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1
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Vats A, Laimins L. How human papillomavirus (HPV) targets DNA repair pathways for viral replication: from guardian to accomplice. Microbiol Mol Biol Rev 2025; 89:e0015323. [PMID: 39868790 PMCID: PMC11948491 DOI: 10.1128/mmbr.00153-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2025] Open
Abstract
SUMMARYHuman papillomaviruses (HPVs) are small DNA viruses that are responsible for significant disease burdens worldwide, including cancers of the cervix, anogenital tract, and oropharynx. HPVs infect stratified epithelia at a variety of body locations and link their productive life cycles to the differentiation of the host cell. These viruses have evolved sophisticated mechanisms to exploit cellular pathways, such as DNA damage repair (DDR), to regulate their life cycles. HPVs activate key DDR pathways such as ATM, ATR, and FA, which are critical for maintaining genomic integrity but are often dysregulated in cancers. Importantly, these DDR pathways are essential for HPV replication in undifferentiated cells and amplification upon differentiation. The ability to modulate these DDR pathways not only enables HPV persistence but also contributes to cellular transformation. In this review, we discuss the recent advances in understanding the mechanisms by which HPV manipulates the host DDR pathways and how these depend upon enhanced topoisomerase activity and R-loop formation. Furthermore, the strategies to manipulate DDR pathways utilized by high-risk HPVs are compared with those used by other DNA viruses that exhibit similarities and distinct differences.
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Affiliation(s)
- Arushi Vats
- Department of Microbiology-Immunology, Northwestern University, Chicago, Illinois, USA
| | - Laimonis Laimins
- Department of Microbiology-Immunology, Northwestern University, Chicago, Illinois, USA
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2
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Ho HY, Chen MK, Lin CC, Lo YS, Chuang YC, Hsieh MJ. Arenobufagin induces cell apoptosis by modulating the cell cycle regulator claspin and the JNK pathway in nasopharyngeal carcinoma cells. Expert Opin Ther Targets 2024; 28:461-471. [PMID: 38659296 DOI: 10.1080/14728222.2024.2348014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2024] [Accepted: 04/23/2024] [Indexed: 04/26/2024]
Abstract
BACKGROUND The high recurrence rate and incidence of distant metastasis of nasopharyngeal carcinoma (NPC) result in poor prognosis. It is necessary to identify natural compounds that can complement combination radiation therapy. Arenobufagin is commonly used for heart diseases and liver cancer, but its effectiveness in NPC is unclear. STUDY DESIGN AND METHODS The effect of arenobufagin-induced apoptosis was measured by a cell viability assay, tumorigenic assay, fluorescence assay, and Western blot assay through NPC-039 and NPC-BM cell lines. The protease array, Western blot assay, and transient transfection were used to investigate the underlying mechanism of arenobufagin-induced apoptosis. An NPC xenograft model was established to explore the antitumor activity of arenobufagin in vivo. RESULTS Our findings indicated that arenobufagin exerted cytotoxic effects on NPC cells, inhibiting proliferation through apoptosis activation. Downregulation of claspin was confirmed in arenobufagin-induced apoptosis. Combined treatment with arenobufagin and mitogen-activated protein kinase inhibitors demonstrated that arenobufagin induced NPC apoptosis through the c-Jun N-terminal kinases (JNK) pathway inhibition. Furthermore, arenobufagin suppressed NPC tumor proliferation in vivo. CONCLUSION Our results revealed the antitumor effect of arenobufagin in vitro and in vivo. Arenobufagin may have clinical utility in treating NPC due to its suppression of claspin and inhibition of the JNK pathway.
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Affiliation(s)
- Hsin-Yu Ho
- Oral Cancer Research Center, Changhua Christian Hospital, Changhua, Taiwan
| | - Mu-Kuan Chen
- Department of Otorhinolaryngology-Head and Neck Surgery, Changhua Christian Hospital, Changhua, Taiwan
- Department of Post-Baccalaureate Medicine, College of Medicine, National Chung Hsing University, Taichung, Taiwan
| | - Chia-Chieh Lin
- Oral Cancer Research Center, Changhua Christian Hospital, Changhua, Taiwan
| | - Yu-Sheng Lo
- Oral Cancer Research Center, Changhua Christian Hospital, Changhua, Taiwan
| | - Yi-Ching Chuang
- Oral Cancer Research Center, Changhua Christian Hospital, Changhua, Taiwan
| | - Ming-Ju Hsieh
- Oral Cancer Research Center, Changhua Christian Hospital, Changhua, Taiwan
- Doctoral Program in Tissue Engineering and Regenerative Medicine, College of Medicine, National Chung Hsing University, Taichung, Taiwan
- Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan
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3
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Shi Y, Wang Y, Niu K, Zhang W, Lv Q, Zhang Y. How CLSPN could demystify its prognostic value and potential molecular mechanism for hepatocellular carcinoma: A crosstalk study. Comput Biol Med 2024; 172:108260. [PMID: 38492457 DOI: 10.1016/j.compbiomed.2024.108260] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2024] [Revised: 02/23/2024] [Accepted: 03/06/2024] [Indexed: 03/18/2024]
Abstract
BACKGROUND & AIMS CLSPN, a critical component of the S-phase checkpoint in response to DNA replication stress, has been implicated in the pathogenesis of multiple tumor types. The rising incidence of hepatocellular carcinoma (HCC) poses a significant challenge to global public health. Despite this, the specific functions of CLSPN in the development of HCC remain poorly understood. METHODS We systematically evaluated the expression of CLSPN, prognosis and immune infiltration in patients with HCC and identified a competing endogenous RNA (ceRNA) network by using public database. The RT-qPCR, western blot, CCK8, transwell, flow cytometry, animal experiments, proteasome inhibition experiment, Co-IP assay and mass spectrometry were applied to explore its biological functions, post-transcriptional modifications and potential molecular mechanisms of CLSPN in HCC. RESULTS We verified the expression of CLSPN, and its high expression is an independent prognostic factor in HCC. The expression of CLSPN is also associated with the immune microenvironment of HCC. CLSPN silencing inhibited the proliferation, migration, invasion and cell cycle progression of HCC cells. We established a PSMA3-AS1/hsa-miR-101-3p/CLSPN regulator axis in HCC. CLSPN was influenced by ubiquitination and was involved in the Wnt/β-catenin pathway to regulate HCC progression. CONCLUSIONS It was the first time to comprehensively discover and identify the expression, prognosis, immunotherapy, RNAs regulator, posttranscriptional modification, and molecular mechanisms of CLSPN in HCC. These novel insights have the potential to expedite the development of personalized treatment strategies and translational medicine approaches for HCC patients.
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Affiliation(s)
- Yanlong Shi
- Hepatopancreatobiliary Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, 210003, China
| | - Yizhu Wang
- Hepatopancreatobiliary Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, 210003, China
| | - Kaiyi Niu
- Hepatopancreatobiliary Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, 210003, China
| | - Wenning Zhang
- Hepatopancreatobiliary Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, 210003, China
| | - Qingpeng Lv
- Hepatopancreatobiliary Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, 210003, China
| | - Yewei Zhang
- Hepatopancreatobiliary Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, 210003, China.
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Heuzé J, Lin YL, Lengronne A, Poli J, Pasero P. Impact of R-loops on oncogene-induced replication stress in cancer cells. C R Biol 2023; 346:95-105. [PMID: 37779381 DOI: 10.5802/crbiol.123] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2023] [Revised: 07/19/2023] [Accepted: 07/20/2023] [Indexed: 10/03/2023]
Abstract
Replication stress is an alteration in the progression of replication forks caused by a variety of events of endogenous or exogenous origin. In precancerous lesions, this stress is exacerbated by the deregulation of oncogenic pathways, which notably disrupts the coordination between replication and transcription, and leads to genetic instability and cancer development. It is now well established that transcription can interfere with genome replication in different ways, such as head-on collisions between polymerases, accumulation of positive DNA supercoils or formation of R-loops. These structures form during transcription when nascent RNA reanneals with DNA behind the RNA polymerase, forming a stable DNA:RNA hybrid. In this review, we discuss how these different cotranscriptional processes disrupt the progression of replication forks and how they contribute to genetic instability in cancer cells.
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5
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Thong KX, Andriesei P, Luo J, Qin M, Ng J, Tagalakis AD, Hysi P, Yu-Wai-Man C. Adrenaline blocks key cell cycle genes and exhibits antifibrotic and vasoconstrictor effects in glaucoma surgery. Exp Eye Res 2023; 233:109561. [PMID: 37429521 DOI: 10.1016/j.exer.2023.109561] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 05/04/2023] [Accepted: 06/26/2023] [Indexed: 07/12/2023]
Abstract
Adrenaline is a sympathomimetic drug used to maintain pupil dilation and to decrease the risk of bleeding. The aim of this study was to demonstrate if adrenaline could exert antifibrotic effects in glaucoma surgery. Adrenaline was tested in fibroblast-populated collagen contraction assays and there was a dose-response decrease in fibroblast contractility: matrices decreased to 47.4% (P = 0.0002) and 86.6% (P = 0.0036) with adrenaline 0.0005% and 0.01%, respectively. There was no significant decrease in cell viability even at high concentrations. Human Tenon's fibroblasts were also treated with adrenaline (0%, 0.0005%, 0.01%) for 24 h and RNA-Sequencing was performed on the Illumina NextSeq 2000. We carried out detailed gene ontology, pathway, disease and drug enrichment analyses. Adrenaline 0.01% upregulated 26 G1/S and 11 S-phase genes, and downregulated 23 G2 and 17 M-phase genes (P < 0.05). Adrenaline demonstrated similar pathway enrichment to mitosis and spindle checkpoint regulation. Adrenaline 0.05% was also injected subconjunctivally during trabeculectomy, PreserFlo Microshunt and Baerveldt 350 tube surgeries, and patients did not experience any adverse effects. Adrenaline is a safe and cheap antifibrotic drug that significantly blocks key cell cycle genes when used at high concentrations. Unless contraindicated, we recommend subconjunctival injections of adrenaline (0.05%) in all glaucoma bleb-forming surgeries.
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Affiliation(s)
- Kai Xin Thong
- Faculty of Life Sciences & Medicine, King's College London, London, SE1 7EH, UK
| | - Petru Andriesei
- Faculty of Life Sciences & Medicine, King's College London, London, SE1 7EH, UK
| | - Jinyuan Luo
- Faculty of Life Sciences & Medicine, King's College London, London, SE1 7EH, UK
| | - Mengqi Qin
- Faculty of Life Sciences & Medicine, King's College London, London, SE1 7EH, UK
| | - Jia Ng
- Faculty of Life Sciences & Medicine, King's College London, London, SE1 7EH, UK
| | | | - Pirro Hysi
- Faculty of Life Sciences & Medicine, King's College London, London, SE1 7EH, UK
| | - Cynthia Yu-Wai-Man
- Faculty of Life Sciences & Medicine, King's College London, London, SE1 7EH, UK; Department of Ophthalmology, Guy's and St Thomas' NHS Foundation Trust, London, SE1 7EH, UK.
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Saldanha J, Rageul J, Patel JA, Kim H. The Adaptive Mechanisms and Checkpoint Responses to a Stressed DNA Replication Fork. Int J Mol Sci 2023; 24:10488. [PMID: 37445667 PMCID: PMC10341514 DOI: 10.3390/ijms241310488] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2023] [Revised: 06/13/2023] [Accepted: 06/20/2023] [Indexed: 07/15/2023] Open
Abstract
DNA replication is a tightly controlled process that ensures the faithful duplication of the genome. However, DNA damage arising from both endogenous and exogenous assaults gives rise to DNA replication stress associated with replication fork slowing or stalling. Therefore, protecting the stressed fork while prompting its recovery to complete DNA replication is critical for safeguarding genomic integrity and cell survival. Specifically, the plasticity of the replication fork in engaging distinct DNA damage tolerance mechanisms, including fork reversal, repriming, and translesion DNA synthesis, enables cells to overcome a variety of replication obstacles. Furthermore, stretches of single-stranded DNA generated upon fork stalling trigger the activation of the ATR kinase, which coordinates the cellular responses to replication stress by stabilizing the replication fork, promoting DNA repair, and controlling cell cycle and replication origin firing. Deregulation of the ATR checkpoint and aberrant levels of chronic replication stress is a common characteristic of cancer and a point of vulnerability being exploited in cancer therapy. Here, we discuss the various adaptive responses of a replication fork to replication stress and the roles of ATR signaling that bring fork stabilization mechanisms together. We also review how this knowledge is being harnessed for the development of checkpoint inhibitors to trigger the replication catastrophe of cancer cells.
