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Maruoka M, Zhang P, Mori H, Imanishi E, Packwood DM, Harada H, Kosako H, Suzuki J. Caspase cleavage releases a nuclear protein fragment that stimulates phospholipid scrambling at the plasma membrane. Mol Cell 2021; 81:1397-1410.e9. [PMID: 33725486 DOI: 10.1016/j.molcel.2021.02.025] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Revised: 12/15/2020] [Accepted: 02/19/2021] [Indexed: 12/25/2022]
Abstract
Phospholipid scrambling in dying cells promotes phosphatidylserine exposure, a critical process for efferocytosis. We previously identified the Xkr family protein Xkr4 as a phospholipid-scrambling protein, but its activation mechanisms remain unknown. Here we show that Xkr4 is activated in two steps: dimer formation by caspase-mediated cleavage and structural change caused by activating factors. To identify the factors, we developed a new screening system, "revival screening," using a CRISPR sgRNA library. Applying this system, we identified the nuclear protein XRCC4 as the single candidate for the Xkr4 activator. Upon apoptotic stimuli, XRCC4, contained in the DNA repair complex, is cleaved by caspases, and its C-terminal fragment with an intrinsically disordered region is released into the cytoplasm. Protein interaction screening showed that the fragment interacts directly with the Xkr4 dimer to activate it. This study demonstrates that caspase-mediated cleavage releases a nuclear protein fragment for direct regulation of lipid dynamics on the plasma membrane.
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Affiliation(s)
- Masahiro Maruoka
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan; Center for Integrated Biosystems, Institute for Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Panpan Zhang
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan; Graduate School of Biostudies, Kyoto University, Konoe-cho, Yoshida, Sakyoku, Kyoto 606-8501, Japan
| | - Hiromi Mori
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan
| | - Eiichi Imanishi
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan
| | - Daniel M Packwood
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan
| | - Hiroshi Harada
- Graduate School of Biostudies, Kyoto University, Konoe-cho, Yoshida, Sakyoku, Kyoto 606-8501, Japan
| | - Hidetaka Kosako
- Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Jun Suzuki
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan; Graduate School of Biostudies, Kyoto University, Konoe-cho, Yoshida, Sakyoku, Kyoto 606-8501, Japan; AMED-FORCE, Japanese Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyodaku, Tokyo 100-0004, Japan; Center for Integrated Biosystems, Institute for Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
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2
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Chen P, Kuang P, Wang L, Li W, Chen B, Liu Y, Wang H, Zhao S, Ye L, Yu F, He Y, Zhou C. Mechanisms of drugs-resistance in small cell lung cancer: DNA-related, RNA-related, apoptosis-related, drug accumulation and metabolism procedure. Transl Lung Cancer Res 2020; 9:768-786. [PMID: 32676338 PMCID: PMC7354133 DOI: 10.21037/tlcr-19-547] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Small-cell lung cancer (SCLC), the highest malignant cancer amongst different types of lung cancer, has the feature of lower differentiation, rapid growth, and poor survival rate. Despite the dramatically initial sensitivity of SCLC to various types of treatment methods, including chemotherapy, radiotherapy and immunotherapy, the emergence of drugs-resistance is still a grandly clinical challenge. Therefore, in order to improve the prognosis and develop new therapeutic approaches, having a better understanding of the complex mechanisms of resistance in SCLC is of great clinical significance. This review summarized recent advances in understanding of multiple mechanisms which are involved in the resistance during SCLC treatment, including DNA-related process, RNA-related process, apoptosis-related mechanism, and the process of drug accumulation and metabolism.
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Affiliation(s)
- Peixin Chen
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China.,Department of Medical School, Tongji University, Shanghai, China
| | - Peng Kuang
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China.,Department of Medical Oncology, The First Affiliated Hospital Of Nanchang University, Nanchang, China
| | - Lei Wang
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China
| | - Wei Li
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China
| | - Bin Chen
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China
| | - Yu Liu
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China.,Department of Medical School, Tongji University, Shanghai, China
| | - Hao Wang
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China.,Department of Medical School, Tongji University, Shanghai, China
| | - Sha Zhao
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China
| | - Lingyun Ye
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China
| | - Feng Yu
- Department of Medical Oncology, The First Affiliated Hospital Of Nanchang University, Nanchang, China
| | - Yayi He
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China
| | - Caicun Zhou
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China
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Koi M, Okita Y, Carethers JM. Fusobacterium nucleatum Infection in Colorectal Cancer: Linking Inflammation, DNA Mismatch Repair and Genetic and Epigenetic Alterations. J Anus Rectum Colon 2018; 2:37-46. [PMID: 30116794 PMCID: PMC6090547 DOI: 10.23922/jarc.2017-055] [Citation(s) in RCA: 53] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/31/2017] [Accepted: 01/30/2018] [Indexed: 12/13/2022] Open
Abstract
It has been recently reported that the population of Fusobacterium, particularly Fusobacterium nucleatum (Fn), is overrepresented in colorectal cancers and adenomas. The promoting effects of Fn infection on adenoma and/or carcinoma formation have been shown in ApcMin/+mice. Characteristics of Fn-associated CRC were identified through studies using human CRC cohorts, and include right-sided colon location, CpG island methylation phenotype-high (CIMP-H), high level of microsatellite instability (MSI-H), and poor patient prognosis. A subset of Fn-associated CRC exhibits a low level of microsatellite instability (MSI-L) and elevated microsatellite alterations in selected tetra-nucleotide repeats (EMAST) induced by translocation of MSH3 from the nucleus to the cytoplasm in response to oxidative DNA damage or inflammatory signals. The association between CIMP/MSI-H and Fn-infection can be explained by the role of the mismatch repair (MMR) protein complex formed between MSH2 and MSH6 (MutSα) to repair aberrant bases generated by ROS to form 7,8-dihydro-8-oxo-guanine (8-oxoG). Clustered 8-oxoGs formed at CpG-rich regions including promoters by ROS is refractory to base excision repair (BER). Under these conditions, MutSα initiates repair in cooperation with DNA methyltransferases (DNMTs) and the polycomb repressive complex 4 (PRC4). DNMTs at damaged sites methylate CpG islands to repress transcription of target genes and promote repair reactions. Thus, continuous generation of ROS through chronic Fn infection may initiate 1) CIMP-positive adenoma and carcinoma in an MSH2/MSH6-dependent manner, and/or 2) MSI-L/EMAST CRC in an MSH3-dependent manner. The poor prognosis of Fn-associated CRC can be explained by Fn-induced immune-evasion and/or chemo-resistance.
