Journal articles on the topic 'FUCCI reporters'
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Singh, Amar M., Robert Trost, Benjamin Boward, and Stephen Dalton. "Utilizing FUCCI reporters to understand pluripotent stem cell biology." Methods 101 (May 2016): 4–10. http://dx.doi.org/10.1016/j.ymeth.2015.09.020.
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Yano, Shuya, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara, and Robert M. Hoffman. "FUCCI Real-Time Cell-Cycle Imaging as a Guide for Designing Improved Cancer Therapy: A Review of Innovative Strategies to Target Quiescent Chemo-Resistant Cancer Cells." Cancers 12, no. 9 (September 17, 2020): 2655. http://dx.doi.org/10.3390/cancers12092655.
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Progress in chemotherapy of solid cancer has been tragically slow due, in large part, to the chemoresistance of quiescent cancer cells in tumors. The fluorescence ubiquitination cell-cycle indicator (FUCCI) was developed in 2008 by Miyawaki et al., which color-codes the phases of the cell cycle in real-time. FUCCI utilizes genes linked to different color fluorescent reporters that are only expressed in specific phases of the cell cycle and can, thereby, image the phases of the cell cycle in real-time. Intravital real-time FUCCI imaging within tumors has demonstrated that an established tumor comprises a majority of quiescent cancer cells and a minor population of cycling cancer cells located at the tumor surface or in proximity to tumor blood vessels. In contrast to most cycling cancer cells, quiescent cancer cells are resistant to cytotoxic chemotherapy, most of which target cells in S/G2/M phases. The quiescent cancer cells can re-enter the cell cycle after surviving treatment, which suggests the reason why most cytotoxic chemotherapy is often ineffective for solid cancers. Thus, quiescent cancer cells are a major impediment to effective cancer therapy. FUCCI imaging can be used to effectively target quiescent cancer cells within tumors. For example, we review how FUCCI imaging can help to identify cell-cycle-specific therapeutics that comprise decoy of quiescent cancer cells from G1 phase to cycling phases, trapping the cancer cells in S/G2 phase where cancer cells are mostly sensitive to cytotoxic chemotherapy and eradicating the cancer cells with cytotoxic chemotherapy most active against S/G2 phase cells. FUCCI can readily image cell-cycle dynamics at the single cell level in real-time in vitro and in vivo. Therefore, visualizing cell cycle dynamics within tumors with FUCCI can provide a guide for many strategies to improve cell-cycle targeting therapy for solid cancers.
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Zheng, Lixia, Zihao Wang, Jianyong Du, Xiaojun Zhu, and Jing-Wei Xiong. "Protocol to identify small molecules promoting rat and mouse cardiomyocyte proliferation based on the FUCCI and MADM reporters." STAR Protocols 3, no. 4 (December 2022): 101903. http://dx.doi.org/10.1016/j.xpro.2022.101903.
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Ippati, Stefania, Yuanyuan Deng, Julia van der Hoven, Celine Heu, Annika van Hummel, Sook Wern Chua, Esmeralda Paric, et al. "Rapid initiation of cell cycle reentry processes protects neurons from amyloid-β toxicity." Proceedings of the National Academy of Sciences 118, no. 12 (March 18, 2021): e2011876118. http://dx.doi.org/10.1073/pnas.2011876118.
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Neurons are postmitotic cells. Reactivation of the cell cycle by neurons has been reported in Alzheimer’s disease (AD) brains and models. This gave rise to the hypothesis that reentering the cell cycle renders neurons vulnerable and thus contributes to AD pathogenesis. Here, we use the fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology to monitor the cell cycle in live neurons. We found transient, self-limited cell cycle reentry activity in naive neurons, suggesting that their postmitotic state is a dynamic process. Furthermore, we observed a diverse response to oligomeric amyloid-β (oAβ) challenge; neurons without cell cycle reentry activity would undergo cell death without activating the FUCCI reporter, while neurons undergoing cell cycle reentry activity at the time of the oAβ challenge could maintain and increase FUCCI reporter signal and evade cell death. Accordingly, we observed marked neuronal FUCCI positivity in the brains of human mutant Aβ precursor protein transgenic (APP23) mice together with increased neuronal expression of the endogenous cell cycle control protein geminin in the brains of 3-mo-old APP23 mice and human AD brains. Taken together, our data challenge the current view on cell cycle in neurons and AD, suggesting that pathways active during early cell cycle reentry in neurons protect from Aβ toxicity.
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Manolopoulou, Marika, Brittany K. Matlock, Stellor Nlandu-Khodo, Alan J. Simmons, Ken S. Lau, Melanie Phillips-Mignemi, Alla Ivanova, Catherine E. Alford, David K. Flaherty, and Leslie S. Gewin. "Novel kidney dissociation protocol and image-based flow cytometry facilitate improved analysis of injured proximal tubules." American Journal of Physiology-Renal Physiology 316, no. 5 (May 1, 2019): F847—F855. http://dx.doi.org/10.1152/ajprenal.00354.2018.