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Affiliation(s)
- Joanne Saldanha
- The Graduate Program in Genetics, Stony Brook University, Stony Brook, NY 11794, USA
- Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA
| | - Julie Rageul
- Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA
| | - Jinal A. Patel
- Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA
| | - Hyungjin Kim
- The Graduate Program in Genetics, Stony Brook University, Stony Brook, NY 11794, USA
- Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA
- Stony Brook Cancer Center, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA
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7
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Patel JA, Kim H. The TIMELESS effort for timely DNA replication and protection. Cell Mol Life Sci 2023; 80:84. [PMID: 36892674 PMCID: PMC9998586 DOI: 10.1007/s00018-023-04738-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2023] [Revised: 02/16/2023] [Accepted: 02/24/2023] [Indexed: 03/10/2023]
Abstract
Accurate replication of the genome is fundamental to cellular survival and tumor prevention. The DNA replication fork is vulnerable to DNA lesions and damages that impair replisome progression, and improper control over DNA replication stress inevitably causes fork stalling and collapse, a major source of genome instability that fuels tumorigenesis. The integrity of the DNA replication fork is maintained by the fork protection complex (FPC), in which TIMELESS (TIM) constitutes a key scaffold that couples the CMG helicase and replicative polymerase activities, in conjunction with its interaction with other proteins associated with the replication machinery. Loss of TIM or the FPC in general results in impaired fork progression, elevated fork stalling and breakage, and a defect in replication checkpoint activation, thus underscoring its pivotal role in protecting the integrity of both active and stalled replication forks. TIM is upregulated in multiple cancers, which may represent a replication vulnerability of cancer cells that could be exploited for new therapies. Here, we discuss recent advances on our understanding of the multifaceted roles of TIM in DNA replication and stalled fork protection, and how its complex functions are engaged in collaboration with other genome surveillance and maintenance factors.
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Affiliation(s)
- Jinal A Patel
- Department of Pharmacological Sciences, State University of New York at Stony Brook, Basic Sciences Tower 8-125, 101 Nicolls Rd, Stony Brook, NY, 11794, USA
| | - Hyungjin Kim
- Department of Pharmacological Sciences, State University of New York at Stony Brook, Basic Sciences Tower 8-125, 101 Nicolls Rd, Stony Brook, NY, 11794, USA.
- Stony Brook Cancer Center and Renaissance School of Medicine, Stony Brook University, Basic Sciences Tower 8-125, 101 Nicolls Rd, Stony Brook, NY, 11794, USA.
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8
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Suominen MI, Knuuttila M, Schatz CA, Schlicker A, Vääräniemi J, Sjöholm B, Alhoniemi E, Haendler B, Mumberg D, Käkönen SM, Scholz A. Enhanced Antitumor Efficacy of Radium-223 and Enzalutamide in the Intratibial LNCaP Prostate Cancer Model. Int J Mol Sci 2023; 24:ijms24032189. [PMID: 36768509 PMCID: PMC9916479 DOI: 10.3390/ijms24032189] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Revised: 01/12/2023] [Accepted: 01/18/2023] [Indexed: 01/24/2023] Open
Abstract
Radium-223 dichloride and enzalutamide are indicated for metastatic castration-resistant prostate cancer and their combination is currently being investigated in a large phase 3 clinical trial. Here, we evaluated the antitumor efficacy of radium-223, enzalutamide, and their combination in the intratibial LNCaP model mimicking prostate cancer metastasized to bone. In vitro experiments revealed that the combination of radium-223 and enzalutamide inhibited LNCaP cell proliferation and showed synergistic efficacy. The combination of radium-223 and enzalutamide also demonstrated enhanced in vivo antitumor efficacy, as determined by measuring serum PSA levels in the intratibial LNCaP model. A decreasing trend in the total area of tumor-induced abnormal bone was associated with the combination treatment. The serum levels of the bone formation marker PINP and the bone resorption marker CTX-I were lowest in the combination treatment group and markedly decreased compared with vehicle group. Concurrent administration of enzalutamide did not impair radium-223 uptake in tumor-bearing bone or the ability of radium-223 to inhibit tumor-induced abnormal bone formation. In conclusion, combination treatment with radium-223 and enzalutamide demonstrated enhanced antitumor efficacy without compromising the integrity of healthy bone. The results support the ongoing phase 3 trial of this combination.
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Affiliation(s)
| | | | | | - Andreas Schlicker
- Research & Development, Pharmaceuticals, Bayer AG, 13353 Berlin, Germany
| | | | | | | | - Bernard Haendler
- Research & Development, Pharmaceuticals, Bayer AG, 13353 Berlin, Germany
| | - Dominik Mumberg
- Research & Development, Pharmaceuticals, Bayer AG, 13353 Berlin, Germany
| | - Sanna-Maria Käkönen
- Aurexel Life Sciences Ltd., 21240 Askainen, Finland
- Institute of Biomedicine, University of Turku, 20520 Turku, Finland
| | - Arne Scholz
- Research & Development, Pharmaceuticals, Bayer AG, 13353 Berlin, Germany
- Correspondence: ; Tel.: +49-30-468-16369
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Caraci F, Fidilio A, Santangelo R, Caruso G, Giuffrida ML, Tomasello MF, Nicoletti F, Copani A. Molecular Connections between DNA Replication and Cell Death in β-Amyloid-Treated Neurons. Curr Neuropharmacol 2023; 21:2006-2018. [PMID: 37021419 PMCID: PMC10514525 DOI: 10.2174/1570159x21666230404121903] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2022] [Revised: 10/18/2022] [Accepted: 10/20/2022] [Indexed: 04/07/2023] Open
Abstract
BACKGROUND Ectopic cell cycle reactivation in neurons is associated with neuronal death in Alzheimer's disease. In cultured rodent neurons, synthetic β-amyloid (Aβ) reproduces the neuronal cell cycle re-entry observed in the Alzheimer's brain, and blockade of the cycle prevents Aβ-induced neurodegeneration. DNA polymerase-β, whose expression is induced by Aβ, is responsible for the DNA replication process that ultimately leads to neuronal death, but the molecular mechanism(s) linking DNA replication to neuronal apoptosis are presently unknown. AIM To explore the role of a conserved checkpoint pathway started by DNA replication stress, namely the ATM-ATR/Claspin/Chk-1 pathway, in switching the neuronal response from DNA replication to apoptosis. METHODS Experiments were carried out in cultured rat cortical neurons challenged with toxic oligomers of Aβ protein. RESULTS Small inhibitory molecules of ATM/ATR kinase or Chk-1 amplified Aβ-induced neuronal DNA replication and apoptosis, as they were permissive to the DNA polymerase-β activity triggered by Aβ oligomers. Claspin, i.e., the adaptor protein between ATM/ATR kinase and the downstream Chk-1, was present on DNA replication forks of neurons early after Aβ challenge, and decreased at times coinciding with neuronal apoptosis. The caspase-3/7 inhibitor I maintained overtime the amount of Claspin loaded on DNA replication forks and, concomitantly, reduced neuronal apoptosis by holding neurons in the S phase. Moreover, a short phosphopeptide mimicking the Chk-1-binding motif of Claspin was able to prevent Aβ-challenged neurons from entering apoptosis. CONCLUSION We speculate that, in the Alzheimer's brain, Claspin degradation by intervening factors may precipitate the death of neurons engaged into DNA replication.
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Affiliation(s)
- Filippo Caraci
- Department of Drug and Health Sciences, University of Catania, Catania, Italy
- UOR of Neuropharmacology and Translational Neurosciences, Oasi Research Institute - IRCCS, Troina, Italy
| | - Annamaria Fidilio
- UOR of Neuropharmacology and Translational Neurosciences, Oasi Research Institute - IRCCS, Troina, Italy
| | - Rosa Santangelo
- Department of Drug and Health Sciences, University of Catania, Catania, Italy
| | - Giuseppe Caruso
- Department of Drug and Health Sciences, University of Catania, Catania, Italy
| | - Maria Laura Giuffrida
- Institute of Crystallography, National Council of Research, Catania Unit, Catania, Italy
| | | | - Ferdinando Nicoletti
- Department of Physiology and Pharmacology, University Sapienza of Rome, Rome, Italy
- IRCCS Neuromed, Pozzilli, Italy
| | - Agata Copani
- Department of Drug and Health Sciences, University of Catania, Catania, Italy
- Institute of Crystallography, National Council of Research, Catania Unit, Catania, Italy
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Yang CC, Masai H. Claspin is Required for Growth Recovery from Serum Starvation through Regulating the PI3K-PDK1-mTOR Pathway in Mammalian Cells. Mol Cell Biol 2023; 43:1-21. [PMID: 36720467 PMCID: PMC9936878 DOI: 10.1080/10985549.2022.2160598] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation, which requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. In the absence of Claspin, cells do not proceed into S phase and eventually die partially in a ROS- and p53-dependent manner. Claspin directly interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4EBP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 physically interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. Thus, Claspin plays a novel role as a key regulator for nutrition-induced proliferation/survival signaling by activating the mTOR pathway. The results also suggest a possibility that Claspin may serve as a common mediator that receives signals from different PI3K-related kinases and transmit them to specific downstream kinases.
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Affiliation(s)
- Chi-Chun Yang
- Department of Basic Medical Sciences, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Hisao Masai
- Department of Basic Medical Sciences, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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11
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Keeping RelApse in Chk: molecular mechanisms of Chk1 inhibitor resistance in lymphoma. Biochem J 2022; 479:2345-2349. [DOI: 10.1042/bcj20220461] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Revised: 10/06/2022] [Accepted: 10/10/2022] [Indexed: 11/24/2022]
Abstract
Chk1 is a member of the DNA damage response pathway, whose loss leads to replication stress and genome instability. Because of its protective role against lethal levels of DNA replication stress, Chk1 has been studied as a valuable and intriguing target for cancer therapy. However, one of the most prominent challenges with this strategy is development of resistance to Chk1 inhibitors, rendering the treatment ineffective. In their recent papers, Hunter and colleagues demonstrate multiple mechanisms by which Chk1 inhibitor resistance can arise in lymphomas. Specifically, this series of papers identify the relationship between dysfunction in NF-κB and the development of Chk1 inhibitor resistance through a loss of Chk1 activity in mouse models of lymphoma. They identify that cells lacking Chk1 activity can compensate for this loss through up-regulation of alternative pathways, such as PI3K/AKT. Finally, this work also identifies a novel role for Claspin, an important Chk1 activator, in female fertility and cancer development, furthering our understanding of how dysfunction in the Claspin/Chk1 signaling pathway affects disease states. These findings improve our understanding of drug resistance in cancer therapy, which has important implications for clinical use of Chk1 inhibitors.
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12
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Guerra B, Doktor TK, Frederiksen SB, Somyajit K, Andresen BS. Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint. Cell Mol Life Sci 2022; 79:339. [PMID: 35661926 PMCID: PMC9166893 DOI: 10.1007/s00018-022-04374-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2021] [Revised: 05/09/2022] [Accepted: 05/12/2022] [Indexed: 11/25/2022]
Abstract
The ataxia telangiectasia mutated and Rad3-related (ATR)-CHK1 pathway is the major signalling cascade activated in response to DNA replication stress. This pathway is associated with the core of the DNA replication machinery comprising CDC45, the replicative MCM2-7 hexamer, GINS (altogether forming the CMG complex), primase-polymerase (POLε, -α, and -δ) complex, and additional fork protection factors such as AND-1, CLASPIN (CLSPN), and TIMELESS/TIPIN. In this study, we report that functional protein kinase CK2α is critical for preserving replisome integrity and for mounting S-phase checkpoint signalling. We find that CDC45, CLSPN and MCM7 are novel CK2α interacting partners and these interactions are particularly important for maintenance of stable MCM7-CDC45, ATRIP-ATR-MCM7, and ATR-CLSPN protein complexes. Consistently, cells depleted of CK2α and treated with hydroxyurea display compromised replisome integrity, reduced chromatin binding of checkpoint mediator CLSPN, attenuated ATR-mediated S-phase checkpoint and delayed recovery of stalled forks. In further support of this, differential gene expression analysis by RNA-sequencing revealed that down-regulation of CK2α accompanies global shutdown of genes that are implicated in the S-phase checkpoint. These findings add to our understanding of the molecular mechanisms involved in DNA replication by showing that the protein kinase CK2α is essential for maintaining the stability of the replisome machinery and for optimizing ATR-CHK1 signalling activation upon replication stress.
