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Perimbeti S, Jamroze A, Gopalakrishnan D, Jain R, Jiang C, Holleran JL, Parise RA, Bies R, Quinn D, Attwood K, Liu X, Kirk JS, Beumer JH, Tang DG, Chatta G. Phase Ib Study of Enzalutamide with Venetoclax in Patients with Metastatic Castration-Resistant Prostate Cancer. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2025:2025.04.22.25326208. [PMID: 40313289 PMCID: PMC12045430 DOI: 10.1101/2025.04.22.25326208] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 05/03/2025]
Abstract
Purpose Castration and enzalutamide induce BCL-2 to drive therapy resistance in prostate cancer (PCa). We conducted a phase Ib trial to test that metastatic castration-resistant PCa (mCRPC) can be effectively targeted by combining enzalutamide with the BCL-2 inhibitor venetoclax. Experimental Design This phase Ib single-arm trial of enzalutamide (160 mg/d) with venetoclax in patients with progressive mCRPC assessed dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Three dose levels (DL) of venetoclax (DL1 400 mg/d; DL2 600 mg/d; and DL3 800 mg) were evaluated using a 3+3 design. We also analyzed enzalutamide and venetoclax pharmacokinetics and conducted pharmacodynamic studies in peripheral blood mononuclear cells (PBMCs) to determine the impact of venetoclax on BCL-2 expression. Results A total of 10 patients were enrolled across 3 DL and no DLT was observed. Mean duration on treatment was 29 weeks (range: 8-140 weeks). Treatment-related adverse events (TRAEs) were mostly grade 1-2, and Grade 3 TRAEs included hypertension (20%), fatigue (10%), and thrombocytopenia (10%). 1/10 (10%) attained PSA50 response and 4/10 (40%) had stable disease. Estimated median overall survival (OS) was 19 months (95% CI 5-28 months) and median time to next systemic therapy (TNST) was 5 months (95% CI 1-35 months). Pharmacokinetic results revealed sub-therapeutic plasma levels of venetoclax. Pharmacodynamic studies demonstrated that venetoclax enhanced BCL-2β generation and promoted BCL-2 degradation. Conclusions Enzalutamide with venetoclax has an acceptable toxicity profile in patients with mCRPC. Despite sub-therapeutic venetoclax levels, the treatment elicited pharmacodynamic and clinical response in a subset of patients.
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Affiliation(s)
- Stuthi Perimbeti
- Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Anmbreen Jamroze
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | | | - Rohit Jain
- Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Changchuan Jiang
- Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Julianne L Holleran
- Cancer Therapeutics Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA
| | - Robert A. Parise
- Cancer Therapeutics Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA
| | - Robert Bies
- Department of Pharmaceutical Sciences, University at Buffalo School of Pharmacy and Pharmaceutical Sciences, State University of New York at Buffalo, Buffalo, NY, USA
- Institute for Computational and Data Sciences, University at Buffalo, State University of New York at Buffalo, Buffalo, NY, USA
| | | | - Kristopher Attwood
- Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Xiaozhuo Liu
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Jason S. Kirk
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Jan H. Beumer
- Cancer Therapeutics Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA
- Division of Hematology/Oncology, Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
- Department of Pharmaceutical Sciences, School of Pharmacy, Pittsburgh, PA, USA
| | - Dean G. Tang
- Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
- Experimental Therapeutics Graduate Program, University at Buffalo and Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Gurkamal Chatta
- Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
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Cheng W, Tan L, Yu S, Song J, Li Z, Peng X, Wei Q, He Z, Zhang W, Yang X. Geniposide reduced oxidative stress-induced apoptosis in HK-2 cell through PI3K/AKT3/FOXO1 by m6A modification. Int Immunopharmacol 2024; 131:111820. [PMID: 38508092 DOI: 10.1016/j.intimp.2024.111820] [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: 01/01/2024] [Revised: 02/28/2024] [Accepted: 03/05/2024] [Indexed: 03/22/2024]
Abstract
Exogenous hydrogen peroxide (H2O2) may generate excessive oxidative stress, inducing renal cell apoptosis related with kidney dysfunction. Geniposide (GP) belongs to the iridoid compound with anti-inflammatory, antioxidant and anti-apoptotic effects. This study aimed to observe the intervention effect of GP on H2O2-induced apoptosis in human kidney-2 (HK-2) cells and to explore its potential mechanism in relation to N6-methyladenosine (m6A) RNA methylation. Cell viability, apotosis rate and cell cycle were tested separately after different treatments. The mRNA and protein levels of m6A related enzymes and phosphoinositide 3-kinase (PI3K)/a serine/threonine-specific protein kinase 3 (AKT3)/forkhead boxo 1 (FOXO1) and superoxide dismutase 2 (SOD2) were detected by reverse transcription-quantitative real-time PCR (RT-qPCR) and Western blot. The whole m6A methyltransferase activity and the m6A content were measured by ELISA-like colorimetric methods. The changes of m6A methylation levels of PI3K/AKT3/FOXO1 and SOD2 were determined by methylated RNA immunoprecipitation (MeRIP)-qPCR. Multiple comparisons were performed by ANOVA with Turkey's post hoc test. Exposed to 400 μmol/L H2O2, cells were arrested in G1 phase and the apoptosis rate increased, which were significantly alleviated by GP. Compared with the H2O2 apoptosis group, both the whole m6A RNA methyltransferase activity and the m6A contents were increased due to GP intervention. Besides, the SOD2 protein was increased, while PI3K and FOXO1 decreased. The m6A methylation level of AKT3 was negatively correlated with its protein level. Taken together, GP affects the global m6A methylation microenvironment and regulates the expression of PI3K/AKT3/FOXO1 signaling pathway via m6A modification, alleviating cell cycle arrest and apoptosis caused by oxidative stress in HK-2 cells with a good application prospect.
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Affiliation(s)
- Wenli Cheng
- Food Safety and Health Research Center, NMPA Key Laboratory for Safety Evaluation of Cosmetics, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou 510515, PR China; Department of Public Health and Preventive Medicine, School of Medicine, Jinan University, Guangzhou, Guangdong 510632, PR China
| | - Luyi Tan
- Department of Public Health and Preventive Medicine, School of Medicine, Jinan University, Guangzhou, Guangdong 510632, PR China
| | - Susu Yu
- Department of Public Health and Preventive Medicine, School of Medicine, Jinan University, Guangzhou, Guangdong 510632, PR China
| | - Jia Song
- Food Safety and Health Research Center, NMPA Key Laboratory for Safety Evaluation of Cosmetics, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou 510515, PR China
| | - Ziyin Li
- Food Safety and Health Research Center, NMPA Key Laboratory for Safety Evaluation of Cosmetics, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou 510515, PR China
| | - Xinyue Peng
- Department of Public Health and Preventive Medicine, School of Medicine, Jinan University, Guangzhou, Guangdong 510632, PR China
| | - Qinzhi Wei
- Food Safety and Health Research Center, NMPA Key Laboratory for Safety Evaluation of Cosmetics, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou 510515, PR China
| | - Zhini He
- Food Safety and Health Research Center, NMPA Key Laboratory for Safety Evaluation of Cosmetics, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou 510515, PR China
| | - Wenjuan Zhang
- Department of Public Health and Preventive Medicine, School of Medicine, Jinan University, Guangzhou, Guangdong 510632, PR China.
| | - Xingfen Yang
- Food Safety and Health Research Center, NMPA Key Laboratory for Safety Evaluation of Cosmetics, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou 510515, PR China.
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Erazo-Oliveras A, Muñoz-Vega M, Salinas ML, Wang X, Chapkin RS. Dysregulation of cellular membrane homeostasis as a crucial modulator of cancer risk. FEBS J 2024; 291:1299-1352. [PMID: 36282100 PMCID: PMC10126207 DOI: 10.1111/febs.16665] [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] [Received: 06/18/2022] [Revised: 09/09/2022] [Accepted: 10/24/2022] [Indexed: 11/07/2022]
Abstract
Cellular membranes serve as an epicentre combining extracellular and cytosolic components with membranous effectors, which together support numerous fundamental cellular signalling pathways that mediate biological responses. To execute their functions, membrane proteins, lipids and carbohydrates arrange, in a highly coordinated manner, into well-defined assemblies displaying diverse biological and biophysical characteristics that modulate several signalling events. The loss of membrane homeostasis can trigger oncogenic signalling. More recently, it has been documented that select membrane active dietaries (MADs) can reshape biological membranes and subsequently decrease cancer risk. In this review, we emphasize the significance of membrane domain structure, organization and their signalling functionalities as well as how loss of membrane homeostasis can steer aberrant signalling. Moreover, we describe in detail the complexities associated with the examination of these membrane domains and their association with cancer. Finally, we summarize the current literature on MADs and their effects on cellular membranes, including various mechanisms of dietary chemoprevention/interception and the functional links between nutritional bioactives, membrane homeostasis and cancer biology.
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Affiliation(s)
- Alfredo Erazo-Oliveras
- Program in Integrative Nutrition and Complex Diseases; Texas A&M University; College Station, Texas, 77843; USA
- Department of Nutrition; Texas A&M University; College Station, Texas, 77843; USA
| | - Mónica Muñoz-Vega
- Program in Integrative Nutrition and Complex Diseases; Texas A&M University; College Station, Texas, 77843; USA
- Department of Nutrition; Texas A&M University; College Station, Texas, 77843; USA
| | - Michael L. Salinas
- Program in Integrative Nutrition and Complex Diseases; Texas A&M University; College Station, Texas, 77843; USA
- Department of Nutrition; Texas A&M University; College Station, Texas, 77843; USA
| | - Xiaoli Wang
- Program in Integrative Nutrition and Complex Diseases; Texas A&M University; College Station, Texas, 77843; USA
- Department of Nutrition; Texas A&M University; College Station, Texas, 77843; USA
| | - Robert S. Chapkin
- Program in Integrative Nutrition and Complex Diseases; Texas A&M University; College Station, Texas, 77843; USA
- Department of Nutrition; Texas A&M University; College Station, Texas, 77843; USA
- Center for Environmental Health Research; Texas A&M University; College Station, Texas, 77843; USA
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Shi S, Wang J, Liu C, Zheng L. Developmental toxicity and inflammatory response induced by Botrytis cinerea in zebrafish (Danio rerio) larvae. Comp Biochem Physiol C Toxicol Pharmacol 2023; 267:109575. [PMID: 36813020 DOI: 10.1016/j.cbpc.2023.109575] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/13/2022] [Revised: 02/05/2023] [Accepted: 02/16/2023] [Indexed: 02/22/2023]
Abstract
Botrytis cinerea can reduce the yield of fruits and vegetables by infecting plants. The conidia produced by Botrytis cinerea can be transmitted to the aquatic environment via air and water, but the effects of Botrytis cinerea on aquatic animals is unknown. In this research, the influence of Botrytis cinerea on the development, inflammation, and apoptosis of zebrafish larvae and the underlying mechanism was evaluated. Results indicated that, compared with the control group, the larvae exposed to 101-103 CFU/mL of Botrytis cinerea spore suspension had a delayed hatching rate, lower head and eye area, shorter body length, and larger yolk sac at 72 h post-fertilization. In addition, the quantitative fluorescence intensity of treated larvae displayed a dose-dependent increase in apoptosis sign, revealing that Botrytis cinerea could generate apoptosis. Subsequently, zebrafish larvae were inflamed after exposure to Botrytis cinerea spore suspension, which was characterized as inflammatory infiltration and macrophage aggregation in the intestine. The enrichment of the pro-inflammatory factor TNF-α activated the NF-κB signaling pathway, generating the increase of the transcription level of target genes (jak3, pi3k, pdk1, akt, and ikk2) and the high expression of major proteins NF-κB (P65) in this pathway. Likewise, elevated content of TNF-α could activate JNK, which turned on the P53 apoptotic pathway, leading to a significant increase in the bax, caspase3, and caspase9 transcript abundances. This study demonstrated that Botrytis cinerea could cause developmental toxicity, morphological malformation, inflammation, and cell apoptosis in zebrafish larvae, which provided data support and a theoretical basis for ecological health risk assessment and filled the gap in biological research of Botrytis cinerea.