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Affiliation(s)
- Minoru Koi
- Division of Gastroenterology, Department of Internal Medicine, and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Yoshiki Okita
- Division of Gastroenterology, Department of Internal Medicine, and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan, United States of America
| | - John M Carethers
- Division of Gastroenterology, Department of Internal Medicine, and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan, United States of America
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Torabi B, Flashner S, Beishline K, Sowash A, Donovan K, Bassett G, Azizkhan-Clifford J. Caspase cleavage of transcription factor Sp1 enhances apoptosis. Apoptosis 2018; 23:65-78. [PMID: 29236199 DOI: 10.1007/s10495-017-1437-4] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Sp1 is a ubiquitous transcription factor that regulates many genes involved in apoptosis and senescence. Sp1 also has a role in the DNA damage response; at low levels of DNA damage, Sp1 is phosphorylated by ATM and localizes to double-strand break sites where it facilitates DNA double-strand-break repair. Depletion of Sp1 increases the sensitivity of cells to DNA damage, whereas overexpression of Sp1 can drive cells into apoptosis. In response to a variety of stimuli, Sp1 can be regulated through proteolytic cleavage by caspases and/or degradation. Here, we show that activation of apoptosis through DNA damage or TRAIL-mediated activation of the extrinsic apoptotic pathway induces caspase-mediated cleavage of Sp1. Cleavage of Sp1 was coincident with the appearance of cleaved caspase 3, and produced a 70 kDa Sp1 product. In vitro analysis revealed a novel caspase cleavage site at aspartic acid 183. Mutation of aspartic acid 183 to alanine conferred resistance to cleavage, and ectopic expression of the Sp1 D183A rendered cells resistant to apoptotic stimuli, indicating that Sp1 cleavage is involved in the induction of apoptosis. The 70 kDa product resulting from caspase cleavage of Sp1 comprises amino acids 184-785. This truncated form, designated Sp1-70C, which retains transcriptional activity, induced apoptosis when overexpressed in normal epithelial cells, whereas Sp1D183A induced significantly less apoptosis. Together, these data reveal a new caspase cleavage site in Sp1 and demonstrate for the first time that caspase cleavage of Sp1 promotes apoptosis.
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Affiliation(s)
- Behzad Torabi
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, 19102, USA
| | - Samuel Flashner
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, 19102, USA
| | - Kate Beishline
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, 19102, USA
| | - Aislinn Sowash
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, 19102, USA
| | - Kelly Donovan
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, 19102, USA
| | - Garrett Bassett
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, 19102, USA
| | - Jane Azizkhan-Clifford
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, 19102, USA.
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Mismatch Repair and Colon Cancer: Mechanisms and Therapies Explored. Trends Mol Med 2016; 22:274-289. [PMID: 26970951 DOI: 10.1016/j.molmed.2016.02.003] [Citation(s) in RCA: 131] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2016] [Revised: 02/16/2016] [Accepted: 02/17/2016] [Indexed: 02/06/2023]
Abstract
Colorectal cancer (CRC) remains one of the most prevalent cancers worldwide. In sporadic CRC, mutations frequently occur in the DNA mismatch repair (MMR) pathway. In addition, germline MMR mutations have been linked to Lynch syndrome, the most common form of hereditary CRC. Although genetic mutations, diet, inflammation, and the gut microbiota can influence CRC, it is unclear how MMR deficiency relates to these factors to modulate disease. In this review, the association of MMR to the etiology of CRC is examined, particularly in the context of microRNAs (miRNAs), inflammation, and the microbiome. We also discuss the most current targeted therapies, methods of prevention, and molecular biomarkers against MMR-deficient CRC, all of which are encouraging advancements in the field.
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Patidar PL, Motea EA, Fattah FJ, Zhou Y, Morales JC, Xie Y, Garner HR, Boothman DA. The Kub5-Hera/RPRD1B interactome: a novel role in preserving genetic stability by regulating DNA mismatch repair. Nucleic Acids Res 2016; 44:1718-31. [PMID: 26819409 PMCID: PMC4770225 DOI: 10.1093/nar/gkv1492] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2015] [Accepted: 12/09/2015] [Indexed: 12/29/2022] Open
Abstract
Ku70-binding protein 5 (Kub5)-Hera (K-H)/RPRD1B maintains genetic integrity by concomitantly minimizing persistent R-loops and promoting repair of DNA double strand breaks (DSBs). We used tandem affinity purification-mass spectrometry, co-immunoprecipitation and gel-filtration chromatography to define higher-order protein complexes containing K-H scaffolding protein to gain insight into its cellular functions. We confirmed known protein partners (Ku70, RNA Pol II, p15RS) and discovered several novel associated proteins that function in RNA metabolism (Topoisomerase 1 and RNA helicases), DNA repair/replication processes (PARP1, MSH2, Ku, DNA-PKcs, MCM proteins, PCNA and DNA Pol δ) and in protein metabolic processes, including translation. Notably, this approach directed us to investigate an unpredicted involvement of K-H in DNA mismatch repair (MMR) where K-H depletion led to concomitant MMR deficiency and compromised global microsatellite stability. Mechanistically, MMR deficiency in K-H-depleted cells was a consequence of reduced stability of the core MMR proteins (MLH1 and PMS2) caused by elevated basal caspase-dependent proteolysis. Pan-caspase inhibitor treatment restored MMR protein loss. These findings represent a novel mechanism to acquire MMR deficiency/microsatellite alterations. A significant proportion of colon, endometrial and ovarian cancers exhibit k-h expression/copy number loss and may have severe mutator phenotypes with enhanced malignancies that are currently overlooked based on sporadic MSI+ screening.