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Affiliation(s)
- Barbara Guerra
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
| | - Thomas K Doktor
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
| | - Sabrina B Frederiksen
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
| | - Kumar Somyajit
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
| | - Brage S Andresen
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
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Sahay O, Barik GK, Sharma T, Pillai AD, Rapole S, Santra MK. Damsel in distress calling on her knights: Illuminating the pioneering role of E3 ubiquitin ligases in guarding the genome integrity. DNA Repair (Amst) 2021; 109:103261. [PMID: 34920250 DOI: 10.1016/j.dnarep.2021.103261] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2021] [Revised: 11/30/2021] [Accepted: 12/07/2021] [Indexed: 11/03/2022]
Abstract
The maintenance of genomic integrity is of utmost importance for the organisms to survive and to accurately inherit traits to their progenies. Any kind of DNA damage either due to defect in DNA duplication and/ or uncontrolled cell division or intracellular insults or environment radiation can result in gene mutation, chromosomal aberration and ultimately genomic instability, which may cause several diseases including cancers. Therefore, cells have evolved machineries for the surveillance of genomic integrity. Enormous exciting studies in the past indicate that ubiquitination (a posttranslational modification of proteins) plays a crucial role in maintaining the genomic integrity by diverse ways. In fact, various E3 ubiquitin ligases catalyse ubiquitination of key proteins to control their central role during cell cycle, DNA damage response (DDR) and DNA repair. Some E3 ligases promote genomic instability while others prevent it, deregulation of both of which leads to several malignancies. In this review, we consolidate the recent findings wherein the role of ubiquitination in conferring genome integrity is highlighted. We also discuss the latest discoveries on the mechanisms utilized by various E3 ligases to preserve genomic stability, with a focus on their actions during cell cycle progression and different types of DNA damage response as well as repair pathways.
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Affiliation(s)
- Osheen Sahay
- National Centre for Cell Science, Ganeshkhind Road, Pune, Maharashtra 411007, India; Department of Biotechnology, S.P. Pune University, Ganeshkhind Road, Pune, Maharashtra 411007, India
| | - Ganesh Kumar Barik
- National Centre for Cell Science, Ganeshkhind Road, Pune, Maharashtra 411007, India; Department of Biotechnology, S.P. Pune University, Ganeshkhind Road, Pune, Maharashtra 411007, India
| | - Tanisha Sharma
- National Centre for Cell Science, Ganeshkhind Road, Pune, Maharashtra 411007, India; Department of Biotechnology, S.P. Pune University, Ganeshkhind Road, Pune, Maharashtra 411007, India
| | - Ajay D Pillai
- National Centre for Cell Science, Ganeshkhind Road, Pune, Maharashtra 411007, India
| | - Srikanth Rapole
- National Centre for Cell Science, Ganeshkhind Road, Pune, Maharashtra 411007, India
| | - Manas Kumar Santra
- National Centre for Cell Science, Ganeshkhind Road, Pune, Maharashtra 411007, India.
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Clinicopathological significance of claspin overexpression and its efficacy as a novel biomarker for the diagnosis of urothelial carcinoma. Virchows Arch 2021; 480:621-633. [PMID: 34842980 DOI: 10.1007/s00428-021-03239-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Revised: 10/27/2021] [Accepted: 11/19/2021] [Indexed: 10/19/2022]
Abstract
We previously reported that claspin is a key regulator in the progression of gastric cancer and renal cell carcinoma. However, the clinicopathological significance of claspin in urothelial carcinoma (UC) has not been investigated. We analyzed the expression and distribution of claspin in UC cases by immunohistochemistry. In the non-neoplastic urothelium, the expression of claspin was either weak or absent, whereas UC tissues showed nuclear staining. The expression of claspin was detected in 58 (42%) of a total of 138 upper tract UC cases treated by radical nephroureterectomy without neoadjuvant chemotherapy. Claspin-positive UC cases were associated with nodular/flat morphology, variant histology, high tumor grade, high pathological T grade, and lymphatic and venous invasion. The expression of claspin was significantly associated with decreased progression-free survival and cancer-specific survival. In addition, claspin was co-expressed with Ki-67, PD-L1, HER2, EGFR, and p53 in consecutive tumor sections of UC. An immunohistochemical analysis of claspin in biopsy specimens revealed that strong to moderate claspin staining was more frequently observed in carcinoma in situ in comparison to dysplasia or the benign urothelium. Furthermore, immunocytochemistry for claspin on urine cytology slides demonstrated that the proportion of claspin-positive cells was significantly greater in high-grade UC than in benign cases. These results suggest that claspin may be a novel prognostic marker and a possible therapeutic target molecule for UC. Moreover, claspin could be a useful diagnostic biomarker of urothelial neoplasia.
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Cai X, Wu S, Mipam T, Luo H, Yi C, Xu C, Zhao W, Wang H, Zhong J. Testis transcriptome profiling identified lncRNAs involved in spermatogenic arrest of cattleyak. Funct Integr Genomics 2021; 21:665-678. [PMID: 34626308 DOI: 10.1007/s10142-021-00806-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2021] [Revised: 08/31/2021] [Accepted: 09/11/2021] [Indexed: 02/07/2023]
Abstract
Cattleyaks are the crossbred offspring between cattle and yaks, exhibiting the prominent adaptability to the harsh environment as yaks and much higher growth performances than yaks around Qinghai-Tibet plateau. Unfortunately, cattleyak cannot be effectively used in yak breeding due to its male infertility resulted from spermatogenic arrest. In this study, we performed RNA sequencing (RNA-seq) and bioinformatics analysis to determine the expression profiles of long noncoding RNA (lncRNA) from cattleyak and yak testis. A total of 604 differentially expressed (DE) lncRNAs (135 upregulated and 469 downregulated) were identified in cattleyak with respect to yak. Through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we identified several DE lncRNAs regulating the mitotic cell cycle processes by targeting the genes significantly associated with the mitotic cell cycle checkpoint and DNA damage checkpoint term and also significantly involved in p53 signaling pathway, mismatch repair and homologous recombination pathway (P < 0.05). The reverse transcription PCR (RT-PCR) and quantitative Real-Time PCR (qRT-PCR) analysis of the randomly selected fourteen DE lncRNAs and the seven target genes validated the RNA-seq data and their true expressions during spermatogenesis in vivo. Molecular cloning and sequencing indicated that the testis lncRNAs NONBTAT012170 and NONBTAT010258 presented higher similarity among different cattleyak and yak individuals. The downregulation of these target genes in cattleyak contributed to the abnormal DNA replication and spermatogenic arrest during the S phase of mitotic cell cycle. This study provided a novel insight into lncRNA expression profile changes associated with spermatogenic arrest of cattleyak.
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Affiliation(s)
- Xin Cai
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu, 610041, Sichuan, China.
| | - Shixin Wu
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu, 610041, Sichuan, China
- School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, 621010, Sichuan, China
| | - TserangDonko Mipam
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu, 610041, Sichuan, China
| | - Hui Luo
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu, 610041, Sichuan, China
- School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, 621010, Sichuan, China
| | - Chuanping Yi
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu, 610041, Sichuan, China
- School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, 621010, Sichuan, China
| | - Chuanfei Xu
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu, 610041, Sichuan, China
- School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, 621010, Sichuan, China
| | - Wangsheng Zhao
- School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, 621010, Sichuan, China
| | - Hongying Wang
- College of Chemistry&Environment, Southwest Minzu University, Chengdu, 610041, Sichuan, China
| | - Jincheng Zhong
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu, 610041, Sichuan, China.
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Russi M, Marson D, Fermeglia A, Aulic S, Fermeglia M, Laurini E, Pricl S. The fellowship of the RING: BRCA1, its partner BARD1 and their liaison in DNA repair and cancer. Pharmacol Ther 2021; 232:108009. [PMID: 34619284 DOI: 10.1016/j.pharmthera.2021.108009] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2021] [Revised: 08/22/2021] [Accepted: 09/20/2021] [Indexed: 12/12/2022]
Abstract
The breast cancer type 1 susceptibility protein (BRCA1) and its partner - the BRCA1-associated RING domain protein 1 (BARD1) - are key players in a plethora of fundamental biological functions including, among others, DNA repair, replication fork protection, cell cycle progression, telomere maintenance, chromatin remodeling, apoptosis and tumor suppression. However, mutations in their encoding genes transform them into dangerous threats, and substantially increase the risk of developing cancer and other malignancies during the lifetime of the affected individuals. Understanding how BRCA1 and BARD1 perform their biological activities therefore not only provides a powerful mean to prevent such fatal occurrences but can also pave the way to the development of new targeted therapeutics. Thus, through this review work we aim at presenting the major efforts focused on the functional characterization and structural insights of BRCA1 and BARD1, per se and in combination with all their principal mediators and regulators, and on the multifaceted roles these proteins play in the maintenance of human genome integrity.
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Affiliation(s)
- Maria Russi
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Domenico Marson
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Alice Fermeglia
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Suzana Aulic
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Maurizio Fermeglia
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Erik Laurini
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy
| | - Sabrina Pricl
- Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTs), DEA, University of Trieste, Trieste, Italy; Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland.
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Alsherbiny MA, Bhuyan DJ, Radwan I, Chang D, Li CG. Metabolomic Identification of Anticancer Metabolites of Australian Propolis and Proteomic Elucidation of Its Synergistic Mechanisms with Doxorubicin in the MCF7 Cells. Int J Mol Sci 2021; 22:ijms22157840. [PMID: 34360606 PMCID: PMC8346082 DOI: 10.3390/ijms22157840] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 07/18/2021] [Accepted: 07/19/2021] [Indexed: 12/11/2022] Open
Abstract
The combination of natural products with standard chemotherapeutic agents offers a promising strategy to enhance the efficacy or reduce the side effects of standard chemotherapy. Doxorubicin (DOX), a standard drug for breast cancer, has several disadvantages, including severe side effects and the development of drug resistance. Recently, we reported the potential bioactive markers of Australian propolis extract (AP-1) and their broad spectrum of pharmacological activities. In the present study, we explored the synergistic interactions between AP-1 and DOX in the MCF7 breast adenocarcinoma cells using different synergy quantitation models. Biochemometric and metabolomics-driven analysis was performed to identify the potential anticancer metabolites in AP-1. The molecular mechanisms of synergy were studied by analysing the apoptotic profile via flow cytometry, apoptotic proteome array and measuring the oxidative status of the MCF7 cells treated with the most synergistic combination. Furthermore, label-free quantification proteomics analysis was performed to decipher the underlying synergistic mechanisms. Five prenylated stilbenes were identified as the key metabolites in the most active AP-1 fraction. Strong synergy was observed when AP-1 was combined with DOX in the ratio of 100:0.29 (w/w) as validated by different synergy quantitation models implemented. AP-1 significantly enhanced the inhibitory effect of DOX against MCF7 cell proliferation in a dose-dependent manner with significant inhibition of the reactive oxygen species (p < 0.0001) compared to DOX alone. AP-1 enabled the reversal of DOX-mediated necrosis to programmed cell death, which may be advantageous to decline DOX-related side effects. AP-1 also significantly enhanced the apoptotic effect of DOX after 24 h of treatment with significant upregulation of catalase, HTRA2/Omi, FADD together with DR5 and DR4 TRAIL-mediated apoptosis (p < 0.05), contributing to the antiproliferative activity of AP-1. Significant upregulation of pro-apoptotic p27, PON2 and catalase with downregulated anti-apoptotic XIAP, HSP60 and HIF-1α, and increased antioxidant proteins (catalase and PON2) may be associated with the improved apoptosis and oxidative status of the synergistic combination-treated MCF7 cells compared to the mono treatments. Shotgun proteomics identified 21 significantly dysregulated proteins in the synergistic combination-treated cells versus the mono treatments. These proteins were involved in the TP53/ATM-regulated non-homologous end-joining pathway and double-strand breaks repairs, recruiting the overexpressed BRCA1 and suppressed RIF1 encoded proteins. The overexpression of UPF2 was noticed in the synergistic combination treatment, which could assist in overcoming doxorubicin resistance-associated long non-coding RNA and metastasis of the MCF7 cells. In conclusion, we identified the significant synergy and highlighted the key molecular pathways in the interaction between AP-1 and DOX in the MCF7 cells together with the AP-1 anticancer metabolites. Further in vivo and clinical studies are warranted on this synergistic combination.
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Affiliation(s)
- Muhammad A. Alsherbiny
- NICM Health Research Institute, Western Sydney University, Penrith, NSW 2751, Australia;
- Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
- Correspondence: (M.A.A.); (D.J.B.); (C.-G.L.)
| | - Deep J. Bhuyan
- NICM Health Research Institute, Western Sydney University, Penrith, NSW 2751, Australia;
- Correspondence: (M.A.A.); (D.J.B.); (C.-G.L.)
| | - Ibrahim Radwan
- Faculty of Science and Technology, University of Canberra, Canberra, ACT 2617, Australia;
| | - Dennis Chang
- NICM Health Research Institute, Western Sydney University, Penrith, NSW 2751, Australia;
| | - Chun-Guang Li
- NICM Health Research Institute, Western Sydney University, Penrith, NSW 2751, Australia;
- Correspondence: (M.A.A.); (D.J.B.); (C.-G.L.)