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Affiliation(s)
- Shengnan Shi
- Engineering Research Center of Bio-Process, Ministry of Education, School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China
| | - Ju Wang
- Engineering Research Center of Bio-Process, Ministry of Education, School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China
| | - Changhong Liu
- Engineering Research Center of Bio-Process, Ministry of Education, School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China.
| | - Lei Zheng
- Engineering Research Center of Bio-Process, Ministry of Education, School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China; Intelligent Interconnected Systems Laboratory of Anhui Province, Hefei University of Technology, Hefei 230009, China.
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BCL-2 isoform β promotes angiogenesis by TRiC-mediated upregulation of VEGF-A in lymphoma. Oncogene 2022; 41:3655-3663. [PMID: 35701534 DOI: 10.1038/s41388-022-02372-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2022] [Revised: 05/25/2022] [Accepted: 05/30/2022] [Indexed: 02/05/2023]
Abstract
Bcl-2 (B-cell lymphoma 2), the first identified anti-apoptosis factor, encodes two transcripts, the long isoform α and the short isoform β. The current understanding of the Bcl-2 function mainly focuses on Bcl-2α, while little is known about the function of Bcl-2β, which lacks the transmembrane domain and contains 10 unique amino acids at the C-terminus instead. Here, we analyzed the expressions of BCL-2 two isoforms in diffused large B-cell lymphoma (DLBCL) and found a significant positive correlation between them. Then, with the CRISPR/Cas9-based transcriptional activator (CRISPRa), we generated mouse B-cell lymphomas with Bcl-2 upregulation from the endogenous locus, in which both Bcl-2α and Bcl-2β levels were increased. Bcl-2β itself promoted angiogenesis both in vitro and in vivo through increased vascular endothelial growth factor A (VEGF-A). Inhibiting VEGF receptors with Axitinib reduced angiogenesis induced by Bcl-2β overexpression. Co-immunoprecipitation and mass spectrometry analysis revealed that Bcl-2β interacted with the T-complex protein ring complex (TRiC). Disruption of TRiC significantly impaired the angiogenesis-promoting activity of Bcl-2β, indicated by reduced VEGF-A protein level and HUVEC tube formation. Thus, our study suggests that Bcl-2 isoform β plays a role in promoting tumor angiogenesis through the Bcl-2β-TRiC-VEGF-A axis.
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BCL-2 family isoforms in apoptosis and cancer. Cell Death Dis 2019; 10:177. [PMID: 30792387 PMCID: PMC6384907 DOI: 10.1038/s41419-019-1407-6] [Citation(s) in RCA: 453] [Impact Index Per Article: 75.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2018] [Revised: 12/17/2018] [Accepted: 01/29/2019] [Indexed: 12/17/2022]
Abstract
The BCl-2 family has long been identified for its role in apoptosis. Following the initial discovery of BCL-2 in the context of B-cell lymphoma in the 1980s, a number of homologous proteins have since been identified. The members of the Bcl-2 family are designated as such due to their BCL-2 homology (BH) domains and involvement in apoptosis regulation. The BH domains facilitate the family members’ interactions with each other and can indicate pro- or anti-apoptotic function. Traditionally, these proteins are categorised into one of the three subfamilies; anti-apoptotic, BH3-only (pro-apoptotic), and pore-forming or ‘executioner’ (pro-apoptotic) proteins. Each of the BH3-only or anti-apoptotic proteins has a distinct pattern of activation, localisation and response to cell death or survival stimuli. All of these can vary across cell or stress types, or developmental stage, and this can cause the delineation of the roles of BCL-2 family members. Added to this complexity is the presence of relatively uncharacterised isoforms of many of the BCL-2 family members. There is a gap in our knowledge regarding the function of BCL-2 family isoforms. BH domain status is not always predictive or indicative of protein function, and several other important sequences, which can contribute to apoptotic activity have been identified. While therapeutic strategies targeting the BCL-2 family are constantly under development, it is imperative that we understand the molecules, which we are attempting to target. This review, discusses our current knowledge of anti-apoptotic BCL-2 family isoforms. With significant improvements in the potential for splicing therapies, it is important that we begin to understand the distinctions of the BCL-2 family, not limited to just the mechanisms of apoptosis control, but in their roles outside of apoptosis.
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Sekhar SC, Venkatesh J, Cheriyan VT, Muthu M, Levi E, Assad H, Meister P, Undyala VV, Gauld JW, Rishi AK. A H2AX⁻CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage. Cancers (Basel) 2019; 11:cancers11020221. [PMID: 30769864 PMCID: PMC6406907 DOI: 10.3390/cancers11020221] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2018] [Revised: 02/07/2019] [Accepted: 02/10/2019] [Indexed: 12/16/2022] Open
Abstract
Cell Cycle and Apoptosis Regulatory Protein (CARP-1/CCAR1) is a peri-nuclear phosphoprotein that regulates apoptosis via chemotherapeutic Adriamycin (doxorubicin) and a novel class of CARP-1 functional mimetic (CFM) compounds. Although Adriamycin causes DNA damage, data from Comet assays revealed that CFM-4.16 also induced DNA damage. Phosphorylation of histone 2AX (γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Adriamycin or CFM-4.16 treatments inhibited cell growth and caused elevated CARP-1 and γH2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished γH2AX, their co-localization, and apoptosis in CFM-4.16- or Adriamycin-treated cells. We found that CARP-1 directly binds with H2AX, and H2AX interacted with CARP-1, but not CARP-1 (Δ600–652) mutant. Moreover, cells expressing CARP-1 (Δ600–652) mutant were resistant to apoptosis, and had diminished levels of γH2AX, when compared with cells expressing wild-type CARP-1. Mutagenesis studies revealed that H2AX residues 1–35 harbored a CARP-1-binding epitope, while CARP-1 amino acids 636–650 contained an H2AX-interacting epitope. Surface plasmon resonance studies revealed that CARP-1 (636–650) peptide bound with H2AX (1–35) peptide with a dissociation constant (Kd) of 127 nM. Cells expressing enhanced GFP (EGFP)-tagged H2AX (1–35) peptide or EGFP-tagged CARP-1 (636–650) peptide were resistant to inhibition by Adriamycin or CFM-4.16. Treatment of cells with transactivator of transcription (TAT)-tagged CARP-1 (636–650) peptide resulted in a moderate, statistically significant abrogation of Adriamycin-induced growth inhibition of cancer cells. Our studies provide evidence for requirement of CARP-1 interaction with H2AX in apoptosis signaling by Adriamycin and CFM compounds.
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Affiliation(s)
- Sreeja C Sekhar
- John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201, USA.
- Department of Oncology, Karmanos Cancer Institute, Detroit, MI 48201, USA.
| | - Jaganathan Venkatesh
- John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201, USA.
- Department of Oncology, Karmanos Cancer Institute, Detroit, MI 48201, USA.
| | - Vino T Cheriyan
- John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201, USA.
- Department of Oncology, Karmanos Cancer Institute, Detroit, MI 48201, USA.
| | - Magesh Muthu
- John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201, USA.
- Department of Oncology, Karmanos Cancer Institute, Detroit, MI 48201, USA.
| | - Edi Levi
- John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201, USA.
| | - Hadeel Assad
- Department of Oncology, Karmanos Cancer Institute, Detroit, MI 48201, USA.
| | - Paul Meister
- Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON N9B 3P4, Canada.
| | - Vishnu V Undyala
- Cardiovascular Research Institute, School of Medicine, Wayne State University, Detroit, MI 48201, USA.
| | - James W Gauld
- Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON N9B 3P4, Canada.
| | - Arun K Rishi
- John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201, USA.
- Department of Oncology, Karmanos Cancer Institute, Detroit, MI 48201, USA.
- Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, MI 48201, USA.
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Wang L, Zhang H, Wang Y, Wang F, Liu X, Wu Y, Hua S, Quan F, Zhang Y. Peroxiredoxin 5 is essential for in vitro development of bovine SCNT embryos. Theriogenology 2017; 92:156-166. [DOI: 10.1016/j.theriogenology.2016.09.020] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2016] [Revised: 09/08/2016] [Accepted: 09/09/2016] [Indexed: 01/13/2023]
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Luzio A, Matos M, Santos D, Fontaínhas-Fernandes AA, Monteiro SM, Coimbra AM. Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures. AQUATIC TOXICOLOGY (AMSTERDAM, NETHERLANDS) 2016; 177:269-284. [PMID: 27337697 DOI: 10.1016/j.aquatox.2016.05.029] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/08/2016] [Revised: 05/24/2016] [Accepted: 05/31/2016] [Indexed: 06/06/2023]
Abstract
Zebrafish (Danio rerio) sex determination seems to involve genetic factors (GSD) but also environmental factors (ESD), such as endocrine disrupting chemicals (EDCs) that are known to mimic endogenous hormones and disrupt gonad differentiation. Apoptosis has also been proposed to play a crucial role in zebrafish gonad differentiation. Nevertheless, the interactions between EDCs and apoptosis have received little attention. Thus, this study aimed to assess if and which apoptotic pathways are involved in zebrafish gonad differentiation and how EDCs may interfere with this process. With these purposes, zebrafish were exposed to 17α-ethinylestradiol (EE2, 4ng/L) and fadrozole (Fad, 50μg/L) from 2h to 35days post-fertilization (dpf). Afterwards, a gene expression analysis by qRT-PCR and a stereological analysis, based on systematic sampling and protein immunohistochemistry, were performed. The death receptors (FAS; TRADD), anti-apoptotic (BCL-2; MDM2), pro-apoptotic (CASP-2 and -6) and cell proliferation (BIRC5/survivin; JUN) genes and proteins were evaluated. In general, apoptosis was inhibited in females through the involvement of anti-apoptotic pathways, while in males apoptosis seemed to be crucial to the failure of the "juvenile ovary" development and the induction of testes transformation. The JUN protein was shown to be necessary in juvenile ovaries, while the BIRC5 protein seemed to be involved in zebrafish spermatogenesis. Both EDCs, EE2 and Fad, increased the apoptosis stimulus in zebrafish gonad. It was noticed that the few females that were resistant to Fad-induced sex reversal had increased anti-apoptotic factor levels, while males exposed to EE2 showed increased pro-apoptotic genes/proteins and were more advanced in gonad differentiation. Overall, our findings show that apoptosis pathways are involved in zebrafish gonad differentiation and that EDCs can disrupt this process.