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Flow cytometry studies on injured kidney tubules are complicated by the low yield of nucleated single cells. Furthermore, cell-specific responses such as cell cycle dynamics in vivo have conventionally relied on indirect immunohistochemistry and proximal tubule markers that may be downregulated in injury. Here, we report a new tissue dissociation protocol for the kidney with an early fixation step that greatly enhances the yield of single cells. Genetic labeling of the proximal tubule with either mT/mG “tomato” or R26Fucci2aR (Fucci) cell cycle reporter mice allows us to follow proximal tubule-specific changes in cell cycle after renal injury. Image-based flow cytometry (FlowSight) enables gating of the cell cycle and concurrent visualization of the cells with bright field and fluorescence. We used the Fucci mouse in conjunction with FlowSight to identify a discrete polyploid population in proximal tubules after aristolochic acid injury. The tissue dissociation protocol in conjunction with genetic labeling and image-based flow cytometry is a tool that can improve our understanding of any discrete cell population after injury.
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Takahashi, Kei, Ryo Tanabe, Shogo Ehata, Shimpei I. Kubota, Yasuyuki Morishita, Hiroki R. Ueda, and Kohei Miyazono. "Visualization of the cancer cell cycle by tissue‐clearing technology using the Fucci reporter system." Cancer Science 112, no. 9 (July 7, 2021): 3796–809. http://dx.doi.org/10.1111/cas.15034.
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Zambon, Alexander C., Tom Hsu, Seunghee Erin Kim, Miranda Klinck, Jennifer Stowe, Lindsay M. Henderson, Donald Singer, et al. "Methods and sensors for functional genomic studies of cell-cycle transitions in single cells." Physiological Genomics 52, no. 10 (October 1, 2020): 468–77. http://dx.doi.org/10.1152/physiolgenomics.00065.2020.
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Much of our understanding of the regulatory mechanisms governing the cell cycle in mammals has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our current understanding of cell proliferation, these approaches mask the genetic signatures of rare subpopulations such as quiescent (G0) and very slowly dividing (SD) cells. Results described in this study and those of others using single-cell analysis reveal that even in clonally derived immortalized cancer cells, ∼1–5% of cells can exhibit G0 and SD phenotypes. Therefore to enable the study of these rare cell phenotypes we established an integrated molecular, computational, and imaging approach to track, isolate, and genetically perturb single cells as they proliferate. A genetically encoded cell-cycle reporter (K67p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67p-FUCCI over time. To enable the further study G0 and SD phenotypes, we retrofitted a live cell imaging system with a micromanipulator to enable single-cell targeting for functional validation studies. Single-cell analysis revealed HT1080 and MCF7 cells had a doubling time of ∼24 and ∼48 h, respectively, with high duration variability in G1 and G2 phases. Direct single-cell microinjection of mRNA encoding (GFP) achieves detectable GFP fluorescence within ∼5 h in both cell types. These findings coupled with the possibility of targeting several hundreds of single cells improves throughput and sensitivity over conventional methods to study rare cell subpopulations.
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Mubarok, Wildan, Kelum Chamara Manoj Lakmal Elvitigala, Masaki Nakahata, Masaru Kojima, and Shinji Sakai. "Modulation of Cell-Cycle Progression by Hydrogen Peroxide-Mediated Cross-Linking and Degradation of Cell-Adhesive Hydrogels." Cells 11, no. 5 (March 3, 2022): 881. http://dx.doi.org/10.3390/cells11050881.
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The cell cycle is known to be regulated by features such as the mechanical properties of the surrounding environment and interaction of cells with the adhering substrates. Here, we investigated the possibility of regulating cell-cycle progression of the cells on gelatin/hyaluronic acid composite hydrogels obtained through hydrogen peroxide (H2O2)-mediated cross-linking and degradation of the polymers by varying the exposure time to H2O2 contained in the air. The stiffness of the hydrogel varied with the exposure time. Human cervical cancer cells (HeLa) and mouse mammary gland epithelial cells (NMuMG) expressing cell-cycle reporter Fucci2 showed the exposure-time-dependent different cell-cycle progressions on the hydrogels. Although HeLa/Fucci2 cells cultured on the soft hydrogel (Young’s modulus: 0.20 and 0.40 kPa) obtained through 15 min and 120 min of the H2O2 exposure showed a G2/M-phase arrest, NMuMG cells showed a G1-phase arrest. Additionally, the cell-cycle progression of NMuMG cells was not only governed by the hydrogel stiffness, but also by the low-molecular-weight HA resulting from H2O2-mediated degradation. These results indicate that H2O2-mediated cross-linking and degradation of gelatin/hyaluronic acid composite hydrogel could be used to control the cell adhesion and cell-cycle progression.
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Yano, Shuya, Hiroshi Tazawa, Hiroyuki Kishimoto, Shunsuke Kagawa, Toshiyoshi Fujiwara, and Robert M. Hoffman. "Real-Time Fluorescence Image-Guided Oncolytic Virotherapy for Precise Cancer Treatment." International Journal of Molecular Sciences 22, no. 2 (January 17, 2021): 879. http://dx.doi.org/10.3390/ijms22020879.