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Pescatori S, Berardinelli F, Albanesi J, Ascenzi P, Marino M, Antoccia A, di Masi A, Acconcia F. A Tale of Ice and Fire: The Dual Role for 17β-Estradiol in Balancing DNA Damage and Genome Integrity. Cancers (Basel) 2021; 13:1583. [PMID: 33808099 PMCID: PMC8036963 DOI: 10.3390/cancers13071583] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2021] [Revised: 03/24/2021] [Accepted: 03/26/2021] [Indexed: 12/21/2022] Open
Abstract
17β-estradiol (E2) regulates human physiology both in females and in males. At the same time, E2 acts as a genotoxic substance as it could induce DNA damages, causing the initiation of cellular transformation. Indeed, increased E2 plasma levels are a risk factor for the development of several types of cancers including breast cancer. This paradoxical identity of E2 undermines the foundations of the physiological definition of "hormone" as E2 works both as a homeostatic regulator of body functions and as a genotoxic compound. Here, (i) the molecular circuitries underlying this double face of E2 are reviewed, and (ii) a possible framework to reconcile the intrinsic discrepancies of the E2 function is reported. Indeed, E2 is a regulator of the DNA damage response, which this hormone exploits to calibrate its genotoxicity with its physiological effects. Accordingly, the genes required to maintain genome integrity belong to the E2-controlled cellular signaling network and are essential for the appearance of the E2-induced cellular effects. This concept requires an "upgrade" to the vision of E2 as a "genotoxic hormone", which balances physiological and detrimental pathways to guarantee human body homeostasis. Deregulation of this equilibrium between cellular pathways would determine the E2 pathological effects.
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Affiliation(s)
- Sara Pescatori
- Department of Sciences, Section Biomedical Sciences, and Technology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146 Rome, Italy; (S.P.); (F.B.); (J.A.); (P.A.); (M.M.)
| | - Francesco Berardinelli
- Department of Sciences, Section Biomedical Sciences, and Technology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146 Rome, Italy; (S.P.); (F.B.); (J.A.); (P.A.); (M.M.)
- Neurodevelopment, Neurogenetics and Molecular Neurobiology Unit, IRCCS Santa Lucia Foundation, 00143 Rome, Italy
| | - Jacopo Albanesi
- Department of Sciences, Section Biomedical Sciences, and Technology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146 Rome, Italy; (S.P.); (F.B.); (J.A.); (P.A.); (M.M.)
| | - Paolo Ascenzi
- Department of Sciences, Section Biomedical Sciences, and Technology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146 Rome, Italy; (S.P.); (F.B.); (J.A.); (P.A.); (M.M.)
- Neuroendocrinology, Metabolism and Neuropharmacology Unit, IRCCS Santa Lucia Foundation, 00143 Rome, Italy
| | - Maria Marino
- Department of Sciences, Section Biomedical Sciences, and Technology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146 Rome, Italy; (S.P.); (F.B.); (J.A.); (P.A.); (M.M.)
- Neuroendocrinology, Metabolism and Neuropharmacology Unit, IRCCS Santa Lucia Foundation, 00143 Rome, Italy
| | - Antonio Antoccia
- Department of Sciences, Section Biomedical Sciences, and Technology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146 Rome, Italy; (S.P.); (F.B.); (J.A.); (P.A.); (M.M.)
| | - Alessandra di Masi
- Department of Sciences, Section Biomedical Sciences, and Technology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146 Rome, Italy; (S.P.); (F.B.); (J.A.); (P.A.); (M.M.)
| | - Filippo Acconcia
- Department of Sciences, Section Biomedical Sciences, and Technology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146 Rome, Italy; (S.P.); (F.B.); (J.A.); (P.A.); (M.M.)
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The Impact of the Deepwater Horizon Oil Spill upon Lung Health-Mouse Model-Based RNA-Seq Analyses. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 2020; 17:ijerph17155466. [PMID: 32751227 PMCID: PMC7432840 DOI: 10.3390/ijerph17155466] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/28/2020] [Revised: 07/22/2020] [Accepted: 07/23/2020] [Indexed: 01/09/2023]
Abstract
We used a transcriptomic approach to interrogate the effects of a saline-accommodated fraction from the Macondo 252 well (MC252) oil and Corexit dispersants on lung tissue. Wild-type C57BL/6 male and female mice were exposed on days 0, 7 and 13 by oropharyngeal aspiration to saline accommodated fractions (SAF) of crude oil from the Macondo (MC252) well, Corexit 9500, Corexit 9527, 9500+oil and 9527+oil or a saline solution as the vehicle control. These treatments did not cause overt toxicity, with the exception of the Corexit exposures which caused brief weight loss after the first exposure. On day 14, total RNA was isolated from the left lung for RNA-seq analyses. KEGG-pathway-based differential expression revealed that Corexit 9527 elicited the strongest changes involving the upregulation of 19 KEGG pathways (FDR < 0.10), followed by Corexit 9500 with the upregulation of seven pathways (FDR < 0.10). As an important signature, pathways related to a response to DNA damage (e.g., p53 signaling and mismatch repair) dominate those upregulated by Corexit 9527 and Corexit 9500. In addition, pro-inflammatory pathways (e.g., cytokine-cytokine receptor interaction, IL-17 signaling pathway and TNF signaling pathways) were upregulated selectively in oil-treated male mice. Surprisingly, oil + dispersant combinations caused lesser effects than the individual treatments at the transcriptomic level. Overall, these findings support potential genotoxicity, inflammation and cell death due to dispersant or oil exposures. Similar exposures to lung tumor bearing K-RasLA1 mice provided evidence for tumor promotion by oil and Corexit dispersant treatments. Our mouse RNA-seq analyses may be relevant to the pulmonary health hazards of MC252 oil and dispersants experienced in exposed populations.
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Ahmed Ezzat H, Price C. Characterisation of unessential genes required for survival under conditions of DNA stress. J Genet Eng Biotechnol 2020; 18:14. [PMID: 32372157 PMCID: PMC7201005 DOI: 10.1186/s43141-020-00025-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2019] [Accepted: 03/11/2020] [Indexed: 11/10/2022]
Abstract
BACKGROUND Genomic instability is a hallmark of cancer. Cancer progression depends on the development and amplification of mutations that alter the cellular response to threats to the genome. This can lead to DNA replication stress and the potential loss of genetic integrity of the newly formed cells. This study utilised fission yeast to map the interactions occurring in some of the most crucial pathways in both DNA replication and checkpoint monitoring involving Rad4, the Schizosaccharomyces pombe (S. pombe) TopBP1 homologue. We have modelled conditions of replication stress in the genetically tractable fission yeast, S. pombe using the hypomorphic rad4-116 allele. Synthetic genetic analysis was used to identify processes required for cell survival under conditions of DNA replication stress. With the aim of mapping the genetic interactions of rad4 and its mutant allele, rad4-116, several genes that could have an interaction with rad4 during replication stress have emerged as attractive. RESULTS Interactions with genes involved in chromatin remodelling, such as hip1, and replication fork stalling resolution, such as mrc1, swi1 and swi3 were explored and confirmed. The interactions of Rad4 with each of the genes provided separate and distinct tumour formation pathways, as evident in the synthetically lethal interactions. Even within the same complex, rad4-116 double mutants behaved differently proving that Rad4 interacts at different levels and functions with the same proteins. CONCLUSION Results from this study provide a novel view of the rad4 interactions, the association of Rad4 with the replisome. The study also provides the groundwork on a theoretical and practical level for the exploration and separation of interactions of TopBP1 with the histone chaperone family and the replisome.
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Affiliation(s)
- Hassan Ahmed Ezzat
- Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, UK.
| | - Clive Price
- Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, UK
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Cassidy KB, Bang S, Kurokawa M, Gerber SA. Direct regulation of Chk1 protein stability by E3 ubiquitin ligase HUWE1. FEBS J 2020; 287:1985-1999. [PMID: 31713291 PMCID: PMC7226928 DOI: 10.1111/febs.15132] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2019] [Revised: 08/19/2019] [Accepted: 11/09/2019] [Indexed: 12/14/2022]
Abstract
The HECT E3 ubiquitin ligase HUWE1 is required for a wide array of important functions in cell biology. Although HUWE1 is known to play a role in DNA damage signaling, the mechanism(s) that underlie this function remain elusive. HUWE1 regulates effectors of DNA replication and genotoxic stress tolerance. However, the loss of HUWE1 can also result in the accrual of significant endogenous DNA damage due to insufficient remediation of replication stress induced by an overabundance of key substrates. We discovered that HUWE1 depletion leads to a significant increase in levels of the single-strand break effector kinase Chk1, independent of the DNA damage response, activation of apical DNA damage repair (DDR) signaling kinases (ATM and ATR), and the tumor suppressor p53. We also identified multiple lysine residues on Chk1 that are polyubiquitinated by HUWE1 in vitro, many of which are within the kinase domain. HUWE1 knockdown also markedly prolonged the protein half-life of Chk1 in steady-state conditions and resulted in greater stabilization of Chk1 protein than depletion of Cul4A, an E3 ubiquitin ligase previously described to control Chk1 abundance. Moreover, prolonged replication stress induced by hydroxyurea or camptothecin resulted in a reduction of Chk1 protein levels, which was rescued by HUWE1 knockdown. Our study indicates that HUWE1 plays a significant role in the regulation of the DDR signaling pathway by directly modulating the abundance of Chk1 protein.
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Affiliation(s)
- Katelyn B. Cassidy
- Department of Molecular & Systems Biology, Geisel School of Medicine, Hanover, NH 03755
| | - Scott Bang
- Department of Biological Sciences, Kent State University, Kent, OH 44242
| | - Manabu Kurokawa
- Department of Molecular & Systems Biology, Geisel School of Medicine, Hanover, NH 03755
- Department of Biological Sciences, Kent State University, Kent, OH 44242
- Norris Cotton Cancer Center, Geisel School of Medicine, Lebanon, NH 03756
| | - Scott A. Gerber
- Department of Molecular & Systems Biology, Geisel School of Medicine, Hanover, NH 03755
- Norris Cotton Cancer Center, Geisel School of Medicine, Lebanon, NH 03756
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Chen Z, Wang C, Lei C, Feng X, Li C, Jung SY, Qin J, Chen J. Phosphoproteomics Analysis Reveals a Potential Role of CHK1 in Regulation of Innate Immunity through IRF3. J Proteome Res 2020; 19:2264-2277. [DOI: 10.1021/acs.jproteome.9b00829] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- Zhen Chen
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United States
| | - Chao Wang
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United States
| | - Caoqi Lei
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United States
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, China
| | - Xu Feng
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United States
| | - Chen Li
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, China
| | - Sung Yun Jung
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, United States
| | - Jun Qin
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, United States
| | - Junjie Chen
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United States
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Feu S, Unzueta F, Llopis A, Semple JI, Ercilla A, Guaita-Esteruelas S, Jaumot M, Freire R, Agell N. OZF is a Claspin-interacting protein essential to maintain the replication fork progression rate under replication stress. FASEB J 2020; 34:6907-6919. [PMID: 32267586 DOI: 10.1096/fj.201901926r] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Revised: 03/10/2020] [Accepted: 03/16/2020] [Indexed: 12/12/2022]
Abstract
DNA replication is essential for cell proliferation and is one of the cell cycle stages where DNA is more vulnerable. Replication stress is a prominent property of tumor cells and an emerging target for cancer therapy. Although it is not directly involved in nucleotide incorporation, Claspin is a protein with relevant functions in DNA replication. It harbors a DNA-binding domain that interacts preferentially with branched or forked DNA molecules. It also acts as a platform for the interaction of proteins related to DNA damage checkpoint activation, DNA repair, DNA replication origin firing, and fork progression. In order to find new proteins potentially involved in the regulation of DNA replication, we performed a two-hybrid screen to discover new Claspin-binding proteins. This system allowed us to identify the zinc-finger protein OZF (ZNF146) as a new Claspin-interacting protein. OZF is also present at replication forks and co-immunoprecipitates not only with Claspin but also with other replisome components. Interestingly, OZF depletion does not affect DNA replication in a normal cell cycle, but its depletion induces a reduction in the fork progression rate under replication stress conditions. Our results suggest that OZF is a Claspin-binding protein with a specific function in fork progression under replication stress.