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Affiliation(s)
- Praveen L Patidar
- Departments of Pharmacology and Radiation Oncology, Program in Cell Stress and Cancer Nanomedicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Edward A Motea
- Departments of Pharmacology and Radiation Oncology, Program in Cell Stress and Cancer Nanomedicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Farjana J Fattah
- Departments of Pharmacology and Radiation Oncology, Program in Cell Stress and Cancer Nanomedicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Yunyun Zhou
- Quantitative Biomedical Center, Department of Clinical Science, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, USA
| | - Julio C Morales
- Department of Neurosurgery, University of Oklahoma Heath Science Center, Oklahoma City, OK, USA
| | - Yang Xie
- Quantitative Biomedical Center, Department of Clinical Science, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, USA
| | - Harold R Garner
- Edward Via College of Osteopathic Medicine and the MITTE Office, Virginia Tech, Blacksburg, VA, USA
| | - David A Boothman
- Departments of Pharmacology and Radiation Oncology, Program in Cell Stress and Cancer Nanomedicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA
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Hassen S, Ali AA, Kilaparty SP, Al-Anbaky QA, Majeed W, Boman BM, Fields JZ, Ali N. Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines. Mol Cell Biochem 2016; 412:297-305. [PMID: 26728996 DOI: 10.1007/s11010-015-2636-3] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2015] [Accepted: 12/23/2015] [Indexed: 01/06/2023]
Abstract
The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an indicator of apoptosis, showed that MLH1 translocation only occurred in MMR proficient (SW480) cells upon induction of apoptosis further suggested a MSH2 dependent role of MLH1 in apoptosis. These data suggest a role of MLH1 in mediation of apoptosis in a MSH2-dependent manner. Taken together, our data supported an interdependence of mismatch repair proteins, particularly MLH1 and MSH2, in the mediation of apoptosis in human colorectal carcinoma cell lines.
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Affiliation(s)
- Samar Hassen
- Department of Biology, College of Arts, Letters and Sciences, University of Arkansas at Little Rock, 2801 S. University Avenue, Little Rock, AR, 72204, USA
| | - Akhtar A Ali
- Department of Biology, College of Arts, Letters and Sciences, University of Arkansas at Little Rock, 2801 S. University Avenue, Little Rock, AR, 72204, USA.,CATX Inc., Gladwyne, PA, 19035, USA
| | - Surya P Kilaparty
- Department of Biology, College of Arts, Letters and Sciences, University of Arkansas at Little Rock, 2801 S. University Avenue, Little Rock, AR, 72204, USA
| | - Qudes A Al-Anbaky
- Department of Biology, College of Arts, Letters and Sciences, University of Arkansas at Little Rock, 2801 S. University Avenue, Little Rock, AR, 72204, USA
| | - Waqar Majeed
- Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little Rock, AR, 72204, USA
| | - Bruce M Boman
- Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, Kimmel Cancer Center, Philadelphia, PA, 19107, USA.,Center for Translational Cancer Research, University of Delaware, Helen F Graham Cancer Center, Newark, DE, 19713, USA.,CATX Inc., Gladwyne, PA, 19035, USA
| | | | - Nawab Ali
- Department of Biology, College of Arts, Letters and Sciences, University of Arkansas at Little Rock, 2801 S. University Avenue, Little Rock, AR, 72204, USA.
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Moghbeli M, Moaven O, Memar B, Raziei HR, Aarabi A, Dadkhah E, Forghanifard MM, Manzari F, Abbaszadegan MR. Role of hMLH1 and E-cadherin promoter methylation in gastric cancer progression. J Gastrointest Cancer 2014; 45:40-7. [PMID: 24022108 DOI: 10.1007/s12029-013-9548-9] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
INTRODUCTION Gastric cancer (GC) is one of the leading causes of cancer-related death in Iran. Genome stability is one of the main genetic issues in cancer biology which is governed via the different repair systems such as DNA mismatch repair (MMR). A clear correlation between MMR defects and tumor progression has been shown. Beside the genetic mutations, epigenetic changes also have a noticeable role in MMR defects. METHODS Here, we assessed promoter methylation status and the level of hMLH1mRNA expression as the main component of MMR system in 51 GC patients using the methylation-specific PCR and real-time PCR, respectively. Moreover, we performed a promoter methylation study of the E-cadherin gene promoter. RESULTS It was observed that, 12 out of 39 cases (23.5%) had hMLH1 overexpression. Hypermethylation of hMLH1 and E-cadherin promoter regions were observed in 25.5 and 36.4%, respectively. Although, there was no significant correlation between hMLH1 mRNA expression and clinicopathological features, there are significant correlations between E-cadherin promoter methylation and tumor stage (p = 0.028) and location (p = 0.025). The rate of hMLH1 promoter methylation in this study was lower than that in the other population, showing the importance of the other mechanisms, in gastric tumorigenesis. CONCLUSION The results of this study indicate that DNA repair system is adversely affected by hypermethylation of hMLH1 in a fraction of gastric cancer patients. Additionally, E-cadherin hypermethylation seen in a subset of our gastric cancer patients is consistent with other reports showing correlation with aggressiveness and metastasis of gastric cancer.