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Oncolytic virotherapy is one of the most promising, emerging cancer therapeutics. We generated three types of telomerase-specific replication-competent oncolytic adenovirus: OBP-301; a green fluorescent protein (GFP)-expressing adenovirus, OBP-401; and Killer-Red-armed OBP-301. These oncolytic adenoviruses are driven by the human telomerase reverse transcriptase (hTERT) promoter; therefore, they conditionally replicate preferentially in cancer cells. Fluorescence imaging enables visualization of invasion and metastasis in vivo at the subcellular level; including molecular dynamics of cancer cells, resulting in greater precision therapy. In the present review, we focused on fluorescence imaging applications to develop precision targeting for oncolytic virotherapy. Cell-cycle imaging with the fluorescence ubiquitination cell cycle indicator (FUCCI) demonstrated that combination therapy of an oncolytic adenovirus and a cytotoxic agent could precisely target quiescent, chemoresistant cancer stem cells (CSCs) based on decoying the cancer cells to cycle to S-phase by viral treatment, thereby rendering them chemosensitive. Non-invasive fluorescence imaging demonstrated that complete tumor resection with a precise margin, preservation of function, and prevention of distant metastasis, was achieved with fluorescence-guided surgery (FGS) with a GFP-reporter adenovirus. A combination of fluorescence imaging and laser ablation using a KillerRed-protein reporter adenovirus resulted in effective photodynamic cancer therapy (PDT). Thus, imaging technology and the designer oncolytic adenoviruses may have clinical potential for precise cancer targeting by indicating the optimal time for administering therapeutic agents; accurate surgical guidance for complete resection of tumors; and precise targeted cancer-specific photosensitization.
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Abe, T., A. Sakaue-Sawano, H. Kiyonari, G. Shioi, K. i. Inoue, T. Horiuchi, K. Nakao, A. Miyawaki, S. Aizawa, and T. Fujimori. "Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter." Development 140, no. 1 (November 22, 2012): 237–46. http://dx.doi.org/10.1242/dev.084111.
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Mathews, Juanita, Franz Kuchling, David Baez-Nieto, Miranda Diberardinis, Jen Q. Pan, and Michael Levin. "Ion Channel Drugs Suppress Cancer Phenotype in NG108-15 and U87 Cells: Toward Novel Electroceuticals for Glioblastoma." Cancers 14, no. 6 (March 15, 2022): 1499. http://dx.doi.org/10.3390/cancers14061499.
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Glioblastoma is a lethal brain cancer that commonly recurs after tumor resection and chemotherapy treatment. Depolarized resting membrane potentials and an acidic intertumoral extracellular pH have been associated with a proliferative state and drug resistance, suggesting that forced hyperpolarization and disruption of proton pumps in the plasma membrane could be a successful strategy for targeting glioblastoma overgrowth. We screened 47 compounds and compound combinations, most of which were ion-modulating, at different concentrations in the NG108-15 rodent neuroblastoma/glioma cell line. A subset of these were tested in the U87 human glioblastoma cell line. A FUCCI cell cycle reporter was stably integrated into both cell lines to monitor proliferation and cell cycle response. Immunocytochemistry, electrophysiology, and a panel of physiological dyes reporting voltage, calcium, and pH were used to characterize responses. The most effective treatments on proliferation in U87 cells were combinations of NS1643 and pantoprazole; retigabine and pantoprazole; and pantoprazole or NS1643 with temozolomide. Marker analysis and physiological dye signatures suggest that exposure to bioelectric drugs significantly reduces proliferation, makes the cells senescent, and promotes differentiation. These results, along with the observed low toxicity in human neurons, show the high efficacy of electroceuticals utilizing combinations of repurposed FDA approved drugs.
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Panagiotakopoulou, Magdalini, Tobias Lendenmann, Francesca Michela Pramotton, Costanza Giampietro, Georgios Stefopoulos, Dimos Poulikakos, and Aldo Ferrari. "Cell cycle–dependent force transmission in cancer cells." Molecular Biology of the Cell 29, no. 21 (October 15, 2018): 2528–39. http://dx.doi.org/10.1091/mbc.e17-12-0726.
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The generation of traction forces and their transmission to the extracellular environment supports the disseminative migration of cells from a primary tumor. In cancer cells, the periodic variation of nuclear stiffness during the cell cycle provides a functional link between efficient translocation and proliferation. However, the mechanical framework completing this picture remains unexplored. Here, the Fucci2 reporter was expressed in various human epithelial cancer cells to resolve their cell cycle phase transition. The corresponding tractions were captured by a recently developed reference-free confocal traction-force microscopy platform. The combined approach was conducive to the analysis of phase-dependent force variation at the level of individual integrin contacts. Detected forces were invariably higher in the G1 and early S phases than in the ensuing late S/G2, and locally colocalized with high levels of paxillin phosphorylation. Perturbation of paxillin phosphorylation at focal adhesions, obtained through the biochemical inhibition of focal adhesion kinase (FAK) or the transfection of nonphosphorylatable or phosphomimetic paxillin mutants, significantly diminished the force transmitted to the substrate. These data demonstrate a reproducible modulation of force transmission during the cell cycle progression of cancer cells, instrumental to their invasion of dense environments. In addition, they delineate a model in which paxillin phosphorylation supports the mechanical maturation of adhesions relaying forces to the substrate.