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Affiliation(s)
- Ana Luzio
- Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal; Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal.
| | - Manuela Matos
- University of Lisbon, Faculty of Sciences, BioISI- Biosystems & Integrative Sciences Institute, Campo Grande, 1749-016 Lisbon, Portugal; Department of Genetics and Biotechnology, Life Sciences and Environment School (ECVA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal
| | - Dércia Santos
- Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal
| | - António A Fontaínhas-Fernandes
- Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal; Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal
| | - Sandra M Monteiro
- Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal; Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal
| | - Ana M Coimbra
- Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal; Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal.
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Liu K, Jiang T, Ouyang Y, Shi Y, Zang Y, Li N, Lu S, Chen D. Nuclear EGFR impairs ASPP2-p53 complex-induced apoptosis by inducing SOS1 expression in hepatocellular carcinoma. Oncotarget 2016; 6:16507-16. [PMID: 25980493 PMCID: PMC4599285 DOI: 10.18632/oncotarget.3757] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2014] [Accepted: 04/15/2015] [Indexed: 01/26/2023] Open
Abstract
ASPP2 can bind to p53 and enhance the apoptotic capabilities of p53 by guiding it to the promoters of pro-apoptotic genes. Here, ASPP2 overexpression for 24 hours transiently induced apoptosis in hepatoma cells by enhancing the transactivation of p53 on pro-apoptotic gene promoters. However, long-term ASPP2 overexpression (more than 48 hours) failed to induce apoptosis because p53 was released from the pro-apoptotic gene promoters. In non-apoptotic cells, nuclear EGFR induced SOS1 expression by directly binding to the SOS1 promoter. SOS1 activated the HRAS/PI3K/AKT pathway and resulted in nuclear translocation of p-AKT and Bcl-2. The interaction between p-AKT and ASPP2 facilitates Bcl-2 binding to p53, which releases p53 from the pro-apoptotic gene promoters. The in vivo assay demonstrated that EGFR/SOS1-promoted growth of nuclear p-AKT+, Bcl-2+ cells results in the resistance of hepatoma cells to ASPP2-p53 complex-induced apoptosis and that blocking nuclear translocation of EGFR dramatically improves and enhances the pro-apoptotic function of ASPP2. Finally, the activation of the HRAS/PI3K/AKT pathway by EGFR-induced SOS1 also inhibits cisplatin-induced apoptosis, suggesting a common apoptosis-evasion mechanism in hepatoma cells. Because evasion of apoptosis contributes to treatment resistance in hepatoma, our results also support further investigation of combined therapeutic blockade of EGFR and SOS1.
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Affiliation(s)
- Kai Liu
- Beijing You'an Hospital, Capital Medical University, Beijing, 100069, China.,Beijing Institute of Hepatology, Beijing, 100069, China
| | - Tao Jiang
- Beijing You'an Hospital, Capital Medical University, Beijing, 100069, China
| | - Yabo Ouyang
- Beijing You'an Hospital, Capital Medical University, Beijing, 100069, China.,Beijing Institute of Hepatology, Beijing, 100069, China
| | - Ying Shi
- Beijing You'an Hospital, Capital Medical University, Beijing, 100069, China.,Beijing Institute of Hepatology, Beijing, 100069, China
| | - Yunjin Zang
- Beijing You'an Hospital, Capital Medical University, Beijing, 100069, China
| | - Ning Li
- Beijing You'an Hospital, Capital Medical University, Beijing, 100069, China
| | - Shichun Lu
- Beijing You'an Hospital, Capital Medical University, Beijing, 100069, China
| | - Dexi Chen
- Beijing You'an Hospital, Capital Medical University, Beijing, 100069, China.,Beijing Institute of Hepatology, Beijing, 100069, China
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11
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Sec6/8 regulates Bcl-2 and Mcl-1, but not Bcl-xl, in malignant peripheral nerve sheath tumor cells. Apoptosis 2016; 21:594-608. [DOI: 10.1007/s10495-016-1230-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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12
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Braun F, de Carné Trécesson S, Bertin-Ciftci J, Juin P. Protect and serve: Bcl-2 proteins as guardians and rulers of cancer cell survival. Cell Cycle 2013; 12:2937-47. [PMID: 23974114 PMCID: PMC3875667 DOI: 10.4161/cc.25972] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
It is widely accepted that anti-apoptotic Bcl-2 family members promote cancer cell survival by binding to their pro-apoptotic counterparts, thereby preventing mitochondrial outer membrane permeabilization (MOMP) and cytotoxic caspase activation. Yet, these proteins do not only function as guardians of mitochondrial permeability, preserving it, and maintaining cell survival in the face of acute or chronic stress, they also regulate non-apoptotic functions of caspases and biological processes beyond MOMP from diverse subcellular localizations and in complex with numerous binding partners outside of the Bcl-2 family. In particular, some of the non-canonical effects and functions of Bcl-2 homologs lead to an interplay with E2F-1, NFκB, and Myc transcriptional pathways, which themselves influence cancer cell growth and survival. We thus propose that, by feedback loops that we currently have only hints of, Bcl-2 proteins may act as rulers of survival signaling, predetermining the apoptotic threshold that they also directly scaffold. This underscores the robustness of the control exerted by Bcl-2 homologs over cancer cell survival, and implies that small molecules compounds currently used in the clinic to inhibit their mitochondrial activity may be not always be fully efficient to override this control.
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Affiliation(s)
- Frédérique Braun
- UMR 892 INSERM/6299 CNRS/Université de Nantes; Team 8 "Cell survival and tumor escape in breast cancer"; Institut de Recherche en Santé de l'Université de Nantes; Nantes, France
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13
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Levi E, Zhang L, Aboukameel A, Rishi S, Mohammad RM, Polin L, Hatfield JS, Rishi AK. Cell cycle and apoptosis regulatory protein (CARP)-1 is a novel, adriamycin-inducible, diffuse large B-cell lymphoma (DLBL) growth suppressor. Cancer Chemother Pharmacol 2010; 67:1401-13. [PMID: 20809119 DOI: 10.1007/s00280-010-1442-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2010] [Accepted: 08/17/2010] [Indexed: 11/27/2022]
Abstract
UNLABELLED Diffuse large B-cell lymphoma (DLCL) accounts for 30-40% of adult non-Hodgkin's Lymphoma (NHL). Current anti-NHL therapies often target cellular growth suppression pathways and include R-CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone plus monoclonal anti-CD20 antibody rituximab). However, since many patients relapse, resistant cells to these therapies remain a significant problem and necessitate development of new intervention strategies. Cell cycle and apoptosis regulatory protein (CARP)-1 functions in a biphasic manner to regulate growth factor as well as chemotherapy (adriamycin, etoposide, or iressa)-dependent signaling. PURPOSE To determine whether CARP-1 is a novel suppressor of lymphoma growth. METHODS Flow cytometric analyses coupled with Western immunoblotting, cell growth, apoptosis, and immunocytochemistry methodologies were utilized to determine CARP-1-dependent lymphoma growth inhibition in vitro and in vivo. RESULTS CARP-1 expression correlated with activated caspase-3 and inversely correlated with activated Akt in DLCL. Exposure to adriamycin stimulated CARP-1 expression and inhibited growth of Raji cells, but not CHOP-resistant WSU-DLCL2 cells. Expression of wild-type CARP-1 or its apoptosis-inducing mutants inhibited growth of Raji as well as CHOP-resistant WSU-DLCL2 cells, in part by activating caspase-9 and apoptosis. Since CARP-1 harbors multiple, apoptosis-promoting subdomains, we investigated whether epigenetic compensation of CARP-1 function by intracellular delivery of trans-activator of transcription (TAT) domain-tagged CARP-1 peptide(s) will inhibit lymphoma growth. Treatments with TAT-tagged CARP-1 peptides suppressed growth of the Raji and WSU-DLCL2 cells by stimulating apoptosis. TAT-CARP-1 (1-198) as well as (896-1150) peptides also suppressed growth of WSU-DLCL2 cell-derived tumor xenografts in SCID mice, while administration of TAT-CARP-1 (1-198) also inhibited growth of WSU-FSCCL cell-derived ascites and prolonged host survival. CONCLUSION CARP-1 is a suppressor of NHL growth and could be exploited for targeting the resistant DLCL.
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Affiliation(s)
- Edi Levi
- Room B4334, Veterans Affairs Medical Center, Wayne State University, 4646 John R, Detroit, MI 48201, USA
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14
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Deerberg A, Sosna J, Thon L, Belka C, Adam D. Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death. Radiat Oncol 2009; 4:41. [PMID: 19818125 PMCID: PMC2764721 DOI: 10.1186/1748-717x-4-41] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2009] [Accepted: 10/09/2009] [Indexed: 01/05/2023] Open
Abstract
Background Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist. Methods We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 ΔTM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology. Results Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD. Conclusion Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide.
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Affiliation(s)
- Andrea Deerberg
- Institut für Immunologie, Christian-Albrechts-Universität Kiel, 24105 Kiel, Germany.