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Affiliation(s)
- Sonia Feu
- Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain
| | - Fernando Unzueta
- Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain
| | - Alba Llopis
- Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain
| | | | - Amaia Ercilla
- Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain
| | - Sandra Guaita-Esteruelas
- Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain
| | - Montserrat Jaumot
- Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain
| | - Raimundo Freire
- Unidad de Investigación, Hospital Universitario de Canarias, FIISC, La Laguna, Spain.,Instituto de Tecnologías Biomédicas, Universidad de La Laguna, La Laguna, Spain.,Facultad de Ciencias de la Salud, Universidad Fernando Pessoa Canarias, Las Palmas de Gran Canaria, Spain
| | - Neus Agell
- Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain
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25
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Posttranscriptional control of the replication stress response via TTP-mediated Claspin mRNA stabilization. Oncogene 2020; 39:3245-3257. [PMID: 32086441 DOI: 10.1038/s41388-020-1220-9] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2019] [Revised: 02/09/2020] [Accepted: 02/11/2020] [Indexed: 11/08/2022]
Abstract
ATR and CHK1 play key roles in the protection and recovery of the stalled replication forks. Claspin, an adaptor for CHK1 activation, is essential for DNA damage signaling and efficient replication fork progression. Here, we show that tristetraprolin (TTP), an mRNA-binding protein, can modulate the replication stress response via stabilization of Claspin mRNA. TTP depletion compromised specifically in the phosphorylation of CHK1, but not p53 or H2AX among other ATR substrates, and produced CHK1-defective replication phenotypes including accumulation of stalled replication forks. Importantly, the expression of siRNA-resistant TTP in TTP-deficient cells restored CHK1 phosphorylation and reduced the number of stalled replication forks as close to the control cells. Besides, we found that TTP was required for efficient replication fork progression even in the absence of exogenous DNA damage in a Claspin-dependent manner. Mechanistically, TTP was able to bind to the 3'-untranslated region of Claspin mRNA to increase the stability of Claspin mRNA which eventually contributed to the subsequent ATR-CHK1 activation upon DNA damage. Taken together, our results revealed an intimate link between TTP-dependent Claspin mRNA stability and ATR-CHK1-dependent replication fork stability to maintain replication fork integrity and chromosomal stability.
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26
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Huang X, Cheng K, Liu L, Hu X, Gao X, Li H, Xu F, Li Z, Hua H, Li D. Design, synthesis and apoptosis-related antiproliferative activities of chelidonine derivatives. Bioorg Med Chem Lett 2020; 30:126913. [PMID: 31883693 DOI: 10.1016/j.bmcl.2019.126913] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2019] [Revised: 12/11/2019] [Accepted: 12/16/2019] [Indexed: 11/25/2022]
Abstract
To get chelidonine derivatives with enhanced antiproliferative activity and selectivity, a series of nitric oxide donating derivatives (10a-f and 11a-j) were designed, synthesized and biologically evaluated. Compared with chelidonine, these compounds exhibited lower IC50 values against human hepatoma cells HepG2, breast cancer cells MCF-7, colon cancer cells HCT-116, as well as leukemia cells K562. Compound 11j displayed the strongest antiproliferative activity with IC50 values of 3.91, 6.90, 4.36 and 1.12 μM against the above four cells, respectively. Nevertheless, it showed an IC50 value >40 μM against human peripheral blood mononuclear cells (PBMCs), which demonstrated high selectivity between normal and cancer blood cells. In further mechanism studies, 11j showed the capability to induce K562 cells apoptosis, S phase cell cycle arrest and mitochondrial membrane potential disorder. Besides, 11j was found to be effective in promoting the expression of proapoptotic protein Bad and suppressing the expression of anti-apoptotic proteins Bcl-xL, catalase, survivin, claspin and clusterin.
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Affiliation(s)
- Xueyan Huang
- Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
| | - Keguang Cheng
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, and School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Raod, Guilin 541004, PR China
| | - Lilin Liu
- Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
| | - Xu Hu
- Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
| | - Xiang Gao
- Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
| | - Haonan Li
- Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
| | - Fanxing Xu
- Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China.
| | - Zhanlin Li
- Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
| | - Huiming Hua
- Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China.
| | - Dahong Li
- Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China.
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27
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Marabitti V, Lillo G, Malacaria E, Palermo V, Pichierri P, Franchitto A. Checkpoint Defects Elicit a WRNIP1-Mediated Response to Counteract R-Loop-Associated Genomic Instability. Cancers (Basel) 2020; 12:cancers12020389. [PMID: 32046194 PMCID: PMC7072626 DOI: 10.3390/cancers12020389] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2020] [Revised: 02/03/2020] [Accepted: 02/05/2020] [Indexed: 12/04/2022] Open
Abstract
Conflicts between replication and transcription are a common source of genomic instability, a characteristic of almost all human cancers. Aberrant R-loops can cause a block to replication fork progression. A growing number of factors are involved in the resolution of these harmful structures and many perhaps are still unknown. Here, we reveal that the Werner interacting protein 1 (WRNIP1)-mediated response is implicated in counteracting aberrant R-loop accumulation. Using human cellular models with compromised Ataxia-Telangiectasia and Rad3-Related (ATR)-dependent checkpoint activation, we show that WRNIP1 is stabilized in chromatin and is needed for maintaining genome integrity by mediating the Ataxia Telangiectasia Mutated (ATM)-dependent phosphorylation of Checkpoint kinase 1 (CHK1). Furthermore, we demonstrated that loss of Werner Syndrome protein (WRN) or ATR signaling leads to formation of R-loop-dependent parental ssDNA upon mild replication stress, which is covered by Radiorestistance protein 51 (RAD51). We prove that Werner helicase-interacting protein 1 (WRNIP1) chromatin retention is also required to stabilize the association of RAD51 with ssDNA in proximity of R-loops. Therefore, in these pathological contexts, ATM inhibition or WRNIP1 abrogation is accompanied by increased levels of genomic instability. Overall, our findings suggest a novel function for WRNIP1 in preventing R-loop-driven genome instability, providing new clues to understand the way replication–transcription conflicts are handled.
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28
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Kobayashi G, Sentani K, Babasaki T, Sekino Y, Shigematsu Y, Hayashi T, Oue N, Teishima J, Matsubara A, Sasaki N, Yasui W. Claspin overexpression is associated with high-grade histology and poor prognosis in renal cell carcinoma. Cancer Sci 2020; 111:1020-1027. [PMID: 31912588 PMCID: PMC7060467 DOI: 10.1111/cas.14299] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Revised: 12/09/2019] [Accepted: 12/27/2019] [Indexed: 12/27/2022] Open
Abstract
Renal cell carcinoma (RCC) is one of the most common human cancers. We previously reported that claspin is a key regulator in the progression of gastric cancer, and it likely plays an important role in cancer stem cells of gastric cancer. However, the significance of claspin in RCC has not been examined. First, we analyzed the expression and distribution of claspin in 95 RCC cases by immunohistochemistry. In the nonneoplastic kidney, the staining of claspin was either weak or absent, whereas RCC tissue showed nuclear staining. In total, claspin expression was detected in 45 (47%) of 95 RCC cases. The claspin staining appeared relatively stronger in high nuclear grade RCC than in low nuclear grade RCC. Claspin-positive RCC cases were associated with higher T grade, tumor stage, nuclear grade, vein invasion, and poorer prognosis. CLSPN siRNA treatment decreased RCC cell proliferation. The levels of phosphorylated Erk and Akt were lower in CLSPN siRNA-transfected RCC cells than in control cells. In addition, claspin was coexpressed with CD44, epidermal growth factor receptor, p53, and programmed death ligand-1. These results suggest that claspin plays an important role in tumor progression in RCC and might be a prognostic marker and novel therapeutic target molecule.
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Affiliation(s)
- Go Kobayashi
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Department of Pathology, Federation of National Public Service Personnel Mutual Aid Associations, Kure-Kyosai Hospital, Hiroshima, Japan
| | - Kazuhiro Sentani
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Takashi Babasaki
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Department of Urology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Yohei Sekino
- Department of Urology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Yoshinori Shigematsu
- Department of Urology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Tetsutaro Hayashi
- Department of Urology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Naohide Oue
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Jun Teishima
- Department of Urology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Akio Matsubara
- Department of Urology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Naomi Sasaki
- Department of Pathology, Federation of National Public Service Personnel Mutual Aid Associations, Kure-Kyosai Hospital, Hiroshima, Japan
| | - Wataru Yasui
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
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29
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Yang CC, Kato H, Shindo M, Masai H. Cdc7 activates replication checkpoint by phosphorylating the Chk1-binding domain of Claspin in human cells. eLife 2019; 8:50796. [PMID: 31889509 PMCID: PMC6996922 DOI: 10.7554/elife.50796] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2019] [Accepted: 12/30/2019] [Indexed: 01/05/2023] Open
Abstract
Replication checkpoint is essential for maintaining genome integrity in response to various replication stresses as well as during the normal growth. The evolutionally conserved ATR-Claspin-Chk1 pathway is induced during replication checkpoint activation. Cdc7 kinase, required for initiation of DNA replication at replication origins, has been implicated in checkpoint activation but how it is involved in this pathway has not been known. Here, we show that Cdc7 is required for Claspin-Chk1 interaction in human cancer cells by phosphorylating CKBD (Chk1-binding-domain) of Claspin. The residual Chk1 activation in Cdc7-depleted cells is lost upon further depletion of casein kinase1 (CK1γ1), previously reported to phosphorylate CKBD. Thus, Cdc7, in conjunction with CK1γ1, facilitates the interaction between Claspin and Chk1 through phosphorylating CKBD. We also show that, whereas Cdc7 is predominantly responsible for CKBD phosphorylation in cancer cells, CK1γ1 plays a major role in non-cancer cells, providing rationale for targeting Cdc7 for cancer cell-specific cell killing.
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Affiliation(s)
- Chi-Chun Yang
- Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Hiroyuki Kato
- Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Mayumi Shindo
- Protein Analyses Laboratory, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Hisao Masai
- Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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30
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Charman M, Herrmann C, Weitzman MD. Viral and cellular interactions during adenovirus DNA replication. FEBS Lett 2019; 593:3531-3550. [PMID: 31764999 DOI: 10.1002/1873-3468.13695] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2019] [Revised: 11/14/2019] [Accepted: 11/15/2019] [Indexed: 12/30/2022]
Abstract
Adenoviruses represent ubiquitous and clinically significant human pathogens, gene-delivery vectors, and oncolytic agents. The study of adenovirus-infected cells has long been used as an excellent model to investigate fundamental aspects of both DNA virus infection and cellular biology. While many key details supporting a well-established model of adenovirus replication have been elucidated over a period spanning several decades, more recent findings suggest that we have only started to appreciate the complex interplay between viral genome replication and cellular processes. Here, we present a concise overview of adenovirus DNA replication, including the biochemical process of replication, the spatial organization of replication within the host cell nucleus, and insights into the complex plethora of virus-host interactions that influence viral genome replication. Finally, we identify emerging areas of research relating to the replication of adenovirus genomes.
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Affiliation(s)
- Matthew Charman
- Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.,Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
| | - Christin Herrmann
- Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.,Cell and Molecular Biology Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
| | - Matthew D Weitzman
- Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.,Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
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31
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Rai R, Gu P, Broton C, Kumar-Sinha C, Chen Y, Chang S. The Replisome Mediates A-NHEJ Repair of Telomeres Lacking POT1-TPP1 Independently of MRN Function. Cell Rep 2019; 29:3708-3725.e5. [PMID: 31825846 PMCID: PMC7001145 DOI: 10.1016/j.celrep.2019.11.012] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2019] [Revised: 09/22/2019] [Accepted: 11/04/2019] [Indexed: 02/07/2023] Open
Abstract
Telomeres use shelterin to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), repressing ataxia-telangiectasia, mutated (ATM) and ATM and Rad3-related (ATR) dependent DNA damage checkpoint responses. The MRE11 nuclease is thought to be essential for the resection of the 5' C-strand to generate the microhomologies necessary for alternative non-homologous end joining (A-NHEJ) repair. In the present study, we uncover DNA damage signaling and repair pathways engaged by components of the replisome complex to repair dysfunctional telomeres. In cells lacking MRN, single-stranded telomeric overhangs devoid of POT1-TPP1 do not recruit replication protein A (RPA), ATR-interacting protein (ATRIP), and RAD 51. Rather, components of the replisome complex, including Claspin, Proliferating cell nuclear antigen (PCNA), and Downstream neighbor of SON (DONSON), initiate DNA-PKcs-mediated p-CHK1 activation and A-NHEJ repair. In addition, Claspin directly interacts with TRF2 and recruits EXO1 to newly replicated telomeres to promote 5' end resection. Our data indicate that MRN is dispensable for the repair of dysfunctional telomeres lacking POT1-TPP1 and highlight the contributions of the replisome in telomere repair.
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Affiliation(s)
- Rekha Rai
- Departments of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA.
| | - Peili Gu
- Departments of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA
| | - Cayla Broton
- Departments of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA; Tri-Institutional MD/PhD Program, Weill Cornell Medical College, New York, NY 10065, USA
| | - Chandan Kumar-Sinha
- Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA
| | - Yong Chen
- National Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China
| | - Sandy Chang
- Departments of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA; Department of Pathology, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA; Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520, USA.