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Affiliation(s)
- Meysam Moghbeli
- Division of Human Genetics, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran
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9
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FANCD2 is a target for caspase 3 during DNA damage-induced apoptosis. FEBS Lett 2014; 588:3778-85. [DOI: 10.1016/j.febslet.2014.08.027] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2014] [Revised: 08/18/2014] [Accepted: 08/21/2014] [Indexed: 01/01/2023]
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10
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Adamkov M, Furjelová M, Horáček J, Benčat M, Kružliak P. Relationship of mismatch repair proteins and survivin in colon polyps and carcinomas. Acta Histochem 2014; 116:1007-14. [PMID: 24852932 DOI: 10.1016/j.acthis.2014.04.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2014] [Revised: 04/02/2014] [Accepted: 04/03/2014] [Indexed: 12/24/2022]
Abstract
Mismatch repair genes (MMR) play an essential role in DNA repair. MMR mutations predominantly in MLH1, MSH2, MSH6, PMS2, and rarely in PMS1, may cause the production of abnormally short or inactivated proteins. The antiapoptotic protein survivin functions in the inhibition of apoptosis, regulation of cell division and also enhances angiogenesis. Both MMRP and survivin are considered to be powerful prognostic parameters. This study was designed to determine the relationship between MMRP and survivin in colon lesions. The study included 113 cases of colon carcinoma and 51 cases of colon polyps. Survivin expression and MMRP status were assessed by immunohistochemistry. In each section, expression, intensity of immunostaining and percentage of labeled cells were analyzed. In carcinomas, immunoreaction was detected in 100/113 cases for MLH1 (88.5%), 112/113 cases for MSH2 (99.1%), 110/113 cases for MSH6 (97.3%), and 103/113 cases for PMS2 (91.2%). Survivin was shown in 47/113 cases (41.6%). The statistical analysis confirmed a significant correlation between the expression of MMRP and survivin in the assessed parameters. All 51 polyp samples were positive for MLH1, MSH2, MSH6 and PMS2. Only 8 of those (15.7%) were positive for survivin. Statistically significant differences were observed between the expression of MMRP and survivin. In conclusion, this study revealed that MMRP may suppress the antiapoptotic function of survivin through p53 inactivation of its promoter in grade 1 and grade 2 colon carcinomas.
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Abstract
Normal cell function requires strict control over the repair of DNA damage, which prevents excessive mutagenesis. An enhanced accumulation of mutations results in the multistep process generally known as carcinogenesis. Defects in repair pathways fuel such mutagenesis by allowing reiterative cycles of mutation, selection, and clonal expansion that drive cancer progression. The repair of mismatches is an important mechanism in the prevention of such genetic instability. In addition, proteins of this pathway have the unique ability to function in DNA damage response by inducing apoptosis when irreparable damage is encountered. Though originally identified primarily in association with a predisposition to hereditary colon cancer, mismatch repair defects have been identified in many other cancer types, including prostate cancer. From the first discovery of microsatellite instability in prostate cancer cell lines and tumor samples, variations in protein levels and a possible association with recurrence and aggression of disease have been described. Current results suggest that the involvement of mismatch repair proteins in prostate cancer may differ from that found in colorectal cancer, in the type of proteins and protein defects involved and the type of causative mutations. Additional work is clearly needed to investigate this involvement and the possibility that such defects may affect treatment response and androgen independence.
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Affiliation(s)
- John Jarzen
- Department of Biology, College of Science and Technology, Georgia Southern University, Statesboro, Georgia, USA
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12
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Jin B, Robertson KD. DNA methyltransferases, DNA damage repair, and cancer. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2013; 754:3-29. [PMID: 22956494 DOI: 10.1007/978-1-4419-9967-2_1] [Citation(s) in RCA: 347] [Impact Index Per Article: 28.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The maintenance DNA methyltransferase (DNMT) 1 and the de novo methyltransferases DNMT3A and DNMT3B are all essential for mammalian development. DNA methylation, catalyzed by the DNMTs, plays an important role in maintaining genome stability. Aberrant expression of DNMTs and disruption of DNA methylation patterns are closely associated with many forms of cancer, although the exact mechanisms underlying this link remain elusive. DNA damage repair systems have evolved to act as a genome-wide surveillance mechanism to maintain chromosome integrity by recognizing and repairing both exogenous and endogenous DNA insults. Impairment of these systems gives rise to mutations and directly contributes to tumorigenesis. Evidence is mounting for a direct link between DNMTs, DNA methylation, and DNA damage repair systems, which provide new insight into the development of cancer. Like tumor suppressor genes, an array of DNA repair genes frequently sustain promoter hypermethylation in a variety of tumors. In addition, DNMT1, but not the DNMT3s, appear to function coordinately with DNA damage repair pathways to protect cells from sustaining mutagenic events, which is very likely through a DNA methylation-independent mechanism. This chapter is focused on reviewing the links between DNA methylation and the DNA damage response.
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Affiliation(s)
- Bilian Jin
- Department of Biochemistry and Molecular Biology, Georgia Health Sciences University Cancer Center, CN-2151, 1410 Laney Walker Blvd, Augusta, GA 30912, USA
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13
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Varshavsky A. Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis. Protein Sci 2012; 21:1634-61. [PMID: 22930402 PMCID: PMC3527701 DOI: 10.1002/pro.2148] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2012] [Accepted: 08/21/2012] [Indexed: 02/05/2023]
Abstract
Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca²⁺ fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca²⁺-activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca²⁺ transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca²⁺ alone or Ca²⁺ and its ionophore (Erickson et al., Science 1978;199:1219-1221; Harris, Pharmacol Biochem Behav 1979;10:527-534; Erickson et al., Pharmacol Biochem Behav 1980;12:651-656). These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep.
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Affiliation(s)
- Alexander Varshavsky
- Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
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Hassen S, Ali N, Chowdhury P. Molecular signaling mechanisms of apoptosis in hereditary non-polyposis colorectal cancer. World J Gastrointest Pathophysiol 2012; 3:71-9. [PMID: 22737591 PMCID: PMC3382705 DOI: 10.4291/wjgp.v3.i3.71] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/17/2011] [Revised: 05/31/2012] [Accepted: 06/12/2012] [Indexed: 02/06/2023] Open
Abstract
Colorectal cancer is the second most leading cause of cancer related deaths in the western countries. One of the forms of colorectal cancer is hereditary non-polyposis colorectal cancer (HNPCC), also known as “Lynch syndrome”. It is the most common hereditary form of cancer accounting for 5%-10% of all colon cancers. HNPCC is a dominant autosomal genetic disorder caused by germ line mutations in mismatch repair genes. Human mismatch repair genes play a crucial role in genetic stability of DNA, the inactivation of which results in an increased rate of mutation and often a loss of mismatch repair function. Recent studies have shown that certain mismatch repair genes are involved in the regulation of key cellular processes including apoptosis. Thus, differential expression of mismatch repair genes particularly the contributions of MLH1 and MSH2 play important roles in therapeutic resistance to certain cytotoxic drugs such as cisplatin that is used normally as chemoprevention. An understanding of the role of mismatch repair genes in molecular signaling mechanism of apoptosis and its involvement in HNPCC needs attention for further work into this important area of cancer research, and this review article is intended to accomplish that goal of linkage of apoptosis with HNPCC. The current review was not intended to provide a comprehensive enumeration of the entire body of literature in the area of HNPCC or mismatch repair system or apoptosis; it is rather intended to focus primarily on the current state of knowledge of the role of mismatch repair proteins in molecular signaling mechanism of apoptosis as it relates to understanding of HNPCC.