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15
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Choi HJ, Lee JH, Park SY, Cho JH, Han JS. STAT3 is involved in phosphatidic acid-induced Bcl-2 expression in HeLa cells. Exp Mol Med 2009; 41:94-101. [PMID: 19287190 DOI: 10.3858/emm.2009.41.2.012] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Abstract
Phosphatidic acid (PA), the product of a PLD-mediated reaction, is a lipid second messenger that participates in various intracellular signaling events and is known to regulate a growing list of signaling proteins. We found that Bcl-2 was upregulated by PA treatment in HeLa cells. However, how PA upregulates Bcl-2 expression has not yet been studied. In this study, we tried to discover the mechanisms of Bcl-2 up-regulation by PA treatment in HeLa cells. Treatment with PA resulted in significantly increased expression of Bcl-2 in HeLa cells. Moreover, PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of PLA2, but not by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Treatment of 1,2-dipalmitoryl-sn-glycero-3- phosphate (DPPA) also increased Bcl-2 expression. These results indicate that Bcl-2 expression is mediated by lysophosphatidic acid (LPA), not by arachidonic acid (AA). Thereafter, we used MEK1/2 inhibitor, PD98059 to investigate the relationship between ERK1/2 MAPK and PA-induced Bcl-2 expression. PA-induced Bcl-2 expression was decreased when ERK1/2 was inhibited by PD98059. The transcription factor such as STAT3 which is controlled by ERK1/2 MAPK was increased along with Bcl-2 expression when the cells were treated with PA. Furthermore, STAT3 siRNA treatments inhibited PA-induced Bcl-2 expression, suggesting that STAT3 (Ser727) is involved in PA-induced Bcl-2 expression. Taken together, these findings indicate that PA acts as an important mediator for increasing Bcl-2 expression through STAT3 (Ser727) activation via the ERK1/2 MAPK pathway.
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Affiliation(s)
- Hye-Jin Choi
- Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul 133-791, Korea
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16
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Spurgers KB, Gold DL, Coombes KR, Bohnenstiehl NL, Mullins B, Meyn RE, Logothetis CJ, McDonnell TJ. Identification of cell cycle regulatory genes as principal targets of p53-mediated transcriptional repression. J Biol Chem 2006; 281:25134-42. [PMID: 16798743 DOI: 10.1074/jbc.m513901200] [Citation(s) in RCA: 94] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Historically, most studies attribute p53 function to the transactivation of target genes. That p53 can selectively repress genes to affect a cellular response is less widely appreciated. Available evidence suggests that repression is important for p53-induced apoptosis and cell cycle arrest. To better establish the scope of p53-repressed target genes and the cellular processes they may affect, a global expression profiling strategy was used to identify p53-responsive genes following adenoviral p53 gene transfer (Ad-p53) in PC3 prostate cancer cells. A total of 111 genes, 0.77% of the 14,500 genes represented on the Affymetrix U133A microarray, were repressed more than 2-fold (p < or = 0.05). Validation of the array data, using reverse transcription-PCR of 20 randomly selected genes, yielded a confirmation rate of >95.5% for the complete data set. Functional over-representation analysis revealed that cell cycle regulatory genes exhibited a highly significant enrichment (p < or = 5 x 10(-28)) within the transrepressed targets. 41% of the repressed targets are cell cycle regulators. A subset of these genes exhibited repression following DNA damage, preceding cell cycle arrest, in LNCaP cells. The use of a p53 small interfering RNA strategy in LNCaP cells and the use of p53-null cell lines demonstrated that this repression is p53-dependent. These findings identify a set of genes not known previously to be down-regulated by p53 and indicate that p53-induced cell cycle arrest is a function of not only the transactivation of cell cycle inhibitors (e.g. p21) but also the repression of targets that regulate proliferation at several distinct phases of the cell cycle.
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Affiliation(s)
- Kevin B Spurgers
- Department of Molecular Pathology, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA
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17
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Itamochi H, Yamasaki F, Sudo T, Takahashi T, Bartholomeusz C, Das S, Terakawa N, Ueno NT. Reduction of radiation-induced apoptosis by specific expression of Bcl-2 in normal cells. Cancer Gene Ther 2005; 13:451-9. [PMID: 16294215 DOI: 10.1038/sj.cgt.7700920] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Radiation-induced apoptosis is thought to underlie the toxicity of radiation to normal tissues as well as to cancer cells. We hypothesized that specific ectopic overexpression of the antiapoptotic molecule Bcl-2 in normal cells would inhibit radiation-induced apoptosis and thereby reduce radiation-induced toxicity in normal cells. To express Bcl-2 specifically in normal cells (which have wild-type (wt) p53) but not in cancer cells (which often have mutated p53), we constructed a Bcl-2 expression plasmid (PG13-Bcl-2) with a minimal promoter regulated by multiple wt p53 DNA-binding sites and found that the presence of wt p53 protein strongly upregulated Bcl-2 expression through this plasmid. Transfection of NIH 3T3 fibroblasts, which express wt p53, with PG13-Bcl-2 increased cell survival and reduced apoptosis; however, transfection of MDA-MB-231 breast cancer cells, which have mutated p53, did not affect survival and apoptosis of those cells. These results indicate that irradiation of normal cells rapidly upregulates the expression of wt p53, which binds to the p53 binding sequence of the PG13-Bcl-2 plasmid and increases the transcriptional activity of Bcl-2. Ectopic expression of Bcl-2 reduced radiation-induced apoptosis only in normal cells (not in cancer cells). Bcl-2 expression was detected in the lung from mice injected via a tail vein with LPD-PG13-Bcl-2 or LPD-CMV-Bcl-2, but did not in the lung from mice treated with DOTAP or LPD-PG13-mock. This novel approach to inhibiting radiation-induced apoptosis in normal cells may allow such cells to be protected from radiation-induced toxicity. Further preclinical in vivo studies are needed.
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Affiliation(s)
- H Itamochi
- Breast Cancer Translational Research Laboratory, Houston, TX, USA
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18
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Thavathiru E, Ludes-Meyers JH, MacLeod MC, Aldaz CM. Expression of common chromosomal fragile site genes, WWOX/FRA16D and FHIT/FRA3B is downregulated by exposure to environmental carcinogens, UV, and BPDE but not by IR. Mol Carcinog 2005; 44:174-82. [PMID: 16187332 PMCID: PMC4166602 DOI: 10.1002/mc.20122] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Common chromosomal fragile sites are unstable genomic loci susceptible to breakage, rearrangement, and are highly recombinogenic. Frequent alterations at these loci in tumor cells led to the hypothesis that they may contribute to cancer development. The two most common chromosomal fragile sites FRA16D and FRA3B which harbor WWOX and FHIT genes, respectively, are frequently altered in human cancers. Here we report that environmental carcinogens, ultraviolet (UV) light, and Benzo[a]pyrene diol epoxide (BPDE), significantly downregulate expression of both genes. On the other hand, we observe that ionizing radiation (IR) does not affect expression of these genes, suggesting that the effect of repression exerted by UV and BPDE is not just a consequence of DNA damage but may be a result of different signaling pathways triggered by specific DNA lesions. Such downregulation correlates with an induction of an S-phase delay in the cell cycle. Treatment of UV-irradiated cells with caffeine abrogates the S-phase delay while concomitantly overcoming the repression phenomenon. This suggests the involvement of unique cell cycle checkpoint mechanisms in the observed repression. Therefore, it is hypothesized that protracted downregulation of the putative tumor suppressor genes WWOX and FHIT by environmental carcinogens may constitute an additional mechanism of relevance in the initiation of tumorigenesis.
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Affiliation(s)
- Elangovan Thavathiru
- Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, USA
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Valverde M, Rojas E, Kala SV, Kala G, Lieberman MW. Survival and cell death in cells constitutively unable to synthesize glutathione. Mutat Res 2005; 594:172-80. [PMID: 16239016 DOI: 10.1016/j.mrfmmm.2005.08.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2005] [Accepted: 08/29/2005] [Indexed: 01/02/2023]
Abstract
We examined the role of GSH in survival and cell death using GCS-2 cells that are deficient in glutamate cysteine ligase (gamma-glutamyl cysteine synthetase, gammaGCS), an enzyme essential for GSH synthesis. Cells maintained in 2.5 mM GSH have GSH levels that are approximately 2% of wild type and grow indefinitely; however, they express both pro- and anti-apoptotic Bcl-2 family members and have detectable levels of cytoplasmic cytochrome C. Withdrawal of GSH from the medium results in a fall in intracellular GSH to undetectable levels, decreased mitochondrial dehydrogenase activity, decreased anti-apoptotic factor RNAs, increased pro-apoptotic factor RNAs, additional cytochrome C release, and a fall in ATP levels; however, cells continue to grow for another 24h. At 48 h, these trends continue with the exception that mitochondrial membrane potential and ATP levels rise; DNA fragmentation begins at 48 h. Thus, severe reduction of GSH to 2% of wild type produces a metastable state compatible with survival, but complete absence of GSH triggers apoptosis.
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Affiliation(s)
- Mahara Valverde
- Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
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20
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Palmer AE, Jin C, Reed JC, Tsien RY. Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci U S A 2004; 101:17404-9. [PMID: 15585581 PMCID: PMC535104 DOI: 10.1073/pnas.0408030101] [Citation(s) in RCA: 511] [Impact Index Per Article: 24.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The endoplasmic reticulum (ER) serves as a cellular storehouse for Ca(2+), and Ca(2+) released from the ER plays a role in a host of critical signaling reactions, including exocytosis, contraction, metabolism, regulation of transcription, fertilization, and apoptosis. Given the central role played by the ER, our understanding of these signaling processes could be greatly enhanced by the ability to image [Ca(2+)](ER) directly in individual cells. We created a genetically encoded Ca(2+) indicator by redesigning the binding interface of calmodulin and a calmodulin-binding peptide. The sensor has improved reaction kinetics and a K(d) ideal for imaging Ca(2+) in the ER and is no longer perturbed by large excesses of native calmodulin. Importantly, it provides a significant improvement over all previous methods for monitoring [Ca(2+)](ER) and has been used to directly show that, in MCF-7 breast cancer cells, the antiapoptotic protein B cell lymphoma 2 (Bcl-2) (i) lowers [Ca(2+)](ER) by increasing Ca(2+) leakage under resting conditions and (ii) alters Ca(2+) oscillations induced by ATP, and that acute inhibition of Bcl-2 by the green tea compound epigallocatechin gallate results in an increase in [Ca(2+)](ER) due to inhibition of Bcl-2-mediated Ca(2+) leakage.