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32
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Li F, Chen J, Leier A, Marquez-Lago T, Liu Q, Wang Y, Revote J, Smith AI, Akutsu T, Webb GI, Kurgan L, Song J. DeepCleave: a deep learning predictor for caspase and matrix metalloprotease substrates and cleavage sites. Bioinformatics 2019; 36:1057-1065. [PMID: 31566664 PMCID: PMC8215920 DOI: 10.1093/bioinformatics/btz721] [Citation(s) in RCA: 78] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2019] [Revised: 08/13/2019] [Accepted: 09/25/2019] [Indexed: 01/31/2023] Open
Abstract
MOTIVATION Proteases are enzymes that cleave target substrate proteins by catalyzing the hydrolysis of peptide bonds between specific amino acids. While the functional proteolysis regulated by proteases plays a central role in the 'life and death' cellular processes, many of the corresponding substrates and their cleavage sites were not found yet. Availability of accurate predictors of the substrates and cleavage sites would facilitate understanding of proteases' functions and physiological roles. Deep learning is a promising approach for the development of accurate predictors of substrate cleavage events. RESULTS We propose DeepCleave, the first deep learning-based predictor of protease-specific substrates and cleavage sites. DeepCleave uses protein substrate sequence data as input and employs convolutional neural networks with transfer learning to train accurate predictive models. High predictive performance of our models stems from the use of high-quality cleavage site features extracted from the substrate sequences through the deep learning process, and the application of transfer learning, multiple kernels and attention layer in the design of the deep network. Empirical tests against several related state-of-the-art methods demonstrate that DeepCleave outperforms these methods in predicting caspase and matrix metalloprotease substrate-cleavage sites. AVAILABILITY AND IMPLEMENTATION The DeepCleave webserver and source code are freely available at http://deepcleave.erc.monash.edu/. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.
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Affiliation(s)
| | | | - André Leier
- Department of Genetics, USA,Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Tatiana Marquez-Lago
- Department of Genetics, USA,Department of Cell, Developmental and Integrative Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Quanzhong Liu
- College of Information Engineering, Northwest A&F University, Yangling 712100, China
| | - Yanze Wang
- College of Information Engineering, Northwest A&F University, Yangling 712100, China
| | - Jerico Revote
- Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia
| | - A Ian Smith
- Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia
| | - Tatsuya Akutsu
- Bioinformatics Center, Institute for Chemical Research, Kyoto University, Kyoto 611-0011, Japan
| | - Geoffrey I Webb
- Monash Centre for Data Science, Faculty of Information Technology, Monash University, Melbourne, VIC 3800, Australia
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Yan Y, Lu Y, Mao K, Zhang M, Liu H, Zhou Q, Lin J, Zhang J, Wang J, Xiao Z. Identification and validation of a prognostic four-genes signature for hepatocellular carcinoma: integrated ceRNA network analysis. Hepatol Int 2019; 13:618-630. [PMID: 31321712 PMCID: PMC6744548 DOI: 10.1007/s12072-019-09962-3] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/20/2019] [Accepted: 06/14/2019] [Indexed: 12/24/2022]
Abstract
BACKGROUND Hepatocellular carcinoma (HCC) is one of the most aggressive malignant tumors, with a poor long-term prognosis worldwide. The functional deregulations of global transcriptome were associated with the genesis and development of HCC, but lacks systematic research and validation. METHODS A total of 519 postoperative HCC patients were included. We built an interactive and visual competing endogenous RNA network. The prognostic signature was established with the least absolute shrinkage and selection operator algorithm. Multivariate Cox regression analysis was used to screen for independent prognostic factors for HCC overall survival. RESULTS In the training set, we identified a four-gene signature (PBK, CBX2, CLSPN, and CPEB3) and effectively predicted the overall survival. The survival times of patients in the high-score group were worse than those in the low-score group (p = 0.0004), and death was also more likely in the high-score group (HR 2.444, p < 0.001). The results were validated in internal validation set (p = 0.0057) and two external validation cohorts (HR 2.467 and 2.6). The signature (AUCs of 1, 2, 3 years were 0.716, 0.726, 0.714, respectively) showed high prognostic accuracy in the complete TCGA cohort. CONCLUSIONS In conclusion, we successfully built a more extensive ceRNA network for HCC and then identified a four-gene-based signature, enabling prediction of the overall survival of patients with HCC.
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Affiliation(s)
- Yongcong Yan
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China.,Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Yanjiang West Road 107#, Guangzhou, 510120, China.,RNA Biomedical Institute, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China
| | - Yingjuan Lu
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China.,Department of Oral and Maxillofacial Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China.,RNA Biomedical Institute, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China
| | - Kai Mao
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China.,Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Yanjiang West Road 107#, Guangzhou, 510120, China.,RNA Biomedical Institute, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China
| | - Mengyu Zhang
- Department of Gastroenterology and Hepatology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 510120, China
| | - Haohan Liu
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China.,Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Yanjiang West Road 107#, Guangzhou, 510120, China.,RNA Biomedical Institute, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China
| | - Qianlei Zhou
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China.,Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Yanjiang West Road 107#, Guangzhou, 510120, China.,RNA Biomedical Institute, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China
| | - Jianhong Lin
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China.,Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Yanjiang West Road 107#, Guangzhou, 510120, China.,RNA Biomedical Institute, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China
| | - Jianlong Zhang
- Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Yanjiang West Road 107#, Guangzhou, 510120, China
| | - Jie Wang
- Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Yanjiang West Road 107#, Guangzhou, 510120, China.
| | - Zhiyu Xiao
- Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Yanjiang West Road 107#, Guangzhou, 510120, China.
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de Boussac H, Bruyer A, Jourdan M, Maes A, Robert N, Gourzones C, Vincent L, Seckinger A, Cartron G, Hose D, De Bruyne E, Kassambara A, Pasero P, Moreaux J. Kinome expression profiling to target new therapeutic avenues in multiple myeloma. Haematologica 2019; 105:784-795. [PMID: 31289205 PMCID: PMC7049359 DOI: 10.3324/haematol.2018.208306] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2018] [Accepted: 07/05/2019] [Indexed: 12/14/2022] Open
Abstract
Multiple myeloma (MM) account for approximately 10% of hematological malignancies and is the second most common hematological disorder. Kinases inhibitors are widely used and their efficiency for the treatment of cancers has been demonstrated. Here, in order to identify kinases of potential therapeutic interest for the treatment of MM, we investigated the prognostic impact of the kinome expression profile in large cohorts of patients. We identified 36 kinome-related genes significantly linked with a prognostic value to MM, and built a kinome index based on their expression. The Kinome Index (KI) is linked to prognosis, proliferation, differentiation, and relapse in MM. We then tested inhibitors targeting seven of the identified protein kinas-es (PBK, SRPK1, CDC7-DBF4, MELK, CHK1, PLK4, MPS1/TTK) in human myeloma cell lines. All tested inhibitors significantly reduced the viability of myeloma cell lines, and we confirmed the potential clinical interest of three of them on primary myeloma cells from patients. In addition, we demonstrated their ability to potentialize the toxicity of conventional treatments, including Melphalan and Lenalidomide. This highlights their potential beneficial effect in myeloma therapy. Three kinases inhibitors (CHK1i, MELKi and PBKi) overcome resistance to Lenalidomide, while CHK1, PBK and DBF4 inhibitors re-sensitize Melphalan resistant cell line to this conventional therapeutic agent. Altogether, we demonstrate that kinase inhibitors could be of therapeutic interest especially in high-risk myeloma patients defined by the KI. CHEK1, MELK, PLK4, SRPK1, CDC7-DBF4, MPS1/TTK and PBK inhibitors could represent new treatment options either alone or in combination with Melphalan or IMiD for refractory/relapsing myeloma patients.
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Affiliation(s)
| | | | - Michel Jourdan
- IGH, CNRS, Université de Montpellier, Montpellier, France
| | - Anke Maes
- Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel, Brussels, Belgium
| | - Nicolas Robert
- CHU Montpellier, Laboratory for Monitoring Innovative Therapies, Department of Biological Hematology, Montpellier, France
| | | | - Laure Vincent
- CHU Montpellier, Department of Clinical Hematology, Montpellier, France
| | - Anja Seckinger
- Medizinische Klinik und Poliklinik V, Universitätsklinikum Heidelberg, Heidelberg, Germany.,Nationales Centrum für Tumorerkrankungen, Heidelberg , Germany
| | - Guillaume Cartron
- CHU Montpellier, Department of Clinical Hematology, Montpellier, France.,Université de Montpellier, UMR CNRS 5235, Montpellier, France.,Université de Montpellier, UFR de Médecine, Montpellier, France
| | - Dirk Hose
- Medizinische Klinik und Poliklinik V, Universitätsklinikum Heidelberg, Heidelberg, Germany.,Nationales Centrum für Tumorerkrankungen, Heidelberg , Germany
| | - Elke De Bruyne
- Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel, Brussels, Belgium
| | | | | | - Jérôme Moreaux
- IGH, CNRS, Université de Montpellier, Montpellier, France .,CHU Montpellier, Laboratory for Monitoring Innovative Therapies, Department of Biological Hematology, Montpellier, France.,Université de Montpellier, UFR de Médecine, Montpellier, France
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35
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Sezer ED, Oktay LM, Karadadaş E, Memmedov H, Selvi Gunel N, Sözmen E. Assessing Anticancer Potential of Blueberry Flavonoids, Quercetin, Kaempferol, and Gentisic Acid, Through Oxidative Stress and Apoptosis Parameters on HCT-116 Cells. J Med Food 2019; 22:1118-1126. [PMID: 31241392 DOI: 10.1089/jmf.2019.0098] [Citation(s) in RCA: 53] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
In recent years, natural products gained popularity with their anti-inflammatory and antioxidant effects mediated by chemical compounds within their composition. Study results offering them as palliative therapy options in cancer or as anticancer agents with high levels of cytotoxicity brought a new approach to combine cancer treatment protocols with these products. From a different perspective, edible types of these products are suggested in daily diets due to their potential cancer preventive effects. Our preliminary work was on blueberry extracts (Vaccinium myrtillus) as a main representative of these natural products, and the contents of the extracts were analyzed with liquid chromatography tandem mass spectrometry (LC MS/MS) to reveal the composition and distribution of polyphenolic compounds within. The most abundant polyphenols detected in V. myrtillus extracts were quercetin, kaempferol, and a phenolic acid, gentisic acid (GA). The compounds were further evaluated on treated HCT-116 cells for their potential anticancer effects by measuring total antioxidant status, total oxidant status, and 8-hydroxydeoxyguanosine levels for evaluation of oxidative stress and through protein array analysis and flow cytometric analysis for evaluation of apoptosis. In analysis of oxidative stress parameters, reduced total oxidant levels and reduced oxidative stress index levels were found in cells treated with the compounds in comparison with untreated cells. In apoptosis-related protein profiles, at least twofold reduction in various apoptotic proteins was observed after quercetin and kaempferol treatment, whereas a different profile was observed for GA. Overall, results of this study showed that quercetin and kaempferol have strong cytotoxic, antioxidant, and apoptotic effects, although GA is mostly effective as an antioxidant polyphenol on HCT-116 cells.
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Affiliation(s)
- Ebru Demirel Sezer
- Department of Medical Biochemistry, Faculty of Medicine, Ege University, Bornova, Turkey
| | - Latife Merve Oktay
- Department of Medical Biology, Faculty of Medicine, Ege University, Bornova, Turkey
| | - Elif Karadadaş
- Department of Medical Biochemistry, Faculty of Medicine, Ege University, Bornova, Turkey
| | - Hikmet Memmedov
- Department of Medical Biochemistry, Faculty of Medicine, Ege University, Bornova, Turkey
| | - Nur Selvi Gunel
- Department of Medical Biology, Faculty of Medicine, Ege University, Bornova, Turkey
| | - Eser Sözmen
- Department of Medical Biochemistry, Faculty of Medicine, Ege University, Bornova, Turkey
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36
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Thutkawkorapin J, Lindblom A, Tham E. Exome sequencing in 51 early onset non-familial CRC cases. Mol Genet Genomic Med 2019; 7:e605. [PMID: 30809968 PMCID: PMC6503031 DOI: 10.1002/mgg3.605] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2018] [Revised: 12/22/2018] [Accepted: 01/16/2019] [Indexed: 12/12/2022] Open
Abstract
Background Colorectal cancer (CRC) cases with an age of onset <40 years suggests a germline genetic cause. In total, 51 simplex cases were included to test the hypothesis of CRC as a mendelian trait caused by either heterozygous autosomal dominant or bi‐allelic autosomal recessive pathogenic variants. Methods The cohort was whole exome sequenced (WES) at 100× coverage. Both a dominant‐ and recessive model were used for searching predisposing genetic factors. In addition, we assayed recessive variants of potential moderate risk that were enriched in our young‐onset CRC cohort. Variants were filtered using a candidate cancer gene list or by selecting variants more likely to be pathogenic based on variant type (e.g., loss‐of‐function) or allele frequency. Results We identified one pathogenic variant in PTEN in a patient subsequently confirmed to have a hereditary hamartoma tumor syndrome (Cowden syndrome) and one patient with a pathogenic heterozygous variant in PMS2 that was originally not identified by WES due to low quality reads resulting from pseudogenes. In addition, we identified three heterozygous candidate missense variants in known cancer susceptibility genes (BMPR1A,BRIP1, and SRC), three truncating variants in possibly novel cancer genes (CLSPN,SEC24B, SSH2) and four candidate missense variants in ACACA, NR2C2, INPP4A, and DIDO1. We also identify five possible autosomal recessive candidate genes: ATP10B,PKHD1,UGGT2,MYH13,TFF3. Conclusion Two clear pathogenic variants were identified in patients that had not been identified clinically. Thus, the chance of detecting a hereditary cancer syndrome in patients with CRC at young age but without family history is 2/51 (4%) and therefore the clinical benefit of genetic testing in this patient group is low. Of note, using stringent filtering, we have identified a total of ten candidate heterozygous variants and five possibly biallelic autosomal recessive candidate genes that warrant further study.