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Expression analysis of TOP2A, MSH2 and MLH1 genes in MCF7 cells at different levels of etoposide resistance. Biomed Pharmacother 2011; 66:29-35. [PMID: 22285073 DOI: 10.1016/j.biopha.2011.09.002] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2011] [Accepted: 09/06/2011] [Indexed: 12/17/2022] Open
Abstract
PURPOSE Development of resistance against anti-cancer drugs is one of the major obstacles of chemotherapy in the treatment of cancer. Etoposide is a topoisomerase II alpha (TOP2A) inhibitor, which is used in the treatment of breast cancer. Alterations in the expression of drug targets or DNA repair genes are among the important resistance mechanisms against TOP2A inhibitors. In this study, expression changes in TOP2A gene and two important mismatch repair (MMR) genes MSH2 and MLH1 were examined in order to understand the relationship between differential expression of these genes and drug resistance against etoposide. METHODS Resistant cell lines were developed from parental MCF7 cell line by stepwise selection in increasing doses of etoposide. Total RNA was isolated from parental and resistant cell lines by using TriReagent. Expression levels of TOP2A, MSH2 and MLH1 were analysed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Statistical analyses were performed by one way ANOVA. RESULTS Etoposide resistant sublines MCF7/1000E, MCF7/1250E and MCF7/2000E were approximately 2, 3 and 4 fold resistant relative to parental MCF7/S cells, respectively. TOP2A, MSH2 and MLH1 expressions decreased in etoposide resistant sublines relative to MCF7/S cells. Expression levels of TOP2A and MLH1 in resistant sublines differed between 10-95 and 18-58 percent of the expression levels in the parental cells, respectively. MSH2 expression levels were decreased 18-82 percent in resistant cells. A transient 15 percent increase in the expression of this gene was observed in subline MCF7/1250E. CONCLUSIONS Decrease in the expression levels of TOP2A, MSH2 and MLH1 may play significant roles in the development of chemotherapeutic resistance to etoposide in breast cancer. These genes may be considered for further development of new strategies to overcome resistance against topoisomerase II inhibitors.
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Loughery JEP, Dunne PD, O'Neill KM, Meehan RR, McDaid JR, Walsh CP. DNMT1 deficiency triggers mismatch repair defects in human cells through depletion of repair protein levels in a process involving the DNA damage response. Hum Mol Genet 2011; 20:3241-55. [PMID: 21636528 DOI: 10.1093/hmg/ddr236] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/01/2025] Open
Abstract
DNA methyltransferase 1 (DNMT1) maintains methylation at CpG dinucleotides, important for transcriptional silencing at many loci. It is also implicated in stabilizing repeat sequences: DNMT1 deficiency causes microsatellite instability in mouse embryonic stem cells, but it is unclear how this occurs, how repeats lacking CpG become unstable and whether the effect is confined to stem cells. To address these questions, we transfected hTERT-immortalized normal human fibroblasts (hTERT-1604) with a short hairpin RNA construct targeting DNMT1 and isolated stable integrants with different levels of protein. DNMT1 expression levels agreed well with methylation levels at imprinted genes. Knockdown cells showed two key characteristics of mismatch repair (MMR) deficiency, namely resistance to the drug 6-thioguanine and up to 10-fold elevated mutation rates at a CA(17) microsatellite reporter, but had limited viability. The likely cause of MMR defects is a matching drop in steady-state protein levels for key repair components in DNMT1 knockdown cells, affecting both the MutLα and MutSα complexes. This indirect effect on MMR proteins was also seen using a different targeting method in HT29 colon cancer cells and did not involve transcriptional silencing of the respective genes. Decreased levels of MMR components follow activation of the DNA damage response and blocking this response, and in particular poly(ADP-ribose) polymerase (PARP) overactivation, rescues cell viability in DNMT1-depleted cells. These results offer an explanation for how and why unmethylated microsatellite repeats can be destabilized in cells with decreased DNMT1 levels and uncover a novel and important role for PARP in this process.
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Affiliation(s)
- Jayne E P Loughery
- Transcriptional Regulation and Epigenetics Group, Centre for Molecular Biosciences, University of Ulster, Coleraine BT52 1SA, UK
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17
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Brieger A, Adam R, Passmann S, Plotz G, Zeuzem S, Trojan J. A CRM1-dependent nuclear export pathway is involved in the regulation of MutLα subcellular localization. Genes Chromosomes Cancer 2011; 50:59-70. [PMID: 21064154 DOI: 10.1002/gcc.20832] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2023] Open
Abstract
MutLα plays an essential role in DNA mismatch repair (MMR) and is additionally involved in other cellular mechanisms such as the regulation of cell cycle checkpoints and apoptosis. Therefore, not only germline MMR gene defects but also the subcellular localization of MutLα might be of importance for the development of Lynch syndrome. Recently, we showed that MutLα contains functional nuclear import sequences and is most frequently localized in the nucleus. Here, we demonstrate that MutLα can move bidirectionally towards the nuclear membrane. Using MutLα transfected HEK293T cells we observed a significant shift of MLH1 and PMS2 from the nucleus to the cytoplasm after irradiation or cisplatin treatment. We analyzed both proteins for potential nuclear export sequences (NES) and identified one functional Rev-type NES (⁵⁷⁸LFDLAMLAL) in the C-terminal part of MLH1 that facilitates export via the CRM1/exportin pathway. Moreover, an MLH1-NES mutation detected in a patient with Lynch syndrome showed normal MMR activity but led to significantly impaired cytoplasmic transport after actinomycin D treatment. These results indicate that MutLα is able to shuttle from the nucleus to the cytoplasm, probably signaling DNA damages to downstream pathways. In conclusion, not only a defective MMR but also impaired nucleo-cytoplasmic shuttling might result in the onset of Lynch syndrome.