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Affiliation(s)
- Amy E Palmer
- Department of Pharmacology and Howard Hughes Medical Institute, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0647, USA
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Massaad CA, Portier BP, Taglialatela G. Inhibition of transcription factor activity by nuclear compartment-associated Bcl-2. J Biol Chem 2004; 279:54470-8. [PMID: 15471874 DOI: 10.1074/jbc.m407659200] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Using a reporter gene assay in PC12, HEK293, HeLa, and NIH-3T3 cells, we show that the anti-apoptotic protein Bcl-2 significantly inhibits transcriptional activation of various transcription factors, including NF kappa B, AP1, CRE, and NFAT. A Bcl-2 mutant lacking its BH4 domain (Delta BH4) also inhibited transcription, whereas a Bcl-2 mutant lacking its transmembrane domain (Delta TM) was ineffective. Furthermore, Bcl-2 chimeric proteins containing transmembrane domains from the mitochondrial protein monoamine oxidase B (MaoB) or the endoplasmic reticulum protein cytochrome b(5) showed no effect on transcription factor activity. Subcellular localization studies showed that under conditions of transient transfection, the active Bcl-2 forms (wild type and Delta BH4) were predominantly found in the nuclear fraction, whereas the non-active forms (Delta TM, MaoB, and cytochrome b(5)) were in the non-nuclear fraction. Additionally, stably expressed Bcl-2 loses its ability to inhibit transcriptional activation and localizes predominantly to the non-nuclear fraction. Expression of FKBP38 (a chaperone that shuttles Bcl-2 to the mitochondria) removes co-expressed Bcl-2 from the nuclear fraction and reverses its effect on transcription factor activity. Finally, using an inducible gene expression system, we show that nuclear compartment-associated Bcl-2 prevents entry of NF kappa B subunits to the nucleus without affecting NF kappa B release from its cytosolic inhibitory sub-unit I kappa B alpha. These results suggest that (a) Bcl-2 suppresses transcriptional activity of multiple transcription factors; (b) Bcl-2 does not interfere with NF kappa B activation but prevents entrance of its active subunits to the nucleus; (c) membrane anchoring is required for this function of Bcl-2; and (d) association of Bcl-2 with the nuclear compartment is also necessary. We speculate that nuclear compartment-associated Bcl-2 may affect nuclear trafficking of multiple factors necessary for transcriptional activity.
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Affiliation(s)
- Cynthia A Massaad
- Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-1043, USA
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Feng H, Xiang H, Mao YW, Wang J, Liu JP, Huang XQ, Liu Y, Liu SJ, Luo C, Zhang XJ, Liu Y, Li DWC. Human Bcl-2 activates ERK signaling pathway to regulate activating protein-1, lens epithelium-derived growth factor and downstream genes. Oncogene 2004; 23:7310-21. [PMID: 15326476 DOI: 10.1038/sj.onc.1208041] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The proto-oncogene, bcl-2, has various functions besides its role in protecting cells from apoptosis. One of the functions is to regulate expression of other genes. Previous studies have demonstrated that Bcl-2 regulates activities of several important transcription factors including NF-kappaB and p53, and also their downstream genes. In our recent studies, we reported that Bcl-2 substantially downregulates expression of the endogenous alphaB-crystallin gene through modulating the transcriptional activity of lens epithelium-derived growth factor (LEDGF). In the present communication, we report that human Bcl-2 can positively regulate expression of the proto-oncogenes c-jun and c-fos. Moreover, it enhances the DNA binding activity and transactivity of the activating protein-1 (AP-1). Furthermore, we present evidence to show that Bcl-2 can also activate both ERK1 and ERK2 MAP kinases. Inhibition of the activities of these kinases or the upstream activating kinases by pharmacological inhibitors or dominant-negative mutants abolishes the Bcl-2-mediated regulation of AP-1, LEDGF and their downstream genes. Together, our results demonstrate that through activation of the ERK kinase signaling pathway, Bcl-2 regulates the transcriptional activities of multiple transcription factors, and hence modulates the expression of their downstream genes. Thus, our results provide a mechanism to explain how Bcl-2 may regulate expression of other genes.
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Affiliation(s)
- Hao Feng
- College of Life Sciences, Hunan Normal University, Changsha, PR China
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Kumar M, Liu ZR, Thapa L, Wang DY, Tian R, Qin RY. Mechanisms of inhibition of growth of human pancreatic carcinoma implanted in nude mice by somatostatin receptor subtype 2. Pancreas 2004; 29:141-51. [PMID: 15257106 DOI: 10.1097/00006676-200408000-00009] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
OBJECTIVES Several studies reported that somatostatin receptor subtypes, especially subtype 2 (SSTR2), exerted their cytostatic and/or cytotoxic effects on various types of tumors. The aim of this study was to investigate the antitumor effect of SSTR2 gene transfer to the pancreatic cancer cell line PC-3 and the mechanisms involved in this effect. METHODS The full-length human SSTR2 cDNA was introduced into pancreatic cancer cell line PC-3 by lipofectamine-mediated transfection; positive clones were screened by G418, and stable expression of SSTR2 was detected by the immunohistochemical SABC method and RT-PCR. Athymic mice were separately xenografted with SSTR2-expressing cells (experimental group), vector control, and mock control cells. TUNEL assay was used to determine the apoptotic index (AI) in the tumors of these groups. The immunohistochemical SP method was used to determine expression of apoptosis-regulating genes Bcl-2 and Bax and re-expression of SSTR2 and to assess intratumoral microvessel density (MVD). Moreover, tumor volume and weight were compared among these 3 groups. RESULTS Restoration of SSTR2 was observed in the experimental group both in vitro and in vivo. The AI was significantly higher in the experimental group (3.39 +/- 0.84%) compared with that in the vector control (0.69 +/- 0.08%) and mock control (0.68 +/- 0.09%) (P < 0.05). MVD was significantly lower in the experimental group (6.30 +/- 1.71) than that in the vector control (12.64 +/- 1.69) and mock control (13.50 +/- 1.86) (P < 0.05). Furthermore, a significant decrease in Bcl-2 and increase in Bax protein expression were detected in the experimental group compared with the vector control and mock control (P < 0.05). A significant negative correlation of protein expression between Bcl-2/Bax ratio and SSTR2 was observed in these tumors (P < 0.05). Tumor volume and weight were significantly decreased in the experimental group compared with the vector control and mock control (P < 0.05) groups. However, no significant differences were observed between the vector control and mock control (P > 0.05). CONCLUSION Re-expression of the SSTR2 gene, the expression of which is frequently lost in human pancreatic adenocarcinoma, induces apoptosis, which may be mediated via down-regulation of Bcl-2 and up-regulation of Bax (alteration of Bcl-2/Bax ratio) and inhibits tumor angiogenesis in pancreatic carcinoma, resulting in inhibition of tumor growth.
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MESH Headings
- Adenocarcinoma/blood supply
- Adenocarcinoma/pathology
- Adenocarcinoma/therapy
- Animals
- Apoptosis
- Gene Expression Regulation, Neoplastic
- Genes, bcl-2
- Genetic Therapy
- Humans
- Male
- Mice
- Mice, Inbred BALB C
- Mice, Nude
- Neovascularization, Pathologic/prevention & control
- Pancreatic Neoplasms/blood supply
- Pancreatic Neoplasms/pathology
- Pancreatic Neoplasms/therapy
- Proto-Oncogene Proteins c-bcl-2/biosynthesis
- Proto-Oncogene Proteins c-bcl-2/genetics
- RNA, Messenger/biosynthesis
- RNA, Messenger/genetics
- Receptors, Somatostatin/biosynthesis
- Receptors, Somatostatin/genetics
- Receptors, Somatostatin/physiology
- Recombinant Fusion Proteins/physiology
- Reverse Transcriptase Polymerase Chain Reaction
- Transfection
- Xenograft Model Antitumor Assays
- bcl-2-Associated X Protein
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Affiliation(s)
- Manoj Kumar
- Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
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Cummings BS, McHowat J, Schnellmann RG. Role of an endoplasmic reticulum Ca2+-independent phospholipase A2 in cisplatin-induced renal cell apoptosis. J Pharmacol Exp Ther 2004; 308:921-8. [PMID: 14634037 DOI: 10.1124/jpet.103.060541] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
It has been demonstrated recently that rabbit renal proximal tubule cells (RPTC) express a novel Ca(2+)-independent phospholipase A(2) (iPLA(2)) whose activity localizes to the endoplasmic reticulum (ER-iPLA(2)) and is similar to group VIB PLA(2). In this study, the expression of group VIB PLA(2) was examined and the role of ER-iPLA(2) in cisplatin-induced apoptosis was determined. Cisplatin induced both time- and concentration-dependent RPTC apoptosis as determined by p53 nuclear localization, annexin V staining, caspase 3 activity, and chromatin condensation. Inhibition of ER-iPLA(2) with bromoenol lactone (5 microM) reduced cisplatin-induced annexin V binding 40%, chromatin condensation 55%, and caspase 3 activity 42%, but had no effect on p53 nuclear localization. Treatment of RPTC with the protein kinase C stimulator phorbol 12-myristate 13-acetate increased the activity of ER-iPLA(2) 2-fold and increased cisplatin-induced RPTC apoptosis. These studies demonstrate that group VIB PLA(2) is expressed in RPTC and suggest that RPTC ER-iPLA(2) is the rabbit homolog of group VIB PLA(2). These data also demonstrate that ER-iPLA(2) acts downstream of p53 and upstream of caspase 3 to mediate cisplatin-induced RPTC apoptosis. Finally, ER-iPLA(2) seems to be regulated by protein kinase C.
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Affiliation(s)
- Brian S Cummings
- Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA
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Kaufmann JA, Perez M, Zhang W, Bickford PC, Holmes DB, Taglialatela G. Free radical-dependent nuclear localization of Bcl-2 in the central nervous system of aged rats is not associated with Bcl-2-mediated protection from apoptosis. J Neurochem 2004; 87:981-94. [PMID: 14622128 DOI: 10.1046/j.1471-4159.2003.02092.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
We have previously reported that Bcl-2 is up-regulated in the CNS of aged F344 rats as a consequence of oxidative stress. In addition to increased levels of expression, we now report that there is a subcellular redistribution of Bcl-2 in the CNS of aged F344 rats. Using western blotting, we found Bcl-2 predominantly located in the cytosol of young rats. However, in aged rats Bcl-2 was found primarily in the nucleus. This distribution, in the hippocampus and cerebellum, was reversed by treatment with the nitrone spin trap N-tert-butyl-alpha-phenylnitrone (PBN). Paradoxically, PBN treatment in young rats had the opposite effect, changing Bcl-2 from predominantly cytosolic to nuclear. We also detected an increase in Bax in aged hippocampal samples (both nuclear and cytosolic), which was reversed by treatment with PBN. The distribution of Bcl-2 and Bax in the cytosol of aged rats dramatically decreased the Bcl-2/Bax ratio, a probable indicator of neuronal vulnerability, which was restored upon treatment with PBN. In order to assess the effect of nuclear association of Bcl-2 we used PC12 cells stably transfected with a Bcl-2 construct to which we added the nuclear localization sequence of the SV40 large T antigen to the N-terminus which resulted in nuclear targeting of Bcl-2. Measurement of cell death using lactate dehydrogenase assays showed that, contrary to wild-type Bcl-2, Bcl-2 localized to the nucleus was not effective in protecting cells from treatment with 250 microm H2O2. These results suggest that nuclear localization of Bcl-2 observed in the aged CNS may not reflect a protective mechanism against oxidative stress, a major component of age-associated CNS impairments.