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Affiliation(s)
| | - Annika Lindblom
- Department of Molecular Medicine and Surgery, Karolinska Institutet and Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden
| | - Emma Tham
- Department of Molecular Medicine and Surgery, Karolinska Institutet and Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden
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37
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Bianco JN, Bergoglio V, Lin YL, Pillaire MJ, Schmitz AL, Gilhodes J, Lusque A, Mazières J, Lacroix-Triki M, Roumeliotis TI, Choudhary J, Moreaux J, Hoffmann JS, Tourrière H, Pasero P. Overexpression of Claspin and Timeless protects cancer cells from replication stress in a checkpoint-independent manner. Nat Commun 2019; 10:910. [PMID: 30796221 PMCID: PMC6385232 DOI: 10.1038/s41467-019-08886-8] [Citation(s) in RCA: 97] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2017] [Accepted: 02/05/2019] [Indexed: 12/31/2022] Open
Abstract
Oncogene-induced replication stress (RS) promotes cancer development but also impedes tumor growth by activating anti-cancer barriers. To determine how cancer cells adapt to RS, we have monitored the expression of different components of the ATR-CHK1 pathway in primary tumor samples. We show that unlike upstream components of the pathway, the checkpoint mediators Claspin and Timeless are overexpressed in a coordinated manner. Remarkably, reducing the levels of Claspin and Timeless in HCT116 cells to pretumoral levels impeded fork progression without affecting checkpoint signaling. These data indicate that high level of Claspin and Timeless increase RS tolerance by protecting replication forks in cancer cells. Moreover, we report that primary fibroblasts adapt to oncogene-induced RS by spontaneously overexpressing Claspin and Timeless, independently of ATR signaling. Altogether, these data indicate that enhanced levels of Claspin and Timeless represent a gain of function that protects cancer cells from of oncogene-induced RS in a checkpoint-independent manner.
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Affiliation(s)
- Julien N Bianco
- Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labellisée Ligue Contre le Cancer, 34396, Montpellier, France.,Cologne Excellence Cluster for Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, Joseph-Stelzmann-Str. 26, 50931, Cologne, Germany
| | - Valérie Bergoglio
- Cancer Research Center of Toulouse, INSERM U1037, CNRS ERL5294, University of Toulouse 3, 31037, Toulouse, France
| | - Yea-Lih Lin
- Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labellisée Ligue Contre le Cancer, 34396, Montpellier, France
| | - Marie-Jeanne Pillaire
- Cancer Research Center of Toulouse, INSERM U1037, CNRS ERL5294, University of Toulouse 3, 31037, Toulouse, France
| | - Anne-Lyne Schmitz
- Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labellisée Ligue Contre le Cancer, 34396, Montpellier, France
| | - Julia Gilhodes
- Clinical trials Office - Biostatistics Unit, Institute Claudius Regaud, Institute Universitaire du Cancer Toulouse-Oncopole (IUCT-O), 31100, Toulouse, France
| | - Amelie Lusque
- Clinical trials Office - Biostatistics Unit, Institute Claudius Regaud, Institute Universitaire du Cancer Toulouse-Oncopole (IUCT-O), 31100, Toulouse, France
| | - Julien Mazières
- Thoracic Oncology Department, Toulouse University Hospital, University Paul Sabatier, 31062, Toulouse, France
| | | | | | | | - Jérôme Moreaux
- Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labellisée Ligue Contre le Cancer, 34396, Montpellier, France
| | - Jean-Sébastien Hoffmann
- Cancer Research Center of Toulouse, INSERM U1037, CNRS ERL5294, University of Toulouse 3, 31037, Toulouse, France
| | - Hélène Tourrière
- Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labellisée Ligue Contre le Cancer, 34396, Montpellier, France.
| | - Philippe Pasero
- Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labellisée Ligue Contre le Cancer, 34396, Montpellier, France.
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38
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Narayan S, Ramisetti S, Jaiswal AS, Law BK, Singh-Pillay A, Singh P, Amin S, Sharma AK. ASR352, A potent anticancer agent: Synthesis, preliminary SAR, and biological activities against colorectal cancer bulk, 5-fluorouracil/oxaliplatin resistant and stem cells. Eur J Med Chem 2019; 161:456-467. [PMID: 30384048 PMCID: PMC7115410 DOI: 10.1016/j.ejmech.2018.10.052] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2018] [Revised: 10/07/2018] [Accepted: 10/22/2018] [Indexed: 12/21/2022]
Abstract
Despite new agent development and short-term benefits in patients with colorectal cancer (CRC), metastatic CRC cure rates have not improved due to high rates of 5-fluorouracil (5-FU)/leucovorin/oxaliplatin (FOLFOX)-resistance and a clinical therapeutic plateau. At the same time, this treatment regime leads to significant toxicity, cost, and patient inconvenience. Drug-resistance is linked to CRC stem cells, which are associated with the epidermal-to-mesenchymal transition (EMT) pathway. Thus, to optimally treat CRC, a therapy that can target the cell survival and EMT pathways in both CRC bulk and stem cell populations is critical. We recently identified a novel small molecule NSC30049 (7a) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC bulk, FOLFOX-resistant, and CRC stem cells both in vitro and in vivo models. In the present study, we report the synthesis and anti-CRC evaluation of several stable and effective 7a analogs. ASR352 (7b) was identified as one of the equipotent 7a analogs that inhibited the growth of CRC bulk cells, sensitized FOLFOX-resistant cells, and reduced the sphere formation capacity of CRC stem cells. It appears that the complex mechanism of cytotoxicity for 7b includes abrogation of 5-FU-induced the S phase, reduction of the phosphorylation of Chk1 at S317P, S345P and S296P, increased γH2AX staining, activation of caspase 3/PARP1 cleavage, and enhancement of Bax/Bcl2 ratio. Further 7b-mediated reduced phosphorylation of Chk1 was an indirect effect, since it did not inhibit Chk1 activity in an in vitro kinase assay. Our findings suggest that 7b as a single agent, or in combination with 5-FU can be developed as a therapeutic agent in CRC bulk, FOLFOX-resistant, and CRC stem cell populations for unmanageable metastatic CRC conditions.
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Affiliation(s)
- Satya Narayan
- Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL, 32610, USA.
| | - Srinivasa Ramisetti
- Department of Pharmacology, Penn State University College of Medicine, Penn State Cancer Institute, Hershey, PA, 17033, USA
| | - Aruna S Jaiswal
- Department of Hematology and Oncology, University of Florida, Gainesville, FL, 32610, USA
| | - Brian K Law
- Department of Pharmacology and Experimental Therapeutics, University of Florida, Gainesville, FL, 32610, USA
| | - Ashona Singh-Pillay
- School of Chemistry and Physics, University of Kwa-Zulu Natal (UKZN), Westville Campus, Durban, 4000, South Africa
| | - Parvesh Singh
- School of Chemistry and Physics, University of Kwa-Zulu Natal (UKZN), Westville Campus, Durban, 4000, South Africa
| | - Shantu Amin
- Department of Pharmacology, Penn State University College of Medicine, Penn State Cancer Institute, Hershey, PA, 17033, USA
| | - Arun K Sharma
- Department of Pharmacology, Penn State University College of Medicine, Penn State Cancer Institute, Hershey, PA, 17033, USA.
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Azenha D, Lopes MC, Martins TC. Claspin: From replication stress and DNA damage responses to cancer therapy. DNA Repair (Amst) 2019; 115:203-246. [DOI: 10.1016/bs.apcsb.2018.10.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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40
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Kobayashi G, Sentani K, Hattori T, Yamamoto Y, Imai T, Sakamoto N, Kuraoka K, Oue N, Sasaki N, Taniyama K, Yasui W. Clinicopathological significance of claspin overexpression and its association with spheroid formation in gastric cancer. Hum Pathol 2018; 84:8-17. [PMID: 30240769 DOI: 10.1016/j.humpath.2018.09.001] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2018] [Revised: 08/31/2018] [Accepted: 09/06/2018] [Indexed: 01/06/2023]
Abstract
Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. Spheroid colony formation is a useful method to identify cancer stem cells (CSCs). The aim of this study was to identify a novel prognostic marker or therapeutic target for GC using a method to identify CSCs. We analyzed the microarray data in spheroid body-forming and parental cells and focused on the CLSPN gene because it is overexpressed in the spheroid body-forming cells in both the GC cell lines MKN-45 and MKN-74. Quantitative reverse-transcription polymerase chain reaction analysis revealed that CLSPN messenger RNA expression was up-regulated in GC cell lines MKN-45, MKN-74, and TMK-1. Immunohistochemistry of claspin showed that 94 (47%) of 203 GC cases were positive. Claspin-positive GC cases were associated with higher T and N grades, tumor stage, lymphatic invasion, and poor prognosis. In addition, claspin expression was coexpressed with CD44, human epidermal growth factor receptor type 2, and p53. CLSPN small interfering RNA treatment decreased GC cell proliferation and invasion. These results indicate that the expression of claspin might be a key regulator in the progression of GC and might play an important role in CSCs of GC.
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Affiliation(s)
- Go Kobayashi
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8551 Japan; Department of Pathology, Kure-Kyosai Hospital, Federation of National Public Service Personnel Mutual Aid Associations, Hiroshima, 737-8505 Japan
| | - Kazuhiro Sentani
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8551 Japan.
| | - Takuya Hattori
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8551 Japan
| | - Yuji Yamamoto
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8551 Japan
| | - Takeharu Imai
- Department of Surgical Oncology, Graduate School of Medicine, Gifu University, Gifu, 501-1194 Japan
| | - Naoya Sakamoto
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8551 Japan
| | - Kazuya Kuraoka
- Department of Pathology, National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Kure-City, Hiroshima, 737-0023 Japan
| | - Naohide Oue
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8551 Japan
| | - Naomi Sasaki
- Department of Pathology, Kure-Kyosai Hospital, Federation of National Public Service Personnel Mutual Aid Associations, Hiroshima, 737-8505 Japan
| | - Kiyomi Taniyama
- Department of Pathology, National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Kure-City, Hiroshima, 737-0023 Japan
| | - Wataru Yasui
- Department of Molecular Pathology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8551 Japan
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41
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Smits VAJ, Cabrera E, Freire R, Gillespie DA. Claspin – checkpoint adaptor and
DNA
replication factor. FEBS J 2018; 286:441-455. [DOI: 10.1111/febs.14594] [Citation(s) in RCA: 47] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2018] [Revised: 06/13/2018] [Accepted: 06/20/2018] [Indexed: 12/16/2022]
Affiliation(s)
- Veronique A. J. Smits
- Hospital Universitario de Canarias Unidad de Investigación La Laguna Tenerife Spain
- Facultad de Medicina Instituto de Tecnologías Biomédicas Centro de Investigaciones Biomédicas de Canarias Universidad de La Laguna Tenerife Spain
| | - Elisa Cabrera
- Hospital Universitario de Canarias Unidad de Investigación La Laguna Tenerife Spain
- Facultad de Medicina Instituto de Tecnologías Biomédicas Centro de Investigaciones Biomédicas de Canarias Universidad de La Laguna Tenerife Spain
| | - Raimundo Freire
- Hospital Universitario de Canarias Unidad de Investigación La Laguna Tenerife Spain
- Facultad de Medicina Instituto de Tecnologías Biomédicas Centro de Investigaciones Biomédicas de Canarias Universidad de La Laguna Tenerife Spain
| | - David A. Gillespie
- Facultad de Medicina Instituto de Tecnologías Biomédicas Centro de Investigaciones Biomédicas de Canarias Universidad de La Laguna Tenerife Spain
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The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11. Oncotarget 2017; 7:15703-24. [PMID: 26872382 PMCID: PMC4941271 DOI: 10.18632/oncotarget.7310] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2015] [Accepted: 01/27/2016] [Indexed: 11/25/2022] Open
Abstract
Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics.
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43
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Chastain M, Zhou Q, Shiva O, Fadri-Moskwik M, Whitmore L, Jia P, Dai X, Huang C, Ye P, Chai W. Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress. Cell Rep 2017; 16:1300-1314. [PMID: 27487043 DOI: 10.1016/j.celrep.2016.06.077] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2015] [Revised: 05/23/2016] [Accepted: 06/17/2016] [Indexed: 11/25/2022] Open
Abstract
The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance.