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Affiliation(s)
- Angela Brieger
- Medical Clinic I, Biomedical Research Laboratory, Goethe-University, Frankfurt a.M., Germany.
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18
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Kastrati I, Edirisinghe PD, Wijewickrama GT, Thatcher GRJ. Estrogen-induced apoptosis of breast epithelial cells is blocked by NO/cGMP and mediated by extranuclear estrogen receptors. Endocrinology 2010; 151:5602-16. [PMID: 20943808 PMCID: PMC2999489 DOI: 10.1210/en.2010-0378] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Estrogen action, via both nuclear and extranuclear estrogen receptors (ERs), induces a variety of cellular signals that are prosurvival or proliferative, whereas nitric oxide (NO) can inhibit apoptosis via caspase S-nitrosylation and via activation of soluble guanylyl cyclase to produce cGMP. The action of 17β-estradiol (E(2)) at ER is known to elicit NO signaling via activation of NO synthase (NOS) in many tissues. The MCF-10A nontumorigenic, mammary epithelial cell line is genetically stable and insensitive to estrogenic proliferation. In this cell line, estrogens or NOS inhibitors alone had no significant effect, whereas in combination, apoptosis was induced rapidly in the absence of serum; the presence of inducible NOS was confirmed by proteomic analysis. The application of pharmacological agents determined that apoptosis was dependent upon NO/cGMP signaling via cyclic GMP (cGMP)-dependent protein kinase and could be replicated by inhibition of the phosphatidylinositol 3 kinase/serine-threonine kinase pathway prior to addition of E(2). Apoptosis was confirmed by nuclear staining and increased caspase-3 activity in E(2) + NOS inhibitor-treated cells. Apoptosis was partially inhibited by a pure ER antagonist and replicated by agonists selective for extranuclear ER. Cells were rescued from E(2)-induced apoptosis after NOS blockade, by NO-donors and cGMP pathway agonists; preincubation with NO donors was required. The NOS and ER status of breast cancer tissues is significant in etiology, prognosis, and therapy. In this study, apoptosis of preneoplastic mammary epithelial cells was triggered by estrogens via a rapid, extranuclear ER-mediated response, after removal of an antiapoptotic NO/cGMP/cGMP-dependent protein kinase signal.
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Affiliation(s)
- Irida Kastrati
- Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, USA
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The small heat shock protein HspB2 is a novel anti-apoptotic protein that inhibits apical caspase activation in the extrinsic apoptotic pathway. Breast Cancer Res Treat 2010; 124:307-15. [PMID: 20087649 DOI: 10.1007/s10549-010-0735-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2009] [Accepted: 01/06/2010] [Indexed: 01/19/2023]
Abstract
Members of the conserved small heat shock protein (sHSP) family, such as αB-crystallin and Hsp27, are constitutively expressed in diverse malignancies and have been linked to several hallmark features of cancer including apoptosis resistance. In contrast, the sHSP HspB2/MKBP, which shares an intergenic promoter with αB-crystallin, was discovered as a chaperone of the myotonic dystrophy protein kinase and has not been previously implicated in apoptosis regulation. Here we describe a new function for HspB2 as a novel inhibitor of apical caspase activation in the extrinsic apoptotic pathway. Specifically, we demonstrate that HspB2 is expressed in a subset of human breast cancer cell lines and that ectopic expression of HspB2 in breast cancer cells confers resistance to apoptosis induced by both TRAIL and TNF-α. We also show that HspB2 inhibits the extrinsic apoptotic pathway by suppressing apical caspases-8 and 10 activation, thereby blocking downstream apoptotic events, such as Bid cleavage and caspase-3 activation. Consistent with these in vitro effects, HspB2 attenuates the anti-tumor activity of TRAIL in an orthotopic xenograft model of breast cancer. Collectively, our results reveal a novel function of HspB2 as an anti-apoptotic protein that negatively regulates apical caspase activation in the extrinsic apoptotic pathway.
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20
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Ding X, Mohd AB, Huang Z, Baba T, Bernardini MQ, Lyerly HK, Berchuck A, Murphy SK, Buermeyer AB, Devi GR. MLH1 expression sensitises ovarian cancer cells to cell death mediated by XIAP inhibition. Br J Cancer 2009; 101:269-77. [PMID: 19603033 PMCID: PMC2720211 DOI: 10.1038/sj.bjc.6605180] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
BACKGROUND The X-linked inhibitor of apoptosis protein (XIAP), an endogenous apoptosis suppressor, can determine the level of caspase accumulation and the resultant response to apoptosis-inducing agents such as cisplatin in epithelial ovarian cancer (EOC). In addition, the mismatch repair protein, hMLH1, has been linked to DNA damage-induced apoptosis by cisplatin by both p53-dependent and -independent mechanisms. METHODS In this study, hMLH1 expression was correlated with clinical response to platinum drugs and survival in advanced stage (III-IV) EOC patients. We then investigated whether MLH1 loss was a determinant in anti-apoptosis response to cisplatin mediated by XIAP in isogenic and established EOC cell lines with differential p53 status. RESULTS The percentage of cells undergoing cisplatin-induced cell killing was higher in MLH1-proficient cells than in MLH1-defective cells. In addition, the presence of wild-type hMLH1 or hMLH1 re-expression significantly increased sensitivity to 6-thioguanine, a MMR-dependent agent. Cell-death response to 6-thioguanine and cisplatin was associated with significant proteolysis of MLH1, with XIAP destabilisation and increased caspase-3 activity. The siRNA-mediated inhibition of XIAP increased MLH1 proteolysis and cell death in MLH1-proficient cells but not in MLH1-defective cells. CONCLUSION These data suggest that XIAP inhibitors may prove to be an effective means of sensitising EOC to MLH1-dependent apoptosis.