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Affiliation(s)
- Joel A Kaufmann
- Department of Anatomy and Neurosciences, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1043, USA
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Kinkel MD, Yagi R, McBurney D, Nugent A, Horton WE. Age-related expression patterns of Bag-1 and Bcl-2 in growth plate and articular chondrocytes. ACTA ACUST UNITED AC 2004; 279:720-8. [PMID: 15278942 DOI: 10.1002/ar.a.20063] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Aging cartilage displays increased chondrocyte apoptosis and decreased responsiveness of chondrocytes to growth factors. The molecular mechanisms responsible for these changes have not been identified. Bag-1 is a Bcl-2-binding protein that promotes cell survival, interacts with a diverse group of cellular proteins, and may integrate multiple pathways involved in controlling cell survival, growth, and phenotype. Bcl-2 is important for maintaining chondrocyte phenotype and delaying terminal differentiation and apoptosis of chondrocytes. Comparatively little is known about the role of Bag-1 in cartilage. Here we show that both growth plate and articular chondrocytes in the mouse express the Bag-1 protein. In the growth plate, Bag-1 expression is prominent in the late proliferative and prehypertrophic chondrocytes, displaying a pattern similar to what has been reported for Bcl-2. Further, the expression of both Bcl-2 and Bag-1 declines with age in the articular cartilage. Growth assays demonstrate that knocking down Bag-1 expression causes a decrease in growth rate. These results suggest that Bag-1 is involved in the regulation of chondrocyte phenotype and cartilage aging.
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Affiliation(s)
- Mary D Kinkel
- Department of Anatomy, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272, USA.
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27
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Spurgers KB, Coombes KR, Meyn RE, Gold DL, Logothetis CJ, Johnson TJ, McDonnell TJ. A comprehensive assessment of p53-responsive genes following adenoviral-p53 gene transfer in Bcl-2-expressing prostate cancer cells. Oncogene 2003; 23:1712-23. [PMID: 14647426 DOI: 10.1038/sj.onc.1207293] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
The p53 protein can induce cell cycle arrest or apoptosis following activation in response to DNA damage. The function of p53 is largely mediated by regulating the expression of downstream target genes. Adenoviral-p53 gene transfer (Ad-p53) is currently being evaluated in clinical trials as a therapeutic intervention. Tumor response is likely to be influenced by context-dependent variables, such as expression of bcl-2. Bcl-2 is upregulated in a variety of neoplasms, and can inhibit p53-dependent apoptosis. It was therefore of interest to use a global genomic strategy to assess gene expression following Ad-p53 gene transfer and to determine if the expression of specific Ad-p53-responsive genes could be modulated in the context of bcl-2 gene deregulation. cDNA arrays were used to identify p53-responsive genes following Ad-p53 gene transfer in control and bcl-2-overexpressing PC3 prostate cancer cells. A total of 40 transcripts were significantly upregulated by Ad-p53 in both control and bcl-2-transfectant PC3 cells. Conversely, 19 transcripts were significantly repressed in both cell lines. These Ad-p53-responsive transcripts included previously identified p53 targets, known genes representing candidate p53 targets, and transcripts identified as expressed sequence tags. A subset of 15 transcripts was differentially modulated by Ad-p53 in the context of bcl-2. Some of these genes were also differentially modulated in LNCaP (wt p53) cells following DNA damage. These results document a number of potential p53 targets and mediators of therapeutically relevant genotoxic stress. The findings further suggest that bcl-2 may inhibit cell death at multiple points downstream of p53 activation.
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Affiliation(s)
- Kevin B Spurgers
- Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
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Pham TD, MacLennan NK, Chiu CT, Laksana GS, Hsu JL, Lane RH. Uteroplacental insufficiency increases apoptosis and alters p53 gene methylation in the full-term IUGR rat kidney. Am J Physiol Regul Integr Comp Physiol 2003; 285:R962-70. [PMID: 12869365 DOI: 10.1152/ajpregu.00201.2003] [Citation(s) in RCA: 184] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Uteroplacental insufficiency causes intrauterine growth retardation (IUGR), which is associated with adult onset diseases such as hypertension. Previous studies demonstrate that growth retardation in humans and rats decreases glomeruli number; however, the molecular mechanisms responsible for this reduction are unknown. Apoptosis plays a key role in renal organogenesis. We therefore hypothesized that the in utero deprivation associated with uteroplacental insufficiency decreases glomeruli, increases apoptosis, and alters the mRNA levels of key apoptosis-related proteins in full-term IUGR kidneys. To prove this hypothesis, we induced asymmetric IUGR through bilateral uterine artery ligation of the pregnant rat. We found that uteroplacental insufficiency significantly reduced glomeruli number while increasing TUNEL staining and caspase-3 activity in the IUGR kidney. A significant decrease in Bcl-2 mRNA and a significant increase in Bax and p53 mRNA further characterized the IUGR kidney. Because altered p53 CpG methylation affects p53 expression, we analyzed p53 promoter CpG methylation using methylation-sensitive restriction enzymes and real-time PCR. Uteroplacental insufficiency specifically decreased CpG methylation of the renal p53 BstU I site promoter without affecting the Hha I or the Aci I sites. Uteroplacental insufficiency also induced a relative hypomethylation from exon 5 to exon 8, which was associated with deceased mRNA levels of DNMT1. We conclude that uteroplacental insufficiency alters p53 DNA CpG methylation, affects mRNA levels of key apoptosis-related proteins, increases renal apoptosis, and reduces glomeruli number in the IUGR kidney. We speculate that these changes represent mechanisms that contribute to the fetal origins of adult disease.
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Affiliation(s)
- Tho D Pham
- Univ. of Utah School of Medicine, Dept. of Pediatrics and Division of Neonatology, 30 North 1900 East Rm. 2A100, Salt Lake City, UT 84132-2202, USA
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Ruiz-Ruiz C, Robledo G, Cano E, Redondo JM, Lopez-Rivas A. Characterization of p53-mediated up-regulation of CD95 gene expression upon genotoxic treatment in human breast tumor cells. J Biol Chem 2003; 278:31667-75. [PMID: 12788915 DOI: 10.1074/jbc.m304397200] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Death receptor CD95 gene expression is frequently low in human breast tumors and is up-regulated by genotoxic treatments in a p53-dependent manner. We have evaluated the relative contribution of promoter and intronic p53 consensus sites to the regulation of the human CD95 gene in breast tumor cells following doxorubicin treatment. Deletion constructs of the promoter region and site-directed mutagenesis of p53 consensus sites in a fragment spanning 1448 bp of the 5'-promoter demonstrate that these sites are not involved in the observed up-regulation of the CD95 gene upon doxorubicin treatment. In contrast, a p53 consensus site located within the first intron of CD95 gene is absolutely required for the inducible expression of CD95 upon genotoxic treatment in breast tumor cells. Analysis of the transcriptional activity of the two most common p53 mutants found in human breast tumors that are associated with resistance to doxorubicin reveals that these mutations completely eliminate the ability of p53 protein to transactivate CD95 gene expression. On the other hand, Bcl-2 overexpression albeit preventing doxorubicin-induced apoptosis, has no effect on p53-mediated CD95 up-regulation in breast tumor cells. Altogether, these results indicate the lack of involvement of p53 consensus sites of the CD95 promoter region and the pivotal role of intronic p53-responsive element in the regulation of human CD95 gene expression in breast tumor cells. Our results also suggest that in breast cancer patients with certain mutations in the p53 gene, expression of death receptor CD95 in response to genotoxic treatments could be severely compromised.
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Affiliation(s)
- Carmen Ruiz-Ruiz
- Instituto de Parasitología y Biomedicina, CSIC, and the Universidad de Granada, Spain
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Chan HC, Kuo SC, Huang LJ, Liu CH, Hsu SL. A phenylacetate derivative, SCK6, inhibits cell proliferation via G1 cell cycle arrest and apoptosis. Eur J Pharmacol 2003; 467:31-9. [PMID: 12706452 DOI: 10.1016/s0014-2999(03)01596-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Phenylacetate is a differentiation agent and has anticancer activity with relatively low toxicity. In the present study, we examined the anticancer effect of six synthetic phenylacetate derivatives in human lung cancer cells in our search for more effective phenylacetate analogous. Results showed that the antiproliferative effects of these synthetic compounds were stronger than those of phenylacetate, and that N-butyl-2-(2-fluorolphenyl)acetamide (SCK6) is the most potent compound. To address the mechanism of the antiproliferative effect of SCK6, cell cycle analysis was performed. Result showed that SCK6 (1 mM) induced G(1) arrest in CH27 cells. Western blot analysis of G(1) phase regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase 2 (Cdk2), Cdk4, Cyclin E and Cyclin D3 were decreased after treatment with SCK6 but not those of Cdk6, Cyclin D1 and D2. In contrast, SCK6 increased the protein levels of p53 and p21(CIP1/WAF1). Data from in situ terminal transferase-mediated dUTP-fluorescensin nick end-labeling (TUNEL) assay and DNA fragmentation analysis demonstrated that SCK6 induced apoptotic cell death in CH27 cells. This SCK6-induced apoptosis was accompanied by a downregulation of Bcl-2 protein and activation of the caspase-9 cascade. Overexpression of Bcl-2 by adeno-Bcl-2 vector infection significantly inhibited SCK6-induced apoptosis. Moreover, treatment with caspase inhibitors also markedly reduced cell death induced by SCK6. Taken together, these results suggest that downregulation of G(1)-associated Cdks and cyclins and upregulation of p53 and p21(CIP1/WAF1) may contribute to SCK6-mediated G(1)-phase arrest. Furthermore, the decrease in Bcl-2 and the activation of caspase-9/caspase-3 may be the effector mechanism through which SCK6 induces apoptosis.