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Duso, Bruno Achutti, Elena Gavilan Dorronzoro, Giulia Tini, Maria de Filippo, Marica Ippolito, Chiara Soriani, Simona Rodighiero, Stefano Santaguida, Pier Giuseppe Pelicci, and Luca Mazzarella. "Abstract 1164: Somatic NF1 loss in breast cancer leads to centrosome amplification, aneuploidy and increased sensitivity to T-DM1." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1164. http://dx.doi.org/10.1158/1538-7445.am2022-1164.
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Abstract Background: Centrosome amplification (CA, the presence of >2 centrosomes) is a hallmark of several tumors. CA perturbs mitosis by generating nonphysiological pulling forces, leading to chromosome missegregation and aneuploidy. This condition may be advantageous for tumor outgrowth providing it gets corrected by clustering centrosomes into a pseudo-bipolar conformation for successful cell division. Molecular determinants of CA and therapeutic opportunities in breast cancer (BC) are still poorly understood. Preliminary data from our group show that somatic NF1 loss of function (LOF), common upon metastatic progression, is associated in vitro and in patients with selective sensitivity to maytansinoids such as T-DM1. Here, we explored the molecular basis of this increased sensitivity. Methods: Multiple CRISPR/Cas9-generated NF1KO or WT clones of HER2+ BC cell lines (BT474, SKBR3 and HCC1954) were assessed for sensitivity to T-DM1 or DM1 in BrdU-based and clonogenic assays, with RNAseq being performed. BT474 cells were furthered engineered with the FUCCI(Ca) reporter for live cell cycle imaging. Ploidy was assessed through flow cytometry. Aneuploidy in the GENIE cohort (restricted to BC patients analyzed with NF1-covering panels) was assessed by generating a segmentation score (sum of the absolute ploidy scores in segments covered by NGS panels). Centrosomes, spindle conformation and chromosome abnormalities were studied by confocal microscopy in cells synchronized both with RO3306 or thymidine block. Results: All lines showed increased sensitivity to T-DM1 in KO vs WT in BrdU-based and clonogenic assays; similar results were obtained with DM1 only, suggesting independence from HER2 targeting. RNAseq differential analysis showed significant enrichment for gene sets involved in mitotic spindle in KO but not WT cells upon T-DM1 treatment. FUCCI analysis showed significantly longer permanence in G2/M in NF1KO compared to WT cells (24.8 vs 17.9% in G2/M, p=1.81E-07). In vehicle-treated synchronized cells, significantly more KO cells showed >2 centrosomes (21.6 vs 4.7%, p<0.00001) and multiple pseudo-bipolar mitotic figures with narrow intercentriolar distances, indicative of efficient clustering. This was associated with more frequent chromosome misalignment (26.7 vs 6.7%, p=0.038). In the GENIE cohort, segmentation score was higher for patients with NF1 LOF mutations vs NF1 WT (median 46.7 vs 41.1, p=0.0023), indicative of more common aneuploidy. Upon T-DM1, KO cells exhibited significantly more non-bipolar spindles with massively wider intercentriolar distances. Conclusions: Somatic NF1 loss causes aneuploidy due to CA. This likely favors metastatization and can be exploited therapeutically. CA associated with other common oncogenic events (RAS and BRCA1/2 mutations, PTEN loss) may represent a general biomarker for drugs inhibiting centrosome clustering in BC. Citation Format: Bruno Achutti Duso, Elena Gavilan Dorronzoro, Giulia Tini, Maria de Filippo, Marica Ippolito, Chiara Soriani, Simona Rodighiero, Stefano Santaguida, Pier Giuseppe Pelicci, Luca Mazzarella. Somatic NF1 loss in breast cancer leads to centrosome amplification, aneuploidy and increased sensitivity to T-DM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1164.
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Stopka, Tomas, Karin Vargova, Pavel Burda, Juraj Kokavec, Nikola Curik, Adela Berkova, Marek Trneny, Arthur Skoultchi, and Jiri Zavadil. "MicroRNA Mir-155 and Myb Proto-Oncogene Family Members Cooperate in Pathogenesis of Chronic Lymphocytic Leukemia." Blood 114, no. 22 (November 20, 2009): 58. http://dx.doi.org/10.1182/blood.v114.22.58.58.