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Affiliation(s)
- Megan Chastain
- Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University, Spokane, WA 99210, USA
| | - Qing Zhou
- Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University, Spokane, WA 99210, USA
| | - Olga Shiva
- Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University, Spokane, WA 99210, USA
| | - Maria Fadri-Moskwik
- Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University, Spokane, WA 99210, USA
| | - Leanne Whitmore
- School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA
| | - Pingping Jia
- Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University, Spokane, WA 99210, USA
| | - Xueyu Dai
- Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University, Spokane, WA 99210, USA
| | - Chenhui Huang
- Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University, Spokane, WA 99210, USA
| | - Ping Ye
- Department of Molecular and Experimental Medicine, Avera Cancer Institute, 1000 E 23rd Street, Suite 370, Sioux Falls, SD 57105, USA; Department of Pharmacy Practice, South Dakota State University, Brookings, SD 57007, USA
| | - Weihang Chai
- Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University, Spokane, WA 99210, USA.
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The drinking water contaminant dibromoacetonitrile delays G1-S transition and suppresses Chk1 activation at broken replication forks. Sci Rep 2017; 7:12730. [PMID: 28986587 PMCID: PMC5630572 DOI: 10.1038/s41598-017-13033-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2017] [Accepted: 09/15/2017] [Indexed: 11/08/2022] Open
Abstract
Chlorination of drinking water protects humans from water-born pathogens, but it also produces low concentrations of dibromoacetonitrile (DBAN), a common disinfectant by-product found in many water supply systems. DBAN is not mutagenic but causes DNA breaks and elevates sister chromatid exchange in mammalian cells. The WHO issued guidelines for DBAN after it was linked with cancer of the liver and stomach in rodents. How this haloacetonitrile promotes malignant cell transformation is unknown. Using fission yeast as a model, we report here that DBAN delays G1-S transition. DBAN does not hinder ongoing DNA replication, but specifically blocks the serine 345 phosphorylation of the DNA damage checkpoint kinase Chk1 by Rad3 (ATR) at broken replication forks. DBAN is particularly damaging for cells with defects in the lagging-strand DNA polymerase delta. This sensitivity can be explained by the dependency of pol delta mutants on Chk1 activation for survival. We conclude that DBAN targets a process or protein that acts at the start of S phase and is required for Chk1 phosphorylation. Taken together, DBAN may precipitate cancer by perturbing S phase and by blocking the Chk1-dependent response to replication fork damage.
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Reyes ED, Kulej K, Pancholi NJ, Akhtar LN, Avgousti DC, Kim ET, Bricker DK, Spruce LA, Koniski SA, Seeholzer SH, Isaacs SN, Garcia BA, Weitzman MD. Identifying Host Factors Associated with DNA Replicated During Virus Infection. Mol Cell Proteomics 2017; 16:2079-2097. [PMID: 28972080 DOI: 10.1074/mcp.m117.067116] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2017] [Revised: 07/14/2017] [Indexed: 01/22/2023] Open
Abstract
Viral DNA genomes replicating in cells encounter a myriad of host factors that facilitate or hinder viral replication. Viral proteins expressed early during infection modulate host factors interacting with viral genomes, recruiting proteins to promote viral replication, and limiting access to antiviral repressors. Although some host factors manipulated by viruses have been identified, we have limited knowledge of pathways exploited during infection and how these differ between viruses. To identify cellular processes manipulated during viral replication, we defined proteomes associated with viral genomes during infection with adenovirus, herpes simplex virus and vaccinia virus. We compared enrichment of host factors between virus proteomes and confirmed association with viral genomes and replication compartments. Using adenovirus as an illustrative example, we uncovered host factors deactivated by early viral proteins, and identified a subgroup of nucleolar proteins that aid virus replication. Our data sets provide valuable resources of virus-host interactions that affect proteins on viral genomes.
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Affiliation(s)
- Emigdio D Reyes
- From the ‡Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.,§Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Katarzyna Kulej
- From the ‡Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.,§Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Neha J Pancholi
- §Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.,¶Cell and Molecular Biology Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania
| | - Lisa N Akhtar
- ‖Division of Infectious Diseases, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Daphne C Avgousti
- From the ‡Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.,§Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Eui Tae Kim
- §Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Daniel K Bricker
- From the ‡Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.,§Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Lynn A Spruce
- **Protein and Proteomics Core, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Sarah A Koniski
- §Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Steven H Seeholzer
- **Protein and Proteomics Core, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Stuart N Isaacs
- ‡‡Division of Infectious Diseases, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania
| | - Benjamin A Garcia
- §§Epigenetics Program, Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania
| | - Matthew D Weitzman
- From the ‡Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania; .,§Division of Protective Immunity and Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
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Azenha D, Lopes MC, Martins TC. Claspin functions in cell homeostasis-A link to cancer? DNA Repair (Amst) 2017; 59:27-33. [PMID: 28942358 DOI: 10.1016/j.dnarep.2017.09.002] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2017] [Accepted: 09/06/2017] [Indexed: 10/18/2022]
Abstract
Cancer remains one of the leading causes of mortality worldwide. Most cancers present high degrees of genomic instability. DNA damage and replication checkpoints function as barriers to halt cell cycle progression until damage is resolved, preventing the perpetuation of errors. Activation of these checkpoints is critically dependent on Claspin, an adaptor protein that mediates the phosphorylation of the effector kinase Chk1 by ATR. However, Claspin also performs other roles related to the protection and maintenance of cell and genome integrity. For instance, following DNA damage and checkpoint activation, Claspin bridges checkpoint responses to DNA repair or to apoptosis. During DNA replication, Claspin acts a sensor and couples DNA unwinding to strand polymerization, and may also indirectly regulate replication initiation at firing origins. As Claspin participates in several processes that are vital to maintenance of cell homeostasis, its function is tightly regulated at multiple levels. Nevertheless, little is known about its role in cancer. Accumulating evidence suggests that Claspin inactivation could be an essential event during carcinogenesis, indicating that Claspin may function as a tumour suppressor. In this review, we will examine the functions of Claspin and how its deregulation may contribute to cancer initiation and progression. To conclude, we will discuss means by which Claspin can be targeted for cancer therapy.
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Affiliation(s)
- Diana Azenha
- Faculdade de Farmácia da Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal; Centro de Neurociências e Biologia Celular, Universidade de Coimbra, Rua Larga, Faculdade de Medicina, Pólo I, 1º andar, 3004-504 Coimbra, Portugal; Instituto Português de Oncologia de Coimbra de Francisco Gentil, Av. Bissaya Barreto 98, Apartado 2005, 3000-651, Coimbra, Portugal.
| | - Maria Celeste Lopes
- Faculdade de Farmácia da Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal; Centro de Neurociências e Biologia Celular, Universidade de Coimbra, Rua Larga, Faculdade de Medicina, Pólo I, 1º andar, 3004-504 Coimbra, Portugal.
| | - Teresa C Martins
- Centro de Neurociências e Biologia Celular, Universidade de Coimbra, Rua Larga, Faculdade de Medicina, Pólo I, 1º andar, 3004-504 Coimbra, Portugal; Instituto Português de Oncologia de Coimbra de Francisco Gentil, Av. Bissaya Barreto 98, Apartado 2005, 3000-651, Coimbra, Portugal.
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Escorcia W, Forsburg SL. Destabilization of the replication fork protection complex disrupts meiotic chromosome segregation. Mol Biol Cell 2017; 28:2978-2997. [PMID: 28855376 PMCID: PMC5662257 DOI: 10.1091/mbc.e17-02-0101] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2017] [Revised: 08/21/2017] [Accepted: 08/23/2017] [Indexed: 12/17/2022] Open
Abstract
The replication fork protection complex (FPC) coordinates multiple processes that are crucial for unimpeded passage of the replisome through various barriers and difficult to replicate areas of the genome. We examine the function of Swi1 and Swi3, fission yeast's primary FPC components, to elucidate how replication fork stability contributes to DNA integrity in meiosis. We report that destabilization of the FPC results in reduced spore viability, delayed replication, changes in recombination, and chromosome missegregation in meiosis I and meiosis II. These phenotypes are linked to accumulation and persistence of DNA damage markers in meiosis and to problems with cohesion stability at the centromere. These findings reveal an important connection between meiotic replication fork stability and chromosome segregation, two processes with major implications to human reproductive health.
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Affiliation(s)
- Wilber Escorcia
- Program in Molecular & Computational Biology, University of Southern California, Los Angeles, CA 90089-2910
| | - Susan L Forsburg
- Program in Molecular & Computational Biology, University of Southern California, Los Angeles, CA 90089-2910
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Seifert BA, Dejosez M, Zwaka TP. Ronin influences the DNA damage response in pluripotent stem cells. Stem Cell Res 2017; 23:98-104. [PMID: 28715716 DOI: 10.1016/j.scr.2017.06.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/29/2017] [Revised: 06/20/2017] [Accepted: 06/29/2017] [Indexed: 12/12/2022] Open
Abstract
Early mammalian embryonic cells must maintain a particularly robust DNA repair system, as mutations at this developmental point have detrimental consequences for the organism. How the repair system can be tuned to fulfill such elevated requirements is largely unknown, but it may involve transcriptional regulation. Ronin (Thap11) is a transcriptional regulator responsible for vital programs in pluripotent cells. Here, we report that this protein also modulates the DNA damage response of such cells. We show that conditional Ronin knockout sensitizes embryonic stem cells (ESCs) to UV-C-induced DNA damage in association with Atr pathway activation and G2/M arrest. Ronin binds to and regulates the genes encoding several DNA repair factors, including Gtf2h4 and Rad18, providing a potential mechanism for this phenotype. Our results suggest that the unique DNA repair requirements of the early embryo are not met by a static system, but rather via highly regulated processes.
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Affiliation(s)
- Bryce A Seifert
- Graduate Program in Molecular and Human Genetics at Baylor College of Medicine, Houston, TX 77030, USA.
| | - Marion Dejosez
- Huffington Center for Cell-Based Research in Parkinson's Disease, Black Family Stem Cell Institute, Department of Cell, Developmental & Regenerative Biology, Graduate School of Biomedical Sciences, New York, NY 10029, USA.
| | - Thomas P Zwaka
- Huffington Center for Cell-Based Research in Parkinson's Disease, Black Family Stem Cell Institute, Department of Cell, Developmental & Regenerative Biology, Graduate School of Biomedical Sciences, New York, NY 10029, USA.
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Ghosh A, Nethery RC, Herring AH, Tarran R. Flavored little cigar smoke induces cytotoxicity and apoptosis in airway epithelia. Cell Death Discov 2017; 3:17019. [PMID: 28496992 PMCID: PMC5402522 DOI: 10.1038/cddiscovery.2017.19] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2016] [Revised: 02/07/2017] [Accepted: 02/23/2017] [Indexed: 11/25/2022] Open
Abstract
Addition of flavors reduces the harsh taste of tobacco, facilitating the initiation and maintenance of addiction among youths. Flavored cigarettes (except menthol) are now banned. However, the legislation on little cigars remains unclear and flavored little cigars are currently available for purchase. Since inhaled tobacco smoke directly exerts toxic effects on the lungs, we tested whether non-flavored and flavored little cigar smoke exposure had the potential for harm in cultured pulmonary epithelia. We cultured Calu-3 lung epithelia on both 96-well plates and at the air–liquid interface and exposed them to smoke from non-flavored Swisher Sweets and flavored (sweet cherry, grape, menthol, peach and strawberry) Swisher Sweets little cigars. Irrespective of flavor, acute little cigar smoke exposure (10×35 ml puffs) significantly increased cell death and decreased the percentage of live cells. Chronic exposure (10×35 ml puffs per day for 4 days) of smoke to Calu-3 cultures significantly increased lactate dehydrogenase release, further indicating toxicity. To determine whether this exposure was associated with increased cell death/apoptosis, a protein array was used. Chronic exposure to smoke from all types of little cigars induced the activation of the two major apoptosis pathways, namely the intrinsic (mitochondrial-mediated) and the extrinsic (death receptor-mediated) pathways. Both flavored and non-flavored little cigar smoke caused similar levels of toxicity and activation of apoptosis, suggesting that flavored and non-flavored little cigars are equally harmful. Hence, the manufacture, advertisement, sale and use of both non-flavored and flavored little cigars should be strictly controlled.
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Affiliation(s)
- Arunava Ghosh
- Marsico Lung Institute, University of North Carolina at Chapel Hill, Marsico Hall, 125 Mason Farm Road, Chapel Hill, NC, USA
| | | | | | - Robert Tarran
- Marsico Lung Institute, University of North Carolina at Chapel Hill, Marsico Hall, 125 Mason Farm Road, Chapel Hill, NC, USA.,Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC, USA
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Mrc1/Claspin: a new role for regulation of origin firing. Curr Genet 2017; 63:813-818. [DOI: 10.1007/s00294-017-0690-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2017] [Revised: 03/20/2017] [Accepted: 03/22/2017] [Indexed: 12/12/2022]
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