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Affiliation(s)
- Hsu-Chin Chan
- Department of Biochemistry, China Medical College, Taichung, Taiwan, ROC
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Everett H, Barry M, Sun X, Lee SF, Frantz C, Berthiaume LG, McFadden G, Bleackley RC. The myxoma poxvirus protein, M11L, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore. J Exp Med 2002; 196:1127-39. [PMID: 12417624 PMCID: PMC2194110 DOI: 10.1084/jem.20011247] [Citation(s) in RCA: 83] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Abstract
M11L, an antiapoptotic protein essential for the virulence of the myxoma poxvirus, is targeted to mitochondria and prevents the loss of mitochondrial membrane potential that accompanies cell death. In this study we show, using a cross-linking approach, that M11L physically associates with the mitochondrial peripheral benzodiazepine receptor (PBR) component of the permeability transition (PT) pore. Close association of M11L and the PBR is also indicated by fluorescence resonance energy transfer (FRET) analysis. Stable expression of M11L prevents the release of mitochondrial cytochrome c induced by staurosporine or protoporphyrin IX (PPIX), a ligand of the PBR. Transiently expressed M11L also prevents mitochondrial membrane potential loss induced by PPIX, or induced by staurosporine in combination with PK11195, another ligand of the PBR. Myxoma virus infection and the associated expression of early proteins, including M11L, protects cells from staurosporine- and Fas-mediated mitochondrial membrane potential loss and this effect is augmented by the presence of PBR. We conclude that M11L regulates the mitochondrial permeability transition pore complex, most likely by direct modulation of the PBR.
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Affiliation(s)
- Helen Everett
- Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G2H7, Canada
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Chen SS, Chang PC, Cheng YW, Tang FM, Lin YS. Suppression of the STK15 oncogenic activity requires a transactivation-independent p53 function. EMBO J 2002; 21:4491-9. [PMID: 12198151 PMCID: PMC126178 DOI: 10.1093/emboj/cdf409] [Citation(s) in RCA: 89] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Using a transactivation-defective p53 derivative as bait, STK15, a centrosome-associated oncogenic serine/threonine kinase, was isolated as a p53 partner. The p53-STK15 interaction was confirmed further by co-immunoprecipitation and GST pull-down studies. In co-transfection experiments, p53 suppressed STK15-induced centrosome amplification and cellular transformation in a transactivation-independent manner. The suppression of STK15 oncogenic activity by p53 might be explained in part by the finding that p53 inhibited STK15 kinase activity via direct interaction with the latter's Aurora box. Taken together, these findings revealed a novel mechanism for the tumor suppressor function of p53.
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Affiliation(s)
- Shih-Shun Chen
- Institute of Biomedical Sciences, Academia Sinica, Taipei 115 and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 100, Taiwan Present address: Institute of Pharmaceutical Science, Taipei Medical University, Taipei, Taiwan Corresponding author e-mail:
| | - Pi-Chu Chang
- Institute of Biomedical Sciences, Academia Sinica, Taipei 115 and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 100, Taiwan Present address: Institute of Pharmaceutical Science, Taipei Medical University, Taipei, Taiwan Corresponding author e-mail:
| | - Yu-Wen Cheng
- Institute of Biomedical Sciences, Academia Sinica, Taipei 115 and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 100, Taiwan Present address: Institute of Pharmaceutical Science, Taipei Medical University, Taipei, Taiwan Corresponding author e-mail:
| | - Fen-Mei Tang
- Institute of Biomedical Sciences, Academia Sinica, Taipei 115 and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 100, Taiwan Present address: Institute of Pharmaceutical Science, Taipei Medical University, Taipei, Taiwan Corresponding author e-mail:
| | - Young-Sun Lin
- Institute of Biomedical Sciences, Academia Sinica, Taipei 115 and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 100, Taiwan Present address: Institute of Pharmaceutical Science, Taipei Medical University, Taipei, Taiwan Corresponding author e-mail:
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Abstract
OBJECTIVE To review the literature published in the past 6 years concerning the role of p53 tumor-suppressor protein in rheumatoid arthritis (RA). METHODS A MEDLINE search was performed to identify all publications that covered the role of p53 in RA. In addition, selected articles related to proto-oncogenes and matrix metalloproteinases were included in this review. RESULTS p53 protein is expressed in RA fibroblast-like synoviocytes (FLSs), and its overexpression is a characteristic feature of RA. The overexpression of p53 is probably induced by DNA strand breaks caused by the genotoxic environment of RA joints, in some cases because of p53 mutations. Independent studies from 3 groups indicated that p53 mutations can and do occur in RA synovial tissue samples derived from a subset of RA patients. Inactivation of p53 may contribute to the invasiveness of FLSs and to the high-level expression of cartilage degradation enzymes as well. Gene transfer or gene knockout studies using a collagen-II-induced RA animal model to examine the role of p53 in RA have been reported. Initial results are positive and indicate that gene transfer of p53 may be clinically useful for the management of RA. CONCLUSIONS p53 protein is expressed in RA FLSs, and its overexpression is a characteristic feature of RA. p53 mutations occur in the synovial tissues derived from a subset of RA patients. The clinical implications of p53 expression and the functional importance of somatic mutations in RA, however, are still unclear. Further research is needed to fully understand the implications of these findings and develop corresponding new therapeutic strategies.
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Affiliation(s)
- Yubo Sun
- Department of Medicine, University of Miami School of Medicine, FL, USA
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Abstract
The tumor suppressor protein, p53, is often referred to as the guardian of the genome. When p53 function is impaired, its ability to preserve genomic integrity is compromised. This may result in an increase in mutation on both a molecular and chromosomal level and contribute to the progression to a malignant phenotype. In order to study the effect of p53 function on the acquisition of mutation, in vitro and in vivo models have been developed in which both the frequency and mechanism of mutation can be analyzed. In human lymphoblastoid cells in which p53 function was impaired, both the spontaneous and induced mutant frequency increased at the autosomal thymidine kinase (TK) locus. The mutant frequency increased to a greater extent in cell lines in which p53 harbored a point mutation than in those lines in which a "null" mutation had been introduced by molecular targeting or by viral degradation indicating a possible "gain-of-function" associated with the mutant protein. Further, molecular analysis revealed that the loss of p53 function was associated with a greater tendency towards loss-of-heterozygosity (LOH) within the TK gene that was due to non-homologous recombination than that found in wild-type cells. Most data obtained from the in vivo models uses the LacI reporter gene that does not efficiently detect mutation that results in LOH. However, studies that have examined the effect of p53 status on mutation in the adenine phosphoribosyl transferase (APRT) gene in transgenic mice also suggest that loss of p53 function results in an increase in mutation resulting from non-homologous recombination. The results of these studies provide clear and convincing evidence that p53 plays a role in modulating the mutant frequency and the mechanism of mutation. In addition, the types of mutation that occur within the p53 gene are also of importance in determining the mutant frequency and the pathways leading to mutation.
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Affiliation(s)
- Suzanne M Morris
- Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA.
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Abstract
Changes in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) translate a variety of extracellular signals into widely diverse intracellular effects, ranging from secretion to movement, proliferation and also cell death. As regards the last one, it has long been known that large [Ca(2+)](c) increases lead cells to death. More recently, experimental evidence has been obtained that the oncogene Bcl-2 reduces the state of filling of intracellular Ca(2+) stores and thus affects the Ca(2+) responses induced by physiological and pathological stimuli. In this contribution, we will discuss this effect and its significance for the mechanism of action of Bcl-2, an important checkpoint of the apoptotic process.
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Affiliation(s)
- Paolo Pinton
- Department of Biomedical Sciences and CNR Center for the Study of Biomembranes, University of Padova, Via Colombo 3, 35121 Padova, Italy
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Mao YW, Xiang H, Wang J, Korsmeyer S, Reddan J, Li DW. Human bcl-2 gene attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis through down-regulation of the alpha B-crystallin gene. J Biol Chem 2001; 276:43435-45. [PMID: 11546795 DOI: 10.1074/jbc.m102195200] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Regarding the mechanism by which BCL-2 prevents cell death, one theory suggests that it acts by protecting cells from oxidative stress. In the lens system, oxidative stress-induced apoptosis is implicated in cataractogenesis. To explore the possibility of anti-apoptotic gene therapy development for cataract prevention and also to further test the anti-oxidative stress theory of BCL-2 action, we have introduced the human bcl-2 gene into an immortalized rabbit lens epithelial cell line, N/N1003A. The stable expression clones of both vector- and bcl-2-transfected cells have been established. Treatment of the two cell lines with H(2)O(2) revealed that bcl-2-transfected cells were less capable of detoxifying H(2)O(2) than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H(2)O(2)-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H(2)O(2)-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alphaB-crystallin gene was distinctly down-regulated in bcl-2-transfected cells compared with that in vector-transfected cells. This down-regulation was specific because a substantial inhibition of BCL-2 expression through antisense bcl-2 RNA significantly restored the level of alphaB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H(2)O(2)-induced apoptosis. Introduction of a mouse alphaB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alphaB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alphaB-crystallin prevents apoptosis through interaction with procaspase-3 and partially processed procaspase-3 to prevent caspase-3 activation. Together, our results reveal that BCL-2 can regulate gene expression in rabbit lens epithelial cells. Through down-regulation of the alphaB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H(2)O(2)-induced apoptosis.
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Affiliation(s)
- Y W Mao
- Department of Molecular Biology, University of Medicine and Dentistry of New Jersey School of Osteopathic Medicine, Stratford, New Jersey 08084, USA
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Kang BN, Jeong KS, Park SJ, Kim SJ, Kim TH, Kim HJ, Ryu SY. Regulation of apoptosis by somatostatin and substance P in peritoneal macrophages. REGULATORY PEPTIDES 2001; 101:43-9. [PMID: 11495678 DOI: 10.1016/s0167-0115(01)00264-6] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Recent studies have shown that somatostatin (SOM) inhibits interleukin 6 (IL-6) and interferon gamma (IFNgamma) production by lymphocytes and peritoneal macrophages, whereas substance P (SP) enhances these cytokines production. To define the mechanism of the cytokine production enhancements and inhibitions by SOM and SP, we examined the expression of apoptosis modulator, p53, Bcl-2, Bax, inducible nitric oxide synthase (iNOS), Fas, caspase-8 and nitric oxide (NO) in thioglycolate-elicited peritoneal macrophages. SOM caused up-regulation of p53, Bcl-2, Fas and caspase-8 activities, and down-regulation of iNOS expression and NO production. On the other hand, SP slightly induces p53 and highly induces Bcl-2, iNOS expression and NO production. These data suggest that apoptosis by SOM may occur by a Bax- and NO-independent p53 accumulation, and through Fas and caspase-8 activation pathways, and that the inducible expression of Bcl-2 and NO production by SP may contribute to prevent the signals of apoptosis by Bax, and via Fas and caspase-8 activation.