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Abstract Abstract 58 Introduction: Role of small non-coding microRNAs (miRNA) in hematopoiesis has been recently established by studies demonstrating increased levels of miR-155 in chronic lymphocytic leukemia (CLL) (Fulci 2007, Marton 2008). PU.1 is an ETS family transcription factor controling myelo-lymphoid differentiation and is directly negatively regulated by miR-155 (Vigorito 2007). Our aim of this study is to determine mechanisms of miR-155 upregulation in CLL pathogenesis and the role of PU.1 downregulation in the process. Methods: miRNA and mRNA levels were determined by qPCR and Affymetrix mRNA expression profiling in peripheral blood mononuclear cells (PBMC) and in purified B cells. Control (N=13) and CLL patients (N=66) were studied. All patients were subgrouped according to cytogenetics (FISH) and Rai status. Protein-DNA localization assays were done using chromatin immunoprecipitation. Results: miR-155 is significantly upregulated in both CLL patient PBMCs and a subset of sorted B-cells whereas PU.1 and its target genes are repressed in all CLL subgroups. Indeed, expression profiling analysis of CLL samples identified a broad repression of ∼80 miR-155 targets (among them key transcriptional regulators FOS, SATB1, MEF2A, MYBL1, SIRT1, MECP2 and CEBPB) and ∼380 repressed PU.1 target genes, among them regulators of hematopoietic homeostasis (FOS, CSF1R, CSF2R, IL4R, IL21R) and apoptosis (BID, BIRC3). Next, we have studied the mechanism of miR-155 gene (also known as BIC) upregulation in CLL. Wehave newly identified a regulatory CpG island (BIC-CpG) upstream of miR-155 BIC gene that contains DNA binding motifs for E-box transcription factors and is not mutated in CLL patients. Two E-box-binding proteins, MYB and MYBL2, are significantly upregulated in CLL patient PBMCs as well as in a subset of sorted B-cells in all CLL subgroups. Furthermore in primary CLL cells, MYB protein presence is significantly enriched at BIC-CpG alongside a marked enrichment with transcriptionally active chromatin mark histone H3K9Acetyl. To further study the role of MYB in transactivation of the BIC-CpG we have prepared reporter constructs and found that MYB indeed activates BIC-CpG and downstream transcription. Apart from miR-155/BIC, expression profiling analysis identified additional ∼50 upregulated MYB targets, among them cancer-related genes such as CA1, MCM4, BCL2, PDCD4, and CXCR4. Functional assays using siRNA inhibition of PU.1 in normal PBMCs result in upregulation of miR-155 and MYB, indicating that silencing of PU.1 and upregulation of MYB and its target miR-155 may represent an important mechanism of CLL pathogenesis. Conclusion: Our data propose a mechanistic relationship between PU.1 and its negative regulator miR-155 in CLL. Our data also demonstrate that miR-155 is transcriptionally activated by MYB family of E-box binding proteins. Manipulation of these mechanistic relationships may harbor a potential for molecular therapies against CLL. (Grants NR9021-4, 10310-3, 2B06077) Disclosures: No relevant conflicts of interest to declare.
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Duso, Bruno A., Elena Gavilán Dorronzoro, Giulia Tini, Maria R. de Filippo, Emanuele Bonetti, Maria R. Ippolito, Chiara Soriani, et al. "Abstract P5-13-04: NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics." Cancer Research 82, no. 4_Supplement (February 15, 2022): P5–13–04—P5–13–04. http://dx.doi.org/10.1158/1538-7445.sabcs21-p5-13-04.
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Abstract Background: Despite major technological and conceptual advancements, treatment decisions in HER2+ metastatic breast cancer (mBC) remain largely based on clinical evidence, with no established predictive biomarkers to direct treatment for individual patients. The tumour suppressor Neurofibromatosis 1 (NF1) has been implicated in endocrine resistance but its role remains incompletely characterized in mBC. NF1 is best known as a GTPase-activating protein (GAP) that attenuates RAS signalling. However, the GAP function is likely not limited to RAS, and NF1 has been involved in other GTP-dependent processes including cytoskeletal dynamics. Data mining and analysis of public mutational registries revealed NF1 mutations as particularly enriched in HER2+ mBC compared to other molecular subtypes. Methods: To investigate the biological consequences of NF1 loss, we generated NF1 KO HER2+ mBC cell lines (BT474 and SKBR3) by CRISPR-Cas9 and both 2D and 3D proliferations assays were used for drug sensitivity profiling; live-cell imaging, high-resolution confocal microscopy and an ad-hoc computational algorithm were employed to study cell fate and microtubule conformational changes. Patient data were obtained from the Northwestern University through a prospective observational study in mBC patients. Results: Screening of several compounds approved for HER2+ mBC showed that response was generally equal or reduced in NF1 KO vs WT cells. However, response to trastuzumab-emtansine (T-DM1) was significantly increased in NF1 KO cells (IC50 ~0,3 vs 1,6 μg/mL in NF1WT). This sensitization was not observed with other antibody drug conjugates (ADCs) like DS-8201 and was reproducible with maytansine alone, suggesting a pharmacologically relevant NF1 activity on microtubules. Using the FUCCI(Ca) reporter, which tracks cell cycle progression at single-cell level, we saw a more prominent G2/M phase arrest and cell death upon T-DM1 treatment in NF1 KO compared to WT cells. Notably, NF1 KO cells exhibited a higher frequency of aberrant mitotic figures (chromosome alignment defects and multipolar spindle formation) and stronger β-galactosidase activity, an established marker of senescence. Collectively, these results suggest that NF1 KO cells become particularly subject to T-DM1-triggered mitotic catastrophe. Dephosphorylation of GTP-bound tubulin is required for appropriate microtubular dynamics; so-called “GTP islands” within the inner microtubule region are prone to rapid repolymerization and are normally kept at low levels. We hypothesize that expanded GTP-tubulin islands generated by the loss of NF1 GAP activity is a major cause of microtubular instability in NF1 KO cells. Preliminary evidence