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Affiliation(s)
- X Ding
- Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
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Ruzov A, Shorning B, Mortusewicz O, Dunican DS, Leonhardt H, Meehan RR. MBD4 and MLH1 are required for apoptotic induction in xDNMT1-depleted embryos. Development 2009; 136:2277-86. [PMID: 19502488 PMCID: PMC2729342 DOI: 10.1242/dev.032227] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/28/2009] [Indexed: 01/24/2023]
Abstract
Loss of the of the maintenance methyltransferase xDNMT1 during Xenopus development results in premature transcription and activation of a p53-dependent apoptotic program that accounts for embryo lethality. Here, we show that activation of the apoptotic response is signalled through the methyl-CpG binding protein xMBD4 and the mismatch repair pathway protein xMLH1. Depletion of xMBD4 or xMLH1 increases the survival rate of xDNMT1-depleted embryos, whereas overexpression of these proteins in embryos induces programmed cell death at the onset of gastrulation. MBD4 interacts directly with both DNMT1 and MLH1, leading to recruitment of the latter to heterochromatic sites that are coincident with DNMT1 localisation. Time-lapse microscopy of micro-irradiated mammalian cells shows that MLH1/MBD4 (like DNMT1) can accumulate at DNA damage sites. We propose that xMBD4/xMLH1 participates in a novel G2 checkpoint that is responsive to xDNMT1p levels in developing embryos and cells.
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Affiliation(s)
- Alexey Ruzov
- Human Genetics Unit, MRC, IGMM, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
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22
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Bolderson E, Richard DJ, Edelmann W, Khanna KK. Involvement of Exo1b in DNA damage-induced apoptosis. Nucleic Acids Res 2009; 37:3452-63. [PMID: 19339515 PMCID: PMC2691832 DOI: 10.1093/nar/gkp194] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
Apoptosis is essential for the maintenance of inherited genomic integrity. During DNA damage-induced apoptosis, mechanisms of cell survival, such as DNA repair are inactivated to allow cell death to proceed. Here, we describe a role for the mammalian DNA repair enzyme Exonuclease 1 (Exo1) in DNA damage-induced apoptosis. Depletion of Exo1 in human fibroblasts, or mouse embryonic fibroblasts led to a delay in DNA damage-induced apoptosis. Furthermore, we show that Exo1 acts upstream of caspase-3, DNA fragmentation and cytochrome c release. In addition, induction of apoptosis with DNA-damaging agents led to cleavage of both isoforms of Exo1. The cleavage of Exo1 was mapped to Asp514, and shown to be mediated by caspase-3. Expression of a caspase-3 cleavage site mutant form of Exo1, Asp514Ala, prevented formation of the previously observed fragment without any affect on the onset of apoptosis. We conclude that Exo1 has a role in the timely induction of apoptosis and that it is subsequently cleaved and degraded during apoptosis, potentially inhibiting DNA damage repair.
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Affiliation(s)
- Emma Bolderson
- Signal Transduction Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland 4006, Australia
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Strohecker AM, Yehiely F, Chen F, Cryns VL. Caspase cleavage of HER-2 releases a Bad-like cell death effector. J Biol Chem 2008; 283:18269-82. [PMID: 18420586 DOI: 10.1074/jbc.m802156200] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Human epidermal growth factor receptor-2 (HER-2/ErbB2/neu), a receptor tyrosine kinase that is amplified/overexpressed in poor prognosis breast carcinomas, confers resistance to apoptosis by activating cell survival pathways. Here we demonstrate that the cytoplasmic tail of HER-2 is cleaved by caspases at Asp(1016)/Asp(1019) to release a approximately 47-kDa product, which is subsequently proteolyzed by caspases at Asp(1125) into an unstable 22-kDa fragment that is degraded by the proteasome and a predicted 25-kDa product. Both the 47- and 25-kDa products translocate to mitochondria, release cytochrome c by a Bcl-x(L)-suppressible mechanism, and induce caspase-dependent apoptosis. The 47- and 25-kDa HER-2 cleavage products share a functional BH3-like domain, which is required for cytochrome c release in cells and isolated mitochondria and for apoptosis induction. Caspase-cleaved HER-2 binds Bcl-x(L) and acts synergistically with truncated Bid to induce apoptosis, mimicking the actions of the BH3-only protein Bad. Moreover, the HER-2 cleavage products cooperate with Noxa to induce apoptosis in cells expressing both Bcl-x(L) and Mcl-1, confirming their Bad-like function. Collectively, our results indicate that caspases activate a previously unrecognized proapoptotic function of HER-2 by releasing a Bad-like cell death effector.
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Affiliation(s)
- Anne M Strohecker
- Cell Death Regulation Laboratory, Departments of Medicine and Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
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25
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Werner ME, Chen F, Moyano JV, Yehiely F, Jones JCR, Cryns VL. Caspase proteolysis of the integrin beta4 subunit disrupts hemidesmosome assembly, promotes apoptosis, and inhibits cell migration. J Biol Chem 2007; 282:5560-9. [PMID: 17178732 PMCID: PMC2819670 DOI: 10.1074/jbc.m603669200] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Caspases are a conserved family of cell death proteases that cleave intracellular substrates at Asp residues to modify their function and promote apoptosis. In this report we identify the integrin beta4 subunit as a novel caspase substrate using an expression cloning strategy. Together with its alpha6 partner, alpha6beta4 integrin anchors epithelial cells to the basement membrane at specialized adhesive structures known as hemidesmosomes and plays a critical role in diverse epithelial cell functions including cell survival and migration. We show that integrin beta4 is cleaved by caspase-3 and -7 at a conserved Asp residue (Asp(1109)) in vitro and in epithelial cells undergoing apoptosis, resulting in the removal of most of its cytoplasmic tail. Caspase cleavage of integrin beta4 produces two products, 1) a carboxyl-terminal product that is unstable and rapidly degraded by the proteasome and 2) an amino-terminal cleavage product (amino acids 1-1109) that is unable to assemble into mature hemidesmosomes. We also demonstrate that caspase cleavage of integrin beta4 sensitizes epithelial cells to apoptosis and inhibits cell migration. Taken together, we have identified a previously unrecognized proteolytic truncation of integrin beta4 generated by caspases that disrupts key structural and functional properties of epithelial cells and promotes apoptosis.
