (mTORC1) and mTORC2, which are involved in different functions such as cell proliferation and cytoskeleton assembly, respectively. In cancer cells, mTOR is hyperactivated in response to metabolic alterations and/or oncogenic signals to overcome the stressful microenvironments. Therefore, recent research progress for mTOR inhibition involves a variety of compounds that have been developed to disturb the metabolic processes of cancer cells through mTOR inhibition. In addition to competitive or allosteric inhibition, a new inhibition strategy that emerged mTOR complexes destabilization has recently been a concern. Here, we review the history of mTOR and its inhibition, along with the timeline of the mTOR inhibitors. We also introduce prospective drug targets to inhibit mTOR by disrupting the complexation of the components with peptides and small molecules.
    Keywords:  Cancer therapy; Drug target; mTOR; mTOR inhibitors generations; mTORC1/2 destabilizers
    DOI:  https://doi.org/10.1016/j.pharmthera.2021.108012
  9. Nat Struct Mol Biol. 2021 Oct;28(10): 847-857
    A structural model of a Ras-Raf signalosome.
    Venkatesh P Mysore, Zhi-Wei Zhou, Chiara Ambrogio, Lianbo Li, Jonas N Kapp, Chunya Lu, Qi Wang, Maxwell R Tucker, Jeffrey J Okoro, Gabriela Nagy-Davidescu, Xiaochen Bai, Andreas Plückthun, Pasi A Jänne, Kenneth D Westover, Yibing Shan, David E Shaw.
      The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.
    DOI:  https://doi.org/10.1038/s41594-021-00667-6
  10. Br J Cancer. 2021 Oct 07.
    Quantifying ERK activity in response to inhibition of the BRAFV600E-MEK-ERK cascade using mathematical modelling.
    Sara J Hamis, Yury Kapelyukh, Aileen McLaren, Colin J Henderson, C Roland Wolf, Mark A J Chaplain.
      BACKGROUND: Simultaneous inhibition of multiple components of the BRAF-MEK-ERK cascade (vertical inhibition) has become a standard of care for treating BRAF-mutant melanoma. However, the molecular mechanism of how vertical inhibition synergistically suppresses intracellular ERK activity, and consequently cell proliferation, are yet to be fully elucidated.METHODS: We develop a mechanistic mathematical model that describes how the mutant BRAF inhibitor, dabrafenib, and the MEK inhibitor, trametinib, affect BRAFV600E-MEK-ERK signalling. The model is based on a system of chemical reactions that describes cascade signalling dynamics. Using mass action kinetics, the chemical reactions are re-expressed as ordinary differential equations that are parameterised by in vitro data and solved numerically to obtain the temporal evolution of cascade component concentrations.
    RESULTS: The model provides a quantitative method to compute how dabrafenib and trametinib can be used in combination to synergistically inhibit ERK activity in BRAFV600E-mutant melanoma cells. The model elucidates molecular mechanisms of vertical inhibition of the BRAFV600E-MEK-ERK cascade and delineates how elevated BRAF concentrations generate drug resistance to dabrafenib and trametinib. The computational simulations further suggest that elevated ATP levels could be a factor in drug resistance to dabrafenib.
    CONCLUSIONS: The model can be used to systematically motivate which dabrafenib-trametinib dose combinations, for treating BRAFV600E-mutated melanoma, warrant experimental investigation.
    DOI:  https://doi.org/10.1038/s41416-021-01565-w
  11. Nat Commun. 2021 Oct 08. 12(1): 5906
    Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry.
    Febe van Maldegem, Karishma Valand, Megan Cole, Harshil Patel, Mihaela Angelova, Sareena Rana, Emma Colliver, Katey Enfield, Nourdine Bah, Gavin Kelly, Victoria Siu Kwan Tsang, Edurne Mugarza, Christopher Moore, Philip Hobson, Dina Levi, Miriam Molina-Arcas, Charles Swanton, Julian Downward.
      Mouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimise and validate image segmentation strategies and automate the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. With these methods we interrogate the remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, highlighting the infiltration and activation of antigen presenting cells and effector cells.
    DOI:  https://doi.org/10.1038/s41467-021-26214-x
  12. Oncogene. 2021 Oct 07.
    CRISPR screens identify cholesterol biosynthesis as a therapeutic target on stemness and drug resistance of colon cancer.
    Shanshan Gao, Fraser Soares, Shiyan Wang, Chi Chun Wong, Huarong Chen, Zhenjie Yang, Weixin Liu, Minnie Y Y Go, Musaddeque Ahmed, Yong Zeng, Catherine Adell O'Brien, Joseph J Y Sung, Housheng Hansen He, Jun Yu.