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Affiliation(s)
- B N Kang
- Laboratory of Veterinary Anatomy, College of Veterinary Medicine, Chungnam National University, 305-764, Taejeon, South Korea
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Park SA, Park HJ, Lee BI, Ahn YH, Kim SU, Choi KS. Bcl-2 blocks cisplatin-induced apoptosis by suppression of ERK-mediated p53 accumulation in B104 cells. BRAIN RESEARCH. MOLECULAR BRAIN RESEARCH 2001; 93:18-26. [PMID: 11532334 DOI: 10.1016/s0169-328x(01)00176-0] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Bcl-2 has been reported to inhibit neurotoxicity induced by cisplatin. However, neither the mechanism of cisplatin-induced neurotoxicity nor the mechanism by which Bcl-2 confers neuroprotection is clear. In this study, the signaling pathways involved in cisplatin-induced neurotoxicity were examined using a rat neuroblastoma cell line, B104. Treatment of B104 cells with cisplatin induced apoptosis, accompanying the accumulation of p53 and Bax protein. Interestingly, extracellular signal-regulated kinase 1/2 (ERK1/2) activities of MAP kinases were markedly enhanced prior to cisplatin-induced accumulation of p53 and Bax. Inhibition of ERK1/2 activities using PD98059, a selective MEK inhibitor, blocked the apoptotic cell death preventing cisplatin-induced accumulation of p53 and Bax. These results suggest that ERK mediates cisplatin-induced p53 activation to trigger apoptosis in B104 cells. Overexpression of Bcl-2 in B104 cells resulted in the complete resistance to cisplatin-induced apoptosis blocking ERK activation and the subsequent signaling pathway of p53. Our study clearly demonstrates that the action site of Bcl-2 localizes upstream of ERK in cisplatin-induced apoptotic signaling pathway.
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Affiliation(s)
- S A Park
- Department of Neurology, Yonsei University College of Medicine, Seoul, South Korea
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39
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Somogyi RD, Wu Y, Orlofsky A, Prystowsky MB. Transient expression of the Bcl-2 family member, A1-a, results in nuclear localization and resistance to staurosporine-induced apoptosis. Cell Death Differ 2001; 8:785-93. [PMID: 11526431 DOI: 10.1038/sj.cdd.4400879] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2000] [Revised: 02/06/2001] [Accepted: 03/08/2001] [Indexed: 11/09/2022] Open
Abstract
The Bcl-2 family of proteins has been characterized by either anti-apoptotic or pro-apoptotic activity. Insight into how Bcl-2 family members function has been gained by determining their intracellular localization. We have generated a monoclonal anti-A1-a antibody and used a COS-7 overexpression system to study the localization of the murine anti-apoptotic Bcl-2 family member, A1-a. A1-a overexpressed in COS-7 cells localized to the nucleus as determined by subcellular fractionation and immunofluorescent microscopy. A1-a in the COS-7 nucleus bound tightly to the nuclear matrix as evidenced by resistance to treatment with DNAse I and RNAse A and sequential extraction with 1.0% Triton X-100, 0.15 M NaCl, 0.25 M HCl, 0.5 M Tris pH 7.4 and 6 M urea. HPLC analysis of A1-a, subsequent to SDS extraction, produced fractions that gave multiple bands when analyzed by Western blot analysis suggesting a propensity to form multimers. COS-7 cells transfected with A1-a were protected from apoptotic induction by staurosporine treatment.
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Affiliation(s)
- R D Somogyi
- Albert Einstein College of Medicine, Department of Pathology, 1300 Morris Park Ave, Bronx, NY 10461, USA
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Roth JA, Grammer SF, Swisher SG, Nemunaitis J, Merritt J, Meyn RE. Gene replacement strategies for treating non-small cell lung cancer. Semin Radiat Oncol 2000; 10:333-42. [PMID: 11040334 DOI: 10.1053/srao.2000.9127] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Fewer than 15% of the 170,000 patients who develop lung cancer each year will survive their disease, which shows the need for novel, more specific, and less toxic therapeutic strategies. Recent advances in molecular biology have made it possible to ascertain which genetic alterations contribute to the etiology of cancer. For example, the tumor-suppressor gene, p53, responsible for directing repair of damaged DNA or committing a cell to apoptosis, is mutated or otherwise altered in more than 50% of cancers, including 40% to 70% of non-small cell lung cancers. Many p53-deficient tumors have proven remarkably resistant to radiotherapy and chemotherapy. The preclinical and clinical studies of gene therapy reviewed in this article show (1) successful transfer and expression of a potentially therapeutic p53 gene construct in tumor cells, (2) observation of antitumor effects in vitro and in vivo, and (3) most critically, a lack of significant toxicity. The results of these studies indicate that gene replacement therapy is a feasible alternative therapy for cancer. In addition, these studies show that transfer of the p53 gene can induce radiation sensitization in previously radiation-resistant tumors, leading to the possibility of new therapeutic protocols combining gene replacement with radiation therapy.
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Affiliation(s)
- J A Roth
- Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA
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del Mar Martínez-Senac M, Corbalán-García S, Gómez-Fernández JC. Study of the secondary structure of the C-terminal domain of the antiapoptotic protein bcl-2 and its interaction with model membranes. Biochemistry 2000; 39:7744-52. [PMID: 10869179 DOI: 10.1021/bi000256h] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Bcl-2 is a protein which inhibits programmed cell death. It is associated to many cell membranes such as mitochondrial outer membrane, endoplasmic reticulum, and nuclear envelope, apparently through a C-terminal hydrophobic domain. We have used infrared spectroscopy to study the secondary structure of a synthetic peptide (a 23mer) with the same sequence as this C-terminal domain (residues 217-239) of Bcl-2. The spectrum of this peptide in D(2)O buffer shows an amide I' band with a maximum at 1622 cm(-1), which clearly indicates its tendency to aggregate in aqueous solvent. However, the peptide incorporated in multilamellar phosphatidylcholine membranes shows a totally different spectrum of the amide I' band, with a maximum at 1655 cm(-)(1), indicating a predominantly alpha-helical structure. Addition of the peptide to unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein. Differential scanning calorimetry of dimyristoylphosphatidylcholine multilamellar vesicles in which the peptide was incorporated revealed that increasing concentrations of the peptide progressively broadened the pretransition and the main transition, as is to be expected for a membrane integral molecule. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in fluid phosphatidylcholine vesicles showed that increasing concentrations of the peptide produced increased polarization values, pointing to an increase in the apparent order of the membrane and indicating that high concentrations of the peptide considerably broaden the phase transition of dimyristoylphosphatidylcholine multilamellar vesicles. Quenching the intrinsic fluorescence of the Tyr-235 of the peptide, by KI, indicated that this aminoacyl residue is highly exposed to aqueous solvent when incorporated in phospholipid vesicles. The results are discussed in terms of their relevance to the proposed topology of insertion of Bcl-2 into biological membranes.
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Affiliation(s)
- M del Mar Martínez-Senac
- Departamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30080 Murcia, Spain
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Liu JS, Kuo SR, McHugh MM, Beerman TA, Melendy T. Adozelesin triggers DNA damage response pathways and arrests SV40 DNA replication through replication protein A inactivation. J Biol Chem 2000; 275:1391-7. [PMID: 10625690 DOI: 10.1074/jbc.275.2.1391] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesin-treated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect on in vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.
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Affiliation(s)
- J S Liu
- Department of Microbiology and the Center for Microbial Pathogenesis, State University of New York School of Medicine and Biomedical Sciences, Buffalo, New York 14214, USA
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Abstract
The p53 tumor suppressor protein plays a crucial role in regulating cell growth following exposure to various stress stimuli. p53 induces either growth arrest, which prevents the replication of damaged DNA, or programmed cell death (apoptosis), which is important for eliminating defective cells. Whether the cell enters growth arrest or undergoes apoptosis, depends on the final integration of incoming signals with antagonistic effects on cell growth. Many factors affect the cellular response to activated p53. These include the cell type, the oncogenic status of the cell with emphasis on the Rb/E2F balance, the extracellular growth and survival stimuli, the intensity of the stress signals, the level of p53 expression and the interaction of p53 with specific proteins. p53 is regulated both at the levels of protein stability and biochemical activities. This complex regulation is mediated by a range of viral and cellular proteins. This review discusses this intriguing complexity which affects the cell response to p53 activation.
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Affiliation(s)
- R V Sionov
- Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem, 91120, Israel
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Affiliation(s)
- T F Burns
- Laboratory of Molecular Oncology, Howard Hughes Medical Institute, Department of Medicine, Philadelphia, Pennsylvania, USA
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Thangaraju M, Sharma K, Leber B, Andrews DW, Shen SH, Srikant CB. Regulation of acidification and apoptosis by SHP-1 and Bcl-2. J Biol Chem 1999; 274:29549-57. [PMID: 10506221 DOI: 10.1074/jbc.274.41.29549] [Citation(s) in RCA: 88] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Recruitment of the SH2 domain containing cytoplasmic protein-tyrosine phosphatase SHP-1 to the membrane by somatostatin (SST) is an early event in its antiproliferative signaling that induces intracellular acidification-dependent apoptosis in breast cancer cells. Fas ligation also induces acidification-dependent apoptosis in a manner requiring the presence of SHP-1 at the membrane. Moreover, we have recently reported that SHP-1 is required not only for acidification, but also for apoptotic events that follow acidification (Thangaraju, M., Sharma, K., Liu, D., Shen, S. H., and Srikant, C. B. (1999) Cancer Res. 59, 1649-1654). Here we show that ectopically expressed SHP-1 was predominantly membrane-associated and amplified the cytotoxic signaling initiated upon SST receptor activation and Fas ligation. The catalytically inactive mutant of SHP-1 (SHP-1C455S) abolished the ability of the SST agonists to signal apoptosis by preventing the recruitment of wild type SHP-1 to the membrane. Overexpression of the anti-apoptotic protein Bcl-2 in MCF-7 cells inhibited SST-induced apoptosis upstream of acidification by inhibiting p53-dependent induction of Bax as well as by raising the resting pH(i) and attenuating SST-induced decrease in pH(i). By contrast, Bcl-2 failed to prevent apoptosis triggered by direct acidification. These data demonstrate that (i) membrane-associated SHP-1 is required for receptor-mediated cytotoxic signaling that causes intracellular acidification and apoptosis, and (ii) Bcl-2 acts distal to SHP-1 and p53 to prevent SST-induced acidification but cannot inhibit the apoptotic events that ensue intracellular acidification.
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Affiliation(s)
- M Thangaraju
- Fraser Laboratories, Department of Medicine, McGill University and Royal Victoria Hospital, Montreal, Quebec, H3A 1A1
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