in support of this model was obtained by quantification of GTP-tubulin with a specific antibody. Finally, we assessed the predictive role of NF1 as a biomarker for T-DM1 response in a cohort of 300 mBC patients with mutational data in circulating tumour DNA (Guardant 360); we identified 13 heavily pretreated patients (>4 prior lines) who received T-DM1, of which 3 had loss-of-function NF1 mutations and 10 were NF1 WT. Median progression-free survival was higher in NF1-mutated than WT patients (334 vs 80 days); given the small sample size, these results cannot yet be considered significant (p=0.14). Conclusions: These results provide preliminary mechanistic and clinical evidence supporting the use of NF1 loss to guide treatment in HER2+ mBC. As novel HER2-specific agents are being rapidly added to the therapeutic arsenal, we propose biology-driven criteria to identify patients that may benefit specifically from T-DM1. In addition, NF1 dependence for correct microtubular dynamics may be exploited by other inhibitors of microtubular polymerization in use as ADC payloads, further extending the potential usefulness of NF1 determination. Citation Format: Bruno A Duso, Elena Gavilán Dorronzoro, Giulia Tini, Maria R de Filippo, Emanuele Bonetti, Maria R Ippolito, Chiara Soriani, Paolo D'Amico, Simona Rodighiero, Giuseppe Curigliano, Stefano Santaguida, Massimo Cristofanilli, Pier Giuseppe Pelicci, Luca Mazzarella. NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-04.
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Davidson-Moncada, Jan K., Taotao Zhang, Piali Mukherjee, Paul Hakimpour, Richard R. Furman, Nina Papavasiliou, and Wayne Tam. "MicroRNA-155 Modulates Transforming Growth Factor-β Signaling In Chronic Lymphocytic Leukemia through Targeting of Casein Kinase γ Isoform 2." Blood 116, no. 21 (November 19, 2010): 3584. http://dx.doi.org/10.1182/blood.v116.21.3584.3584.
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Abstract Abstract 3584 Chronic lymphocytic leukemia (CLL) is typically characterized by defects in programmed cell death rather than alterations in cell cycle regulation. Transforming growth factor β (TGFβ), a ubiquitously expressed growth factor, regulates multiple normal cellular responses including proliferation, differentiation, migration and apoptosis. Loss of growth inhibition by TGFβ is thought to contribute to the development and progression of a variety of tumors including CLL (DeCoteau et al., PNAS 1997). Approximately 40% of patients contain mutations in the signal sequence of TGFβ receptor 1 (TBR-1) in the form of substitutions or deletions (Schiemann et al., Cancer Detect Prev 2004). In the wild type form, the signal sequence contains a nine alanine stretch, which if truncated has been shown to impair signaling through the receptor and specifically, a truncated, six alanine form is associated with increased cancer risk (Pasche et al., Cancer Res 1999). TGFβ signaling can regulate expression of micoRNAs (miRNA), which are ~22 nucleotide-long RNA gene regulators. Deregulated miRNA expression has been implicated in tumorigenesis, including CLL. Several miRNAs have been shown to be over-expressed in CLL as compared to normal B cells (Fulci et al., Blood 2007). This includes miR-155, which is part of a 13-miRNA signature that has prognostic implications, including a shorter need-for-treatment interval (Calin et al., N Engl J Med 2005). Interestingly, miR-155 has been shown to be upregulated by TGFβ in murine mammary gland cells (Kong et al., Mol Cell Biol 2008). The goals of our study are to investigate the link between TGFβ signaling and miR-155 in CLL and to determine how the interaction between the two may contribute to the pathogenesis of CLL. Here we show that miR-155 is in fact upregulated by TGFβ in mouse splenic B cells and in human peripheral blood B cells. In CLL, miR-155 expression inversely correlates with the proportion of CLL cells harboring signal sequence mutation in TBR-1, consistent with miR155 regulation by TGFβ in vivo. To understand the role of TGFβ-induced miR-155 in CLL pathobiology, identification of specific target genes in the context of this disease is essential. To this end, we compared the gene (cDNA) expression profile between CLL with high miR-155 vs. low miR-155 expression and identified putative miR-155 target genes by selecting those genes that are differentially expressed in SAM analysis with lower expression in the high miR-155 group, and which harbor predicted miR-155 binding sites in their 3’ untranslated region (UTR). Based on this algorithm, we have identified casein kinase 1 gamma 2 (CSK1γ2) as a target for miR155 in CLL. CSK1γ2 is a negative modulator of the TGFβ signaling pathway by targeting the phosphorylated form of SMAD3 for degradation (Guo et al., Oncogene 2008). MiR-155 represses luciferase reporter gene expression by specific binding to the miR-155 site in the CSK1γ2 3’UTR. In addition, we found that CSK1γ2 itself is upregulated in B cells upon TGFβ stimulation, and treatment of human B cells with PNA miR-155 inhibitor (Fabani et al., Nucleic Acids Research 2010) further increases CSK1γ2 mRNA levels. Surprisingly, comparison of CSK1γ2 protein levels between CLLs with high or low miR-155 by Western blotting revealed higher CSK1γ2 protein expression despite lower CSK1γ2 mRNA levels, suggesting that miR-155 may enhance CSK1γ2 translation in CLL cells and implying an intriguing regulatory interaction between miR-155 and CSK1γ2. In summary, our data indicates that the variation of miR-155 seen in CLL is primarily a function of TGFβ signaling activity. Moreover, miR-155 is an important player in a complex auto-regulatory network in TGFβ signaling by fine-tuning the negative feedback mechanism on TGFβ signaling mediated by CSK1γ2. In CLL cells harboring TBR-1 with wild-type signal sequence, higher miR-155 levels may help modulate the TGFβ signaling activity to a level optimal for the survival or other pathobiological functions of CLL. Furthermore, since CLL cells are predominantly non-proliferating, our findings that miR-155 may enhance translation of CSK1γ2 provide support to the model of cell cycle dependence of microRNA functions (Vasudevan et al., Cell Cycle 2008). Disclosures: No relevant conflicts of interest to declare.