      Cancer stem cells (CSCs) are responsible for tumor progression, recurrence, and drug resistance. To identify genetic vulnerabilities of colon cancer, we performed targeted CRISPR dropout screens comprising 657 Drugbank targets and 317 epigenetic regulators on two patient-derived colon CSC-enriched spheroids. Next-generation sequencing of pooled genomic DNAs isolated from surviving cells yielded therapeutic candidates. We unraveled 44 essential genes for colon CSC-enriched spheroids propagation, including key cholesterol biosynthetic genes (HMGCR, FDPS, and GGPS1). Cholesterol biosynthesis was induced in colon cancer tissues, especially CSC-enriched spheroids. The genetic and pharmacological inhibition of HMGCR/FDPS impaired self-renewal capacity and tumorigenic potential of the spheroid models in vitro and in vivo. Mechanistically, HMGCR or FDPS depletion impaired cancer stemness characteristics by activating TGF-β signaling, which in turn downregulated expression of inhibitors of differentiation (ID) proteins, key regulators of cancer stemness. Cholesterol and geranylgeranyl diphosphate (GGPP) rescued the growth inhibitory and signaling effect of HMGCR/FDPS blockade, implying a direct role of these metabolites in modulating stemness. Finally, cholesterol biosynthesis inhibitors and 5-FU demonstrated antitumor synergy in colon CSC-enriched spheroids, tumor organoids, and xenografts. Taken together, our study unravels novel genetic vulnerabilities of colon CSC-enriched spheroids and suggests cholesterol biosynthesis as a potential target in conjunction with traditional chemotherapy for colon cancer treatment.
    DOI:  https://doi.org/10.1038/s41388-021-01882-7
  13. Cancer Treat Rev. 2021 Sep 06. pii: S0305-7372(21)00135-3. [Epub ahead of print]100 102287
    Targeting ARID1A mutations in cancer.
    Jaren Mullen, Shumei Kato, Jason K Sicklick, Razelle Kurzrock.
      Genes encoding SWI/SNF chromatin remodeling complex subunits are collectively mutated in approximately 20% of human cancers. ARID1A is a SWI/SNF subunit gene whose protein product binds DNA. ARID1A gene alterations result in loss of function. It is the most commonly mutated member of the SWI/SNF complex, being aberrant in ∼6% of cancers overall, including ovarian clear cell cancers (∼45% of patients) and uterine endometrioid cancers (∼37%). ARID1A has a crucial role in regulating gene expression that drives oncogenesis or tumor suppression. In particular, ARID1A participates in control of the PI3K/AKT/mTOR pathway, immune responsiveness to cancer, EZH2 methyltransferase activity, steroid receptor modulation, DNA damage checkpoints, and regulation of p53 targets and KRAS signaling. A variety of compounds may be of benefit in ARID1A-altered cancers: immune checkpoint blockade, and inhibitors of mTOR, EZH2, histone deacetylases, ATR and/or PARP. ARID1A alterations may also mediate resistance to platinum chemotherapy and estrogen receptor degraders/modulators.
    Keywords:  ARID1A; Chromatin remodeling; SWI/SNF
    DOI:  https://doi.org/10.1016/j.ctrv.2021.102287
  14. J Coll Physicians Surg Pak. 2021 Oct;31(10): 1174-1178
    Detection of Kras Gene in Colorectal Cancer Patients through Liquid Biopsy: A Cost-effective Method.
    Hafiz Syed Muhammad Osama Jafri, Shamim Mushtaq, Saeeda Baig.
      OBJECTIVE: To detect the Kras gene through liquid biopsy, a less invasive technique in diagnosed colorectal cancer patients.STUDY DESIGN: Cross-sectional study.
    PLACE AND DURATION OF STUDY: Department of Oncology, Dr. Ziauddin Hospital and Bait-us-Sukoon Cancer Hospital, Karachi, from 2019 to 2020.
    METHODOLOGY: Circulating tumor DNA (ctDNA) in colorectal cancer patients was extracted through magnetic bead technique using MagMAX cell free DNA kit (Thermofisher, Uk). The frequency of Kras gene was quantified using a real-time polymerase chain reaction (RT-PCR) assay (qPCR). ANOVA and Chi-square tests were utilised for statistical analysis.