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Affiliation(s)
- Michael E. Werner
- Cell Death Regulation Laboratory, Northwestern University, Chicago, Illinois 60611
- Department of Medicine, Northwestern University, Chicago, Illinois 60611
- Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611
- Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
| | - Feng Chen
- Cell Death Regulation Laboratory, Northwestern University, Chicago, Illinois 60611
- Department of Medicine, Northwestern University, Chicago, Illinois 60611
- Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611
- Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
| | - Jose V. Moyano
- Cell Death Regulation Laboratory, Northwestern University, Chicago, Illinois 60611
- Department of Medicine, Northwestern University, Chicago, Illinois 60611
- Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611
- Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
| | - Fruma Yehiely
- Cell Death Regulation Laboratory, Northwestern University, Chicago, Illinois 60611
- Department of Medicine, Northwestern University, Chicago, Illinois 60611
- Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611
- Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
| | - Jonathan C. R. Jones
- Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611
- Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
| | - Vincent L. Cryns
- Cell Death Regulation Laboratory, Northwestern University, Chicago, Illinois 60611
- Department of Medicine, Northwestern University, Chicago, Illinois 60611
- Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois 60611
- Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
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Valenti A, Napoli A, Ferrara MC, Nadal M, Rossi M, Ciaramella M. Selective degradation of reverse gyrase and DNA fragmentation induced by alkylating agent in the archaeon Sulfolobus solfataricus. Nucleic Acids Res 2006; 34:2098-108. [PMID: 16617150 PMCID: PMC1440885 DOI: 10.1093/nar/gkl115] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Reverse gyrase is a peculiar DNA topoisomerase, specific of hyperthermophilic Archaea and Bacteria, which has the unique ability of introducing positive supercoiling into DNA molecules. Although the function of the enzyme has not been established directly, it has been suggested to be involved in DNA protection and repair. We show here that the enzyme is degraded after treatment of Sulfolobus solfataricus cells with the alkylating agent MMS. MMS-induced reverse gyrase degradation is highly specific, since (i) neither hydroxyurea (HU) nor puromycin have a similar effect, and (ii) topoisomerase VI and two chromatin components are not degraded. Reverse gyrase degradation does not depend on protein synthesis. Experiments in vitro show that direct exposure of cell extracts to MMS does not induce reverse gyrase degradation; instead, extracts from MMS-treated cells contain some factor(s) able to degrade the enzyme in extracts from control cells. In vitro, degradation is blocked by incubation with divalent metal chelators, suggesting that reverse gyrase is selectively degraded by a metal-dependent protease in MMS-treated cells. In addition, we find a striking concurrence of extensive genomic DNA degradation and reverse gyrase loss in MMS-treated cells. These results support the hypothesis that reverse gyrase plays an essential role in DNA thermoprotection and repair in hyperthermophilic organisms.
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Affiliation(s)
| | | | | | - Marc Nadal
- Université de Versailles-Saint-Quentin-en-Yvelines, Laboratoire de Génétique et Biologie Cellulaire, CNRSFRE 2445, Equipe MicrobiologieBâtiment Buffon, 45 Avenue des Etats-Unis 78035 Versailles Cedex, France
| | | | - Maria Ciaramella
- To whom correspondence should be addressed. Tel: 390816132247; Fax: 390816132248;
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Abstract
Postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by up to three orders of magnitude, through correcting DNA polymerase errors that escaped proofreading. MMR also controls homologous recombination (HR) by aborting strand exchange between divergent DNA sequences. In recent years, MMR has also been implicated in the response of mammalian cells to DNA damaging agents. Thus, MMR-deficient cells were shown to be around 100-fold more resistant to killing by methylating agents of the S(N)1type than cells with functional MMR. In the case of cisplatin, the sensitivity difference was lower, typically two- to three-fold, but was observed in all matched MMR-proficient and -deficient cell pairs. More controversial is the role of MMR in cellular response to other DNA damaging agents, such as ionizing radiation (IR), topoisomerase poisons, antimetabolites, UV radiation and DNA intercalators. The MMR-dependent DNA damage signalling pathways activated by the above agents are also ill-defined. To date, signalling cascades involving the Ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), as well as the stress-activated kinases JNK/SAPK and p38alpha have been linked with methylating agent and 6-thioguanine (TG) treatments, while cisplatin damage was reported to activate the c-Abl and JNK/SAPK kinases in MMR-dependent manner. MMR defects are found in several different cancer types, both familiar and sporadic, and it is possible that the involvement of the MMR system in DNA damage signalling play an important role in transformation. The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.
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Affiliation(s)
- Lovorka Stojic
- Institute of Molecular Cancer Research, University of Zurich, August Forel-Strasse 7, 8008 Zurich, Switzerland
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28
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Godovac-Zimmermann J, Kleiner O, Brown LR, Drukier AK. Perspectives in spicing up proteomics with splicing. Proteomics 2005; 5:699-709. [PMID: 15693068 DOI: 10.1002/pmic.200401051] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
In the post-genomics era there has been an acceleration of understanding of cellular and organismal biology and this acceleration has moved the goalposts for proteomics. Higher eukaryotes use alternative promoters, alternative splicing, RNA editing and post-translational modification to produce multiple isoforms of proteins from single genes. Switching amongst these isoforms is a major mechanism for control of cellular function. At present fundamental limitations in sensitivity, in absolute quantitation of proteins and in the characterization of protein structure at functionally important levels strongly limit the applicability of proteomics to higher eukaryotes. Recent developments suggest that quantitative, top-down proteomics analyses of complete proteins at sub-attomole levels are necessary for physiologically relevant studies of higher eukaryotes. New proteomics technologies which will ensure the future of proteomics as an important technology in medicine and cellular biology of higher eukaryotes are becoming available.
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