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Wong, Ming-Kin, Vincy Wing Sze Ho, Xiaotai Huang, Lu-Yan Chan, Dongying Xie, Runsheng Li, Xiaoliang Ren, et al. "Initial characterization of gap phase introduction in every cell cycle of C. elegans embryogenesis." Frontiers in Cell and Developmental Biology 10 (October 25, 2022). http://dx.doi.org/10.3389/fcell.2022.978962.
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Early embryonic cell cycles usually alternate between S and M phases without any gap phase. When the gap phases are developmentally introduced in various cell types remains poorly defined especially during embryogenesis. To establish the cell-specific introduction of gap phases in embryo, we generate multiple fluorescence ubiquitin cell cycle indicators (FUCCI) in C. elegans. Time-lapse 3D imaging followed by lineal expression profiling reveals sharp and differential accumulation of the FUCCI reporters, allowing the systematic demarcation of cell cycle phases throughout embryogenesis. Accumulation of the reporters reliably identifies both G1 and G2 phases only in two embryonic cells with an extended cell cycle length, suggesting that the remaining cells divide either without a G1 phase, or with a brief G1 phase that is too short to be picked up by our reporters. In summary, we provide an initial picture of gap phase introduction in a metazoan embryo. The newly developed FUCCI reporters pave the way for further characterization of developmental control of cell cycle progression.
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Dolfi, Luca, Roberto Ripa, Adam Antebi, Dario Riccardo Valenzano, and Alessandro Cellerino. "Cell cycle dynamics during diapause entry and exit in an annual killifish revealed by FUCCI technology." EvoDevo 10, no. 1 (November 8, 2019). http://dx.doi.org/10.1186/s13227-019-0142-5.
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Abstract Background Annual killifishes are adapted to surviving and reproducing over alternating dry and wet seasons. During the dry season, all adults die and desiccation-resistant embryos remain encased in dry mud for months or years in a state of diapause where their development is halted in anticipation of the months that have to elapse before their habitats are flooded again. Embryonic development of annual killifishes deviates from canonical teleost development. Epiblast cells disperse during epiboly, and a “dispersed phase” precedes gastrulation. In addition, annual fish have the ability to enter diapause and block embryonic development at the dispersed phase (diapause I), mid-somitogenesis (diapause II) and the final phase of development (diapause III). Developmental transitions associated with diapause entry and exit can be linked with cell cycle events. Here we set to image this transition in living embryos. Results To visibly explore cell cycle dynamics during killifish development in depth, we created a stable transgenic line in Nothobranchius furzeri that expresses two fluorescent reporters, one for the G1 phase and one for the S/G2 phases of the cell cycle, respectively (Fluorescent Ubiquitination-based Cell Cycle Indicator, FUCCI). Using this tool, we observed that, during epiboly, epiblast cells progressively become quiescent and exit the cell cycle. All embryos transit through a phase where dispersed cells migrate, without showing any mitotic activity, possibly blocked in the G1 phase (diapause I). Thereafter, exit from diapause I is synchronous and cells enter directly into the S phase without transiting through G1. The developmental trajectories of embryos entering diapause and of those that continue to develop are different. In particular, embryos entering diapause have reduced growth along the medio-lateral axis. Finally, exit from diapause II is synchronous for all cells and is characterized by a burst of mitotic activity and growth along the medio-lateral axis such that, by the end of this phase, the morphology of the embryos is identical to that of direct-developing embryos. Conclusions Our study reveals surprising levels of coordination of cellular dynamics during diapause and provides a reference framework for further developmental analyses of this remarkable developmental quiescent state.
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Chavkin, Nicholas W., Gael Genet, Mathilde Poulet, Erin D. Jeffery, Corina Marziano, Nafiisha Genet, Hema Vasavada, et al. "Endothelial cell cycle state determines propensity for arterial-venous fate." Nature Communications 13, no. 1 (October 6, 2022). http://dx.doi.org/10.1038/s41467-022-33324-7.