    RESULTS: Mean threshold cycle (CT) of Kras gene showed significantly higher expression 15.6 ± 1.82 (p=0.001) in stage IV CRC cases compared to early stages (19.53 ± 18.223.7 ± 2.9 and 19.8 ± 2.69 of stage 1, 2 and 3, respectively. Similarly, ΔCT mean of Kras gene at stage IV showed significantly higher expression of 2.48 ± 1.40 (0.048), compared to 2.39 ± 0.6, 3.12 ± 0.68 and 3.15 ± 0.41 of stage 1, 2 and 3, respectively. Males (n=40, 55%) showed significant association (p=0.001) with CRC compared to females (n=33, 45%). Categorisation of tumor types within different age groups revealed that colon cancer was more frequent (n=11, 15.1%) in the 41-50 age group, while rectal cancer was more frequent (n= 11, 15.1%) in the 41-50 age group, while rectal cancer was more in the 51-60 age group (n=11, 15.1%).
    CONCLUSION: Kras gene was detected with significantly increased levels in plasma of CRC patients at advanced stages. This confirms that liquid biopsy can be used to detect Kras gene in ctDNA of CRC patients through a magnetic bead based technique. Key Words: Liquid biopsy, Circulating tumor DNA, KRAS, Colorectal cancer, Real-time polymerase chain reaction.
    DOI:  https://doi.org/10.29271/jcpsp.2021.10.1174
  15. Front Genet. 2021 ;12 724149
    Cholesterol-Lowering Drugs on Akt Signaling for Prevention of Tumorigenesis.
    Navneet Kumar, Chandi C Mandal.
      Cholesterol has been reported to be accumulated in cancer cells. The metabolic dysregulation of the cholesterol is associated with tumor development and progression. The cholesterol-lowering drugs have been found to be involved in the prevention and treatment of various cancers. Akt, a serine/threonine kinase, can modulate the role of several downstream proteins involved in cell proliferation, migration, invasion, metabolism, and apoptosis. Since its involvement in several signaling pathways, its dysregulation is commonly reported in several cancers. Thus, targeting Akt could be an effective approach for cancer prevention and therapy. Cholesterol-lowering drugs have been found to affect the expression of Akt, and its activation in the cancer cells and thus have shown anticancer activity in different type of cancers. These drugs act on various signaling pathways such as PTEN/Akt, PI3k/Akt, Akt/NF-κB, Akt/FOXO1, Akt/mTOR, etc., which will be discussed in this article. This review article will discuss the significance of cholesterol in cancer cells, cholesterol-lowering drugs, the role of Akt in cancer cells, and the effects of cholesterol-lowering drugs on Akt in the prevention of therapy resistance and metastasis.
    Keywords:  Akt signaling; cancer; cholesterol; cholesterol-lowering drugs; fenofibrate; statins
    DOI:  https://doi.org/10.3389/fgene.2021.724149
  16. Anal Chim Acta. 2021 Oct 16. pii: S0003-2670(21)00769-8. [Epub ahead of print]1182 338943
    Single-nucleotide variant of PIK3CA H1047R gene assay by CRISPR/Cas12a combined with rolling circle amplification.
    Gaihua Cao, Xiaolong Chen, Yuanyi Deng, Fuping Nie, Yin Liu, Guoming Wang, Danqun Huo, Changjun Hou.
      PIK3CA H1047R gene plays an important role in the PI3K/Akt/mTOR signaling pathway, and its mutation is closely related to the occurrence and development of breast cancer and Lipoblastoma. Therefore, it is of great value to detect the PIK3CA H1047R mutant gene. Here, an analytical method coupled CRISPR/Cas12a with rolling circle amplification (RCA) technology was constructed for ultra-sensitive and specific detection of the single-nucleotide variant (SNV) of the PIK3CA H1047R gene. With efficient amplification of RCA and CRISPR/Cas12a, the detection limit of the mutant target and mixture of the mutant with wild-type target were as low as 10 aM and 0.036%, respectively. The detection limit of the RCA-CRISPR/Cas12a method was lower than that of allelic specific PCR (AS-PCR) for detecting SNV of the PIK3CA H1047R gene. Hence, this RCA-CRISPR/Cas12a method is sensitive and specific for the detection of SNV. What's more, this strategy provides a new idea for medical diagnosis and lays a technical foundation for the research of PI3K/Akt/mTOR signaling pathways.
    Keywords:  Allelic specific PCR (AS-PCR); CRISPR/Cas12a; Fluorescence; PIK3CA (H1047R) mutation Gene; Rolling circle amplification (RCA); Single-nucleotide variant (SNV)
    DOI:  https://doi.org/10.1016/j.aca.2021.338943
  17. J Vis Exp. 2021 09 17.
    Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.
    Aline Stedman, Antonin Levy, Philippe J Sansonetti, Giulia Nigro.
      Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.
    DOI:  https://doi.org/10.3791/62880
This page is being maintained by Thomas Krichel. It was last updated on 2021‒10‒24 at 15:06:14.