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AbstractDuring blood vessel development, endothelial cells become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Arterial-venous specification occurs in conjunction with suppression of endothelial cell cycle progression; however, the mechanistic role of cell cycle state is unknown. Herein, using Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous endothelial cells are enriched for the FUCCI-Negative state (early G1) and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state (late G1) and TGF-β signaling. Furthermore, early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-β1-induced arterial gene expression. Pharmacologically induced cell cycle arrest prevents arterial-venous specification defects in mice with endothelial hyperproliferation. Collectively, our results show that distinct endothelial cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous fate.
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Madrigal, Pedro, Siwei Deng, Yuliang Feng, Stefania Militi, Kim Jee Goh, Reshma Nibhani, Rodrigo Grandy, et al. "Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation." Nature Communications 14, no. 1 (January 25, 2023). http://dx.doi.org/10.1038/s41467-023-36116-9.
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AbstractStem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.
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Ungai-Salánki, Rita, Eleonóra Haty, Tamás Gerecsei, Barbara Francz, Bálint Béres, Milán Sztilkovics, Inna Székács, Bálint Szabó, and Robert Horvath. "Single-cell adhesion strength and contact density drops in the M phase of cancer cells." Scientific Reports 11, no. 1 (September 16, 2021). http://dx.doi.org/10.1038/s41598-021-97734-1.
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AbstractThe high throughput, cost effective and sensitive quantification of cell adhesion strength at the single-cell level is still a challenging task. The adhesion force between tissue cells and their environment is crucial in all multicellular organisms. Integrins transmit force between the intracellular cytoskeleton and the extracellular matrix. This force is not only a mechanical interaction but a way of signal transduction as well. For instance, adhesion-dependent cells switch to an apoptotic mode in the lack of adhesion forces. Adhesion of tumor cells is a potential therapeutic target, as it is actively modulated during tissue invasion and cell release to the bloodstream resulting in metastasis. We investigated the integrin-mediated adhesion between cancer cells and their RGD (Arg-Gly-Asp) motif displaying biomimetic substratum using the HeLa cell line transfected by the Fucci fluorescent cell cycle reporter construct. We employed a computer-controlled micropipette and a high spatial resolution label-free resonant waveguide grating-based optical sensor calibrated to adhesion force and energy at the single-cell level. We found that the overall adhesion strength of single cancer cells is approximately constant in all phases except the mitotic (M) phase with a significantly lower adhesion. Single-cell evanescent field based biosensor measurements revealed that at the mitotic phase the cell material mass per unit area inside the cell-substratum contact zone is significantly less, too. Importantly, the weaker mitotic adhesion is not simply a direct consequence of the measured smaller contact area. Our results highlight these differences in the mitotic reticular adhesions and confirm that cell adhesion is a promising target of selective cancer drugs as the vast majority of normal, differentiated tissue cells do not enter the M phase and do not divide.
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De Chiara, Letizia, Elena Lazzeri, and Paola Romagnani. "MO288: Kidney Tubule Polyploidization Preserves Residual Kidney Function and Assures Survival During Acute Kidney Injury." Nephrology Dialysis Transplantation 37, Supplement_3 (May 2022). http://dx.doi.org/10.1093/ndt/gfac067.087.
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Abstract BACKGROUND AND AIMS Acute Kidney Injury (AKI) is characterized by a rapid deterioration of kidney function. Recently, we showed that tubular epithelial cells (TC) respond to AKI by triggering polyploidy, a condition in which a normally diploid cell acquires additional sets of chromosomes. Polyploidy offers several advantages, but in the kidney the biological significance of polyploidization remains unclear. In this study we hypothesized that polyploidy (i) is the predominant cellular response early during AKI and (ii) that is an adaptive stress response required to maintain a residual kidney function assuring survival. METHOD To address these hypotheses, we employed in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) technology in combination with YAP1 downregulation. In these models, mice were subjected to unilateral ischemia reperfusion injury (IRI) or glycerol-induced rhabdomyolysis to induce AKI. Polyploid cells have been then characterized by single cell-RNA sequencing analysis, cell sorting, FACS analysis, super-resolution and transmission electron microscopy. RESULTS After AKI, YAP1 is activated triggering TC polyploidization. Polyploid TC increase in parallel to massive cell death triggered by AKI suggesting that polyploidization could be a means to escape cell death. Indeed, we found that polyploid TC tends to accumulate genome instability and survive, while diploid TC do not. Of note, virtually all dying cells were cycling cells based on the Fucci2aR reporter suggesting that TC death occurred during the S or G2/M phase. As polyploid TC increase immediately following AKI, they may be required to survive injury and damage by sustaining renal function. In order to evaluate the functional role of polyploid cells during AKI, we generated mice where YAP1 is knocked-out specifically in TC (YAP1ko mice). Indeed, after AKI, YAP1ko mice showed a reduced number of polyploid cells, worsened kidney function and a dramatic reduction of mouse survival, proving that polyploidization is required to survive AKI. CONCLUSION We demonstrated that (i) after AKI TC accumulate genome instability and die or become polyploidy; (ii) TC polyploidy is essential to preserve residual kidney function immediately